ABSTRACT

THE ROLE OF MECHANICAL AND CHEMICAL PROCESSES IN RHODALGAL SEDIMENT PRODUCTION AND IMPLICATIONS FOR ACIDIFICATION (BAJA CALIFORNIA, MÉXICO)

Rhodoliths are free-living that produce carbonate sediments in shallow water marine systems worldwide. Rhodalgal sediments, which result from rhodolith breakage and chemical weathering, mix together with living rhodoliths and form shallow water habitats known as rhodolith beds, support a diverse assemblage of organisms. Rhodoliths and sediment cores collected from the El Requesón rhodolith bed in Bahía Concepción, México were used to study the basic mechanical and chemical processes involved in rhodalgal sediment production and basic framework of a rhodolith bed. Results showed four major groups of rhodalgal sediments produced from rhodolith breakdown: "cores," "branches," "crumbs," and “dust” that ranges in size from pebbles, sands, to silts. Dissolution of rhodoliths was evident at seawater below pH 7.5 and at 30% dissolution, core breakdown was accelerated and smaller branches were produced. The general vertical trend of coarse-fine-coarse sedimentary texture indicates the temporal dynamics of a rhodolith bed, suggesting movement in south-north-south direction or expansion-shrinkage-expansion of the active part of the bed from past to present. The effect of 30% dissolution seen in the experiment suggests that dissolution occurring in future high CO2 ocean conditions would cause structural changes to shift towards more compacted framework with smaller interstitial spaces, hence changing the habitat quality of the bed.

Elsie Dekawati Tanadjaja December 2010

THE ROLE OF MECHANICAL AND CHEMICAL PROCESSES IN RHODALGAL SEDIMENT PRODUCTION AND IMPLICATIONS FOR (BAJA CALIFORNIA, MÉXICO)

by Elsie Dekawati Tanadjaja

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Marine Science in the College of Science and Mathematics California State University, Fresno December 2010 APPROVED For the Department of Biology:

We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.

Elsie Dekawati Tanadjaja Thesis Author

Ivano Aiello (Chair) Moss Landing Marine Laboratories

Kenneth Coale Moss Landing Marine Laboratories

Diana Steller Moss Landing Marine Laboratories

For the University Graduate Committee:

Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS

X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.

Permission to reproduce this thesis in part or in its entirety must be obtained from me.

:rohtua siseht fo erutangiS fo siseht :rohtua ACKNOWLEDGMENTS The completion and success of this thesis have depended upon continuous support, generous help, and encouragement of my advisors, mentors, colleagues, friends, and family. My sincere appreciation goes out to my advisors Dr. Ivano Aiello, Dr. Kenneth Coale, and Dr. Diana Steller for their enthusiasm, encouragement, and belief that I could overcome any challenge. Dr. Aiello’s broad knowledge and interest has encouraged me to explore new areas of multi-disciplinary research. Dr. Coale’s patience and unique perspectives about the natural world has inspired and magnify my commitment to science. Dr. Steller’s dedication to the rhodolith world, Baja, and her many field experiences were a source of inspiration and have been instrumental in completing my project. A deep thanks to my dedicated team of interns: Alex Olson, Amber Stroeder, Rachel Pitts, and Ekow Edzie for their hard work, refreshing curiosity, dedication, and the motivation for me to be a good mentor. A sincere thanks to folks in Monterey Bay Aquarium Research Institute for their valuable help, support, and mentorship. In particular, I thank Dr. Jim Barry for letting me use his lab facility to run my dissolution experiment, Gernot Friederich for his continuous support and generous advice and laughter, Chris Lovera for his time and effort with helping me develop and troubleshoot the dissolution experiment, Marguerite Blum for her expertise with the DIC machine, and Kurt Buck for his resourcefulness and friendship. A warm thanks to Dr. Shannon Bros for her wisdoms, guidance with statistics, and optimistic outlook of complicated situations. A sincere thanks to Dr. Simona Bartl for providing me with opportunities to teach and share my vi excitement about the marine world and for her generous advice. Deep appreciation to Dr. Jim Harvey, Aurora Alifano, Selena McMillan, Megan Wehrenberg, Paul Chua, Colleen Young, Jasmine Ruvalcaba, Rosemary Romero, Thew Suskiewicz, Brian Hoover, Gabriel Rodriguez, Paul Tompkins, Hilary Hayford, Marina Salonga, Didi Tanadjaja, Joelle Sweeney, and Dan Strain for helping me with field work and processing large amounts of sediment samples and data. Special thanks to Derek Larson for his expertise with computer programming and statistics coupled with laughter and silliness. A sincere thanks to rhodolith lovers, Dr. Michael Foster and Dr. Rafael Riosmena-Rodríguez, for their insights into rhodoliths. A wholehearted thanks to my friends who were the caretakers of my sanity and a much needed source of inspiration. A mighty thanks to my best friend and personal editor Cassandra Brooks for her deep engagement in my life. A heartfelt thanks to Marina Salonga for being my best friend my first exploration into the marine world. A deep thanks to Eleonora Morelli for her deep friendship and continued love and support from near and far. Thanks to Audrey Ching for reliable cheerleading and comic relief. Thanks to Erin Loury for dependable motivational speeches. Thanks to Tracy Lerman and Leon Vehaba for providing a comfortable space to come home to and healthy food that nurtured my soul. Thanks to the twins, Ashley Greenley and Kristen Green, for bringing delightful ridiculousness to my life. Thanks also to Hilary Hayford, Hideyo Hattori, Chris Scianni, Charlie and Ann Endris, Lily Dayton, Joelle Sweeney, Phil Hoos, and Vijitha Ariyaratne for their endless friendship. Special thanks to folks at MLML including members of Geological Oceanography, the excellent faculty, the library staff, the IT staff, the vii administrative staff, and the Shop guys. Thanks also to the administrative staff in CSU Fresno who are so competent in what they do. My heartfelt gratitude goes out to my family in Indonesia and California. This thesis is a gift to my mama and papa who have been my number one fans. It is such a blessing to always have their wholehearted support and patience with anything I decide to pursue and explore in life. I deeply thank my brother, Didi Tanadjaja, who has been my role-model since I was in the womb. I thank my immediate family for giving me a sense of adventure, practical life skills, generous laughter, and a hard work ethic. I thank cousins Swie Lee and Olivia for inspiring me to continue discovering the wonders of this world and appreciate every unique opportunity in life. I thank the rest of my extended family for their love, support, and stable harbor. This study was supported by numerous grants and awards including the Graduate Student Research and Creative Activities Merit Award, the Earl and Ethel Myers Oceanography Trust, the NOAA Environmental Entrepreneurship Program, and the Tanadjaja Scholarship. PREFACE

This thesis presents research on the mechanical and chemical processes involved in the sediment production of coralline algae rhodoliths, the framework of a rhodolith bed, and the implications of ocean acidification on this carbonate system. Chapter one presents the results of a mechanical breakdown experiment, the development of a sedimentologic classification system for rhodalgal sediments, and the carbonate framework of the El Requeson rhodolith bed. Chapter two presents the results of chemical dissolution and chemical dissolution- mechanical breakdown experiments, and discusses the implications of ocean acidification on the breaking processes and the framework of the bed. Chapter three summarizes the overall conclusions and achievements of this study. Chapter one and two are in journal submission format. Chapter one is intended for Marine Geology, while the journal for chapter two is still undecided. TABLE OF CONTENTS Page

PREFACE ...... viii

LIST OF TABLES...... xi

LIST OF FIGURES ...... xii CHAPTER 1: THE MECHANICAL BREAKDOWN OF RHODOLITHS AND CARBONATE FRAMEWORK OF EL REQUESÓN BED IN BAJA CALIFORNIA, MÉXICO ...... 1

Abstract ...... 1

Introduction ...... 1

Methods...... 6

Results...... 11

Discussion ...... 18

Conclusions ...... 32

References ...... 34 CHAPTER 2: CHEMICAL DISSOLUTION OF RHODOLITHS AND THE EFFECT ON THEIR MECHANICAL BREAKDOWN...... 41

Abstract ...... 41

Introduction ...... 41

Methods...... 44

Results...... 49

Discussion ...... 52

Conclusions ...... 60

References ...... 61

CHAPTER 3: THE OVERALL CONCLUSIONS OF THIS STUDY...... 66

Methodology Contributions ...... 66

Research Findings...... 67 x Page

APPENDICES...... 69

APPENDIX A: CHAPTER 1 TABLES ...... 70

APPENDIX B: CHAPTER 1 FIGURES ...... 78

APPENDIX C: CHAPTER 2 TABLE...... 88

APPENDIX D: CHAPTER 2 FIGURES...... 90

APPENDIX E: DATA ...... 96 LIST OF TABLES

Page

CHAPTER 1 Table 1. Sediments cores collected on 29 March 2007 from the El Requeson bed, Baja California Sur, Mexico. Relative distance was to the beginning live margin (point 0 m) of the bed. Two cores (S3-D and S4-A) were collected outside the upper bed margin...... 71 Table 2. Summary showing similarities and differences of Dunham-Embry- Klovan carbonate classification (1961, 1971) and rhodalgal carbonate classification (this study)...... 72 Table 3. The percentage of rhodalgal sediment constituents (“branches,” “crumbs,” and “dusts”), sediment origin from rhodolith, and the rhodalgal carbonate sedimentologic classification (G = grainstone, P = packstone, W = wackestone, fW = finer-wackestone) from the sediment core samples. Vertical distance is from the top of each core... 74 Table 4. The percentage of living rhodalgal sediments from the top 7.5 cm of the sediment cores...... 77 CHAPTER 2

Table 1. Changes in total inorganic carbon (TCO2), total alkalinity (TA), pH values, initial hydrogen concentration, and rhodolith group weight (grams) from the chemical dissolution experiment. Dissolution in rhodolith groups was detected by TCO2 and TA. Error is expressed as standard error (SE) of the TCO2 and pH measurements. Confidence intervals of TCO2 and TA were derived from combination of nine delta pairs...... 89 LIST OF FIGURES

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CHAPTER 1 Fig. 1. (A) Map of Baja California Peninsula showing location of (B) El Requesón rhodolith bed and transects where (C) rhodoliths and cores of (D) rhodalgal sediments were collected. Transect notation was relative to a previous study (Steller, 1993)...... 79 Fig. 2. Image sequence to illustrate sediment size processing in ImageJ of one sediment sample. (A) Initial image taken using Leica® Z6 APO zoom macroscope with attached Leica® DFC 320 camera. (B) Black and white binary image converted in ImageJ. (C) Each particle was outlined and measured for size. (D) Result of size measurements saved as a text file...... 80 Fig. 3. The mechanical breakdown of individual rhodoliths and rhodolith groups (consisting of 5 individuals per group). Starting rhodoliths were roughly 1 cm, 2 cm, and 3 cm for individuals and 2 cm for rhodolith groups. Vertical error bars represent 2 standard error for the analytical uncertainty associated with measuring area...... 81 Fig. 4. Trimodal pattern of rhodolith breakdown in the mechanical experiment of an individual rhodolith with initial length roughly 2 cm. (A) The breakdown process of the “cores” and “branches” are shown as changes of area (mm2) on the left y-axis and the production of the “crumbs” are shown as changes in weight (mg) on the right y-axis plotted against time (second) in a logarithmic scale. The number 1 and 2 (orange) on the graph refer the “cores” at (B) 1 and (C) 1000 seconds indicated by the orange circles in the pictures. The orange bars in the pictures mark a 1-cm scale...... 82 Fig. 5. Trimodal pattern of rhodolith breakdown in the mechanical experiment of a rhodolith group (consisting of 5 individuals) with initial length roughly 2 cm. (A) The breakdown process of the “cores” and “branches” are shown as changes of area (mm2) on the left y-axis and the production of the “crumbs” are shown as changes in weight (mg) on the right y-axis plotted against time (second) in a logarithmic scale. The number 1 and 2 (orange) on the graph refer the “cores” at (B) 1 and (C) 1000 seconds indicated by the orange circles in the pictures. The orange bars in the pictures mark a 1-cm scale...... 83 xiii Page

Fig. 6. A closer look of the changes in area (mm2) of (A) the “cores” and (B) the “branches” of individual rhodoliths and rhodolith groups (consisting of 5 individuals per group) at the first 30 seconds. Initial lengths of rhodoliths were roughly 1 cm, 2 cm, and 3 cm for the individuals and 2 cm for the rhodolith groups. Vertical error bars represent 2 standard error for the analytical uncertainty associated with measuring area...... 84 Fig. 7. Detailed sediment cores analyses from the middle bed transect S3 showing rhodalgal carbonate sedimentologic classification, percentage of living rhodolith, and percentage of >2 mm particles originated from rhodolith. Relative distance and depth are given respectively below core label. Rulers indicate vertical distance (cm) from the top of cores...... 85 Fig. 8. Detailed sediment cores analyses from the southern edge transect S4 showing rhodalgal carbonate sedimentologic classification, percentage of living rhodolith, and percentage of >2 mm particles originated from rhodolith. Relative distance and depth are given respectively below core label. Rulers indicate vertical distance (cm) from the top of cores...... 86 Fig. 9. Simplified cross section of transect S3 (top) and S4 (bottom) and interpretation of relative depostional energy (high or low). Transects were ~350 m apart...... 87 CHAPTER 2

Fig. 1. Mean changes in (A) total inorganic carbon (TCO2) and (B) total alkalinity (TA) in relation to pH (graphed in logaritmic scale) for the duration of 90-hour incubation in the chemical dissolution experiments. Vertical bars represent 2 confidence intervals. Overlapping confidence intervals between control and treatment at pH ~7.8 is detailed in the blue box...... 91 Fig. 2. Changes in mean area (mm2) of the “cores” for 0% and 30% dissolved (A) individual rhodoliths and (B) rhodolith groups at the first 30 seconds. Vertical error bars represent 2 SE for the analytical uncertainty associated with measuring area...... 92 Fig. 3. Size (area) frequency distribution of rhodalgal fragments produced from 0% and 30% dissolved individual rhodoliths over time (0-1000 seconds). Pictures are showing the fragments produced at each time intervals for both 0% and 30% dissolved rhodoliths. The orange bars in the pictures mark a1-cm scale...... 93 Fig. 4. Size (area) frequency distribution of rhodalgal sediment produced from 0% and 30% dissolved rhodolith groups over time (0-1000 seconds). .. 94 xiv Page

Fig. 5. (A) Rate of CaCO3 dissolution and (B) residence time extrapolated from TCO2 and TA in the rhodolith groups from the chemical dissolution experiment. This plot predicts how quickly a rhodolith bed could dissolve or how long it would last under different ocean pH scenarios based on the initial pH of the rhodolith groups in the experiment. The mark of 30% dissolution indicates early signs of changing rhodalgal sediment production...... 95 CHAPTER 1: THE MECHANICAL BREAKDOWN OF RHODOLITHS AND CARBONATE FRAMEWORK OF EL REQUESÓN BED IN BAJA CALIFORNIA, MÉXICO

Abstract Rhodolith beds are shallow water carbonate reefs resulting from the accumulation of free-living coralline algal rhodoliths and rhodalgal sediments over time. Occurring worldwide, rhodolith beds are unique habitats with high biodiversity. Living rhodolith communities cover the surface of the rhodalgal carbonate framework. Accumulated past rhodolith beds and rhodalgal sediment form the supporting framework beneath. Experimental results showed mechanical breakdown of rhodoliths into rhodalgal sediments, producing distinct sediment groups: cores, branches, and crumbs. Various mixtures of these groups created different sediment textures in the rhodalgal assemblages found in the bed, indicating different depositional environments. Rhodalgal carbonate sedimentologic classification was developed as a tool to interpret these rhodalgal assemblages. The vertical pattern of coarse-fine-coarse sediment texture generally found across the bed was an indicator of rhodolith bed dynamic, suggesting movement in south-north-south direction or expansion-shrinkage-expansion of the active part of the bed from past to present.

Introduction Geologically, shallow water carbonate systems such as reefs, play an important role in the global carbon cycle. Although they only represent a small part in the global carbon cycle, the carbonate system is closely connected to the atmospheric CO2 and therefore other carbon systems (Broecker and Peng, 1982). sediments are the second most abundant sediment type in the 2 global ocean, in which about half come from shallow waters (Milliman and Syvitski, 1992). Biologically, these systems are habitats with high biodiversity (Reaka-Kudla, 1996; Foster, 2001) such as the well-researched coral reefs (Reaka- Kudla, 1996). Coral reefs are carbonate systems that can be distinguished into two ways: geologically, as structural reefs, and biologically, as coral communities (Wainwright, 1965). Similar to coral reefs but less known are the rhodolith “reefs” referred to as rhodolith beds, formed from spherical, branching, unattached coralline (Foster, 2001). Also referred to as ‘maerl’ beds, these beds are unique habitats with their own endemic species (Scoffin et al., 1985; Ballesteros, 1988; Freiwald et al., 1991; Reyes-Bonilla et al., 1997; DeGrave, 1999; Clark, 2000; James et al., 2006) and highly diverse inhabitants (Barbera et. al., 2003; Steller et al., 2003; Hinojosa-Arango and Riosmena-Rodríguez, 2004; Foster et al., 2007). Rhodoliths beds are the result of accumulation of free-living coralline algae rhodoliths (Weber-Van Bosse and Foslie, 1904; Foster, 2001; Hinojosa-Arango and Riosmena-Rodríguez, 2004), extending from the tropic to the polar region (Bosence, 1976; Scoffin et al., 1985; Littler et al., 1991; Freiwald, 1993; Foster, 2001). Like coral reefs, with veneers of living growing on the older reef structures (Wainwright, 1965), the living layer of the rhodoliths are found on the surface of the carbonate framework of the bed (Steller and Foster, 1995; Foster, 2001). The supporting framework underneath the live rhodoliths is the product of accumulation of rhodoliths and their broken down fragments, the rhodalgal sediments, over time. Hence, the current living layer is currently occurring on top of preceding rhodolith beds. Unlike stationary corals that remain fixed at one spot to form a reef, the free-living nature of rhodoliths allows them to behave as living sediments. Like 3 sediments underwater, these ‘tumbleweeds’ are transported and reworked by physical processes and accumulated through deposition. Studies of modern rhodolith beds have primarily been focused on the live rhodoliths, which only comprise the top few centimeters of a rhodolith bed (Foster, 2001). Yet both the rhodoliths and rhodoalgal sediments collectively make up the bed framework and support the live rhodolith communities. As a consequence of the free rolling nature of rhodoliths, the rhodolith beds are also non-stationary, these rhodolith reefs rock and roll around and the extent of the bed expands and contracts (Foster, 2001; D. Steller and M. Foster, personal communications, 2009). Studying the framework of the rhodolith bed can provide information to the past depositional environment of rhodalgal sediments and the dynamic of these moving rhodolith beds from past to present. Dunham’s classification of carbonate sedimentary texture (1962), modified by Embry and Klovan (1971), has been successfully applied in interpreting depositional environments of limestone and carbonate assemblages (e.g., Specht and Brenner, 1979; Twiggs and Collins, 2010). Similarly, investigating the textures of rhodalgal carbonate assemblages found in rhodolith beds may be used to interpret the depositional environment of these carbonate sediments from past to present. The process of rhodalgal sediment production creates the building materials for this framework. Understanding the mechanics of how rhodoliths break down into smaller fragments of rhodalgal sediments will help with the interpretation of the rhodalgal assemblages formed in a rhodolith bed. The aims of this study were to: 1) examine how whole rhodoliths mechanically breakdown into rhodalgal fragments, 2) describe the carbonate framework of a rhodolith bed including the surface living layer and the underlying 4 supporting framework of rhodalgal sediments, and 3) examine the past depositional environments and the occurrence of preceding rhodolith beds.

Strategy In order to complete the objectives mentioned above, new methods were developed, and laboratory experiments and data collection from the field were performed. Of the numerous beds in the Gulf of California, México, the El Requesón bed was chosen because more research has been done here hence more was known about this bed. This bed has been previously studied and was a good representation of other rhodolith beds in the Gulf of California, México (Steller and Foster, 1995), and was easily accessible for sample collection. The mechanical breakdown experiment was developed to examine how rhodoliths break down into smaller fragments and finer sediments. Using a mechanical shaker the breakdown evolution of rhodoliths was tracked by collecting data and information at continuous time intervals. At first, the basic breakdown of rhodoliths was studied by subjecting individual rhodoliths, one at a time, in the experiment. Then, a more complex breakdown process was studied using a group of five rhodoliths. This provided a better picture of how rhodoliths break down in a natural setting, where they are in high concentration. The sediment size of rhodoliths and resulting fragments in the mechanical breakdown were tracked by image analysis due to their complex three-dimensional branching structure. Sediment sieves and a laser particle sizer were not the best tools to assess the size of rhodalgal sediments because they were composed of mostly coarse, pebble-sized sediments. Instead, ImageJ software (Rasband, 1997- 2009) was used to process the photos of these sediments and measure efficiently 5 each particle in the photos. The development of this image analysis method made size measurement of coarser sediments (>2 mm) in this study possible. In order to construct the framework model of the rhodolith bed, sediment cores were collected along two transects representing different depths at two locations. Two transects were set up, one spanned the middle of the bed and one at the edge of the bed in order to capture variation occurring between locations. Various sediment analyses such as size distribution, percentage of living rhodolith, and sediment origins were conducted and the results were used as part of the construction of this framework. Classifying the texture of the rhodalgal sediments found in the sediment cores was necessary to reconstruct this framework. This classification helped with sediment core interpretation regarding the depositional environment of the rhodolith bed and explaining the framework of the bed. No prior sedimentologic classifications exist for rhodalgal sediments, hence this classification had to be developed to match specifically the sediment texture in the sediment cores. The classic and commonly used Dunham carbonate classification (Dunham, 1962) with Embry and Klovan (1971) modification was used as the starting point in developing this classification. Results from the mechanical breakdown experiment also helped with this classification development and the bed framework interpretation.

Study Settings Living rhodolith beds are found throughout the Gulf of California coast in Baja California, México (Foster et al., 1997; Steller and Foster, 1995; Riosmena- Rodriguez et al., 2010). Arid conditions, lack of river system, and steep fault- controlled shoreline and seafloor may be important factors in the accumulation of rhodalgal sediments in this area (Meldahl et al., 1997; Halfar et al., 2004; Halfar et 6 al., 2006). The rhodalgal sands commonly form the beaches in the Gulf of California (Ledesma-Vázquez et al., 2007; Sewell et al., 2007). The El Requesón bed is situated in Conception Bay (Fig. 1A), connected to the tectonically active Gulf of California, where the east pacific rise is still actively spreading (Lonsdale, 1989; Stock and Hodges, 1989). The bed is about 0.5 km by1 km, occurs in 3-12 m of water, (Steller and Foster, 1995; Marrack, 1999) and is dominated by Lithophyllum margaritae rhodoliths (Steller et al., 2003). The bed is located on the windward (east side) of Isla Requesón, which is connected to the mainland by a 350 m long tombolo at low tide (Hayes et al., 1993). The tombolo and the beaches nearby are composed of carbonate sediments primarily from rhodoliths (McFall, 1968; Hayes et al., 1993). The El Requesón rhodolith bed is categorized as a “wave” bed (Foster et al., 1997) due to it’s high energy environment and is influenced by stronger hydraulic action (e.g. waves and storms), as opposed to beds that are exposed only to low-energy tidally induced currents. Particularly during the winter months, winds tend to blow out of the north-west creating wave action (Merrifield et al., 1987; Marrack, 1999). Periodic storms and hurricanes, common in the southwestern part of Baja California, may also periodically influence this bed (Schalanger and Johnson, 1969; Gutiérrez-Sánchez, 1987; Galli- Oliver et al., 1995).

Methods

Rhodoliths Collection, Storage, and Preparation On 29 March 2007, a total of 13 sediment cores were collected from the El Requesón bed (Table 1). SCUBA divers collected cores at different depths along two transects that ran perpendicular to the shore (Fig. 1B) using cylindrical hand- 7 cores made from clear acrylic, 2 in. diameter by 1ft. length. Transect S4 (26° 38.456 N; 111° 49.659 W) was near the southern edge of the bed and located about 350 m southeastern of transect S3 (26° 38.572 N; 111° 49.823 W) that was placed in the middle of the bed. From each transect, five to six hand-cores were collected inside the bed along a depth gradient. Transect notation was relative to a previous study conducted in this bed (Steller, 1993). One core was taken from above the upper, shallow edge of the living portion of the bed from each transect. Random numbers were generated based on the width of the bed (the length from the nearshore end of the bed to the offshore end of the bed) to designate sampling points within each transect. Relative depth and distance from the start of the bed (relative point 0 m, at ~11 m from the edge of rocky shore) were recorded for each core. Cores were then stored and transported vertically within a compartmentalized crate to preserve their stratigraphic layers. Sediment cores were brought back to the laboratory and stored in a cold room (10° C). Whole rhodoliths (Fig. 1C) used for the laboratories experiments were collected from transect S4 at 7 m of depth on 10 October 2008. These rhodoliths were brought back to the laboratory, desiccated in air for a week, and stored for later usage in the experiments. Prior to using them in the experiments, these rhodoliths were put in a seawater sonicated bath for 30 minutes and then in a 3%

H2O2 sonicated bath for 20 minutes to get rid of excess sediment debris and organic matter adhering to them.

Terminology In this study, the term rhodalgal sediment was used to describe the mixture of living and dead rhodoliths, fragmented skeletal remains mainly from rhodoliths with a small portion of carbonate materials from other marine calcifiers, found in 8 the rhodolith bed. This term was also used in the mechanical experiments to describe the sediments produced only from rhodoliths. The words ‘sediment,’ ‘particle,’ ‘grain,’ and ‘fragment’ are used interchangeably.

Mechanical Breakdown Experiment The length (longest axis) of rhodoliths used in this mechanical breakdown were measured to the nearest centimeters and categorized in three different size classes: 1 cm, 2 cm, and 3 cm. Two individual rhodoliths from each of the three size classes and two groups of five rhodoliths from the 2 cm size class were used. First, the experiment was conducted on individual rhodoliths in order to learn the basic breaking pattern of one rhodolith. Then, the same experiment was conducted on rhodolith groups to study the breaking pattern of interacting rhodoliths. Rhodoliths were placed in a 50 mL capped bottle that was filled with filtered seawater and shaken in a mechanical shaker (SPEX® Mixer/Mill 8000) over time (0-1000 seconds, Appendix E). Image analyses method (See image analysis section) was used to track the sediment size distribution of rhodolith fragments larger than 1 mm at each time interval. For those smaller than 1 mm, dry weight was measured at each time interval.

Sediment Cores Analyses Sediment cores were collected and analyzed to construct a simple model of rhodolith bed framework. Several analyses performed on the sediment cores to acquired percent of living rhodoliths, sediment size distribution, and sediment origin. Percent of living rhodoliths were recorded for the top 7.5 cm section at every 2.5 cm from the top of the core, based on pink pigmentation. Digital images of the cores were taken before and after opening the cores. These images were used as visual support during core interpretation. Apparent stratigraphic sections 9 in the cores, roughly every 2 to 3 cm, were noted and described along with other preliminary observations of the sediment cores. Rhodalgal sediment sub-samples (Fig. 1D) were collected from these sections, oven dried, weighed, and analyzed for size distribution and sediment origin. Due to the broad range of rhodalgal sediment sizes (pebbles to silt) found in the sediment cores, the sizes were analyzed using two methods: 1) Image analysis methods for the coarse sediments fraction above 2 mm; and 2) Laser particle sizer for the fraction below 2 mm. The two sizing methods required sediments sampled from the cores to be separated into two groups above 2 mm and below 2 mm. Sediment samples were dried in the oven (~70o C for 30-40 minutes) and the total dry weight (grams) was measured. Sediment sieves were subsequently use to sort sediments into three intermediate groups: above 2 mm, between 1-2 mm, and below 1 mm. Due to the elongated shape of most rhodalgal sediments, some of those above 2 mm did not sorted to the above 2 mm group. Hence, those between 1-2 mm were then laid out on a paper with 2x2 mm grid and visually sorted into above and below 2 mm under a 5x magnifying glass. The final results of these sorting processes were two sediment groups of above and below 2 mm. The dry weights of these sediment groups were measured. Sediments above 2 mm were sorted further into two categories of sediment origin: rhodoliths and non- rhodoliths. Rhodolith sediment weight was measured and then the percentage of the whole sample was calculated. Sediment size distribution of those above 2 mm was analyzed using the image analysis methods (See image analysis section). Following these methods, digital images of the sediments were taken using Leica® Z6 APO zoom macroscope with a Leica® DFC 320 camera attached. Then, the sizes (area and longest axis) of each sediment in these pictures were measured in the image 10 analysis software, ImageJ. All sediments were imaged four times to increase the measurement precision and in order to better represent the sizes of 3-dimensional sediments. For sediments below 2 mm, a Beckman Coulter® LS12 320 particle size analyzer with aqueous module was used to measure their sizes (Beckman Coulter Inc., 2003).

Image Analyses Each sediment sample from either the sediment cores or the mechanical breakdown experiment contained several rhodalgal pieces or fragments (i.e. particles). An image analysis method was developed to measure the size of every particle in the sample at once. Digital images were taken of each sample that include every particle in the sample. The area and length (longest axis) of each particle in an image were then measured using an image analysis software (open source) called ImageJ (Rasband, 1997-2009). From each sample, all particles were poured randomly on a black paper background and arranged so that not one particle was touching another (Fig. 2A). As size reference, a millimeter scale was used in every image taken. Leica® Z6 APO zoom macroscope with attached Leica® DFC 320 camera was used to take digital images of this sediment arrangement. Each arrangement would give result to a sediment size distribution. However, these images can only capture the two- dimension plane of these three-dimensional particles. Therefore, to get a better representation of their sizes, an average were calculated from four different arrangements. For some samples, several photo frames were taken to include all particles. These frames were stitched later in Photoshop® to make up one photo montage representing one arrangement for that particular sample. This photo montage was used for ImageJ particle size analysis instead of the original photo 11 frames. So, as the final results, for every sample there would be four photos (or photo montages) corresponding with the four sediment arrangements. In ImageJ, each photo was converted into a binary image of black particles on white background (Fig. 2B) and afterward, each particle was outlined (Fig. 2C). Then, the “Analyze Particles” feature in ImageJ was run to measure the area and length (i.e., Feret’s diameter) of each sediment particle outlined in this image. The list of these measurements for each photo was saved as text file (Fig. 2D). Batch processing of final photos from several samples were done by creating a macro containing a program script to call each image, converting it to a binary image, measuring the sizes of all particles, and saving the size data in a text file identified to the correct sample. These processes created a large amount sediment size data that required computer programs, written using Matlab programming software, to further process, sort, analyze, and graph these data.

Results

Mechanical Breakdown Experiment During the mechanical breakdown experiment for both individual rhodoliths and rhodolith groups, the breaking patterns of rhodoliths into rhodalgal sediments were consistent. At the beginning of the breaking process, during the first few seconds, rhodoliths broke down rapidly and their sizes reduced quickly. Afterwards, the breakdown tapered off, rhodalgal fragments reached more stable condition and their sizes decreased gradually. These two different conditions separated rhodolith breakdown into two phases: the initial phase and the stable phase. The initial phase occurred during the first few seconds when rapid breakdown and rapid size reduction happened. The stable phase occurred when the breakdown had slowed down, the fragments’ sizes were reduced much more 12 gradually reaching stable sizes that fall within a narrow size range. The mean size, measured as area, showed this general breaking pattern of initial rapid size reduction followed by gradual size decrease reaching a more stable size about 10 mm2 (Fig. 3). The rhodolith size reduction during breakdown varied relative to the starting size of the rhodoliths. The mean area of rhodoliths with larger initial sizes (initial length of ~2 and ~3 cm) decreased more than the mean area of rhodoliths with smaller initial sizes (initial length of ~1 cm). Hence, the larger initial size rhodoliths (~2 cm in length) broke the most (from 540-430 mm2) to reach their stable sizes (~10 mm2), losing more than 95% of their initial sizes. The smallest initial size rhodoliths (~1 cm in length) broke the least (from ~70 mm2) to reach their stable sizes (~10 mm2), losing ~70% of their initial sizes. The breakdown of rhodolith groups with initial length of ~2 cm followed closely the breakdown pattern of individual rhodoliths with initial length of ~2 cm. They broke down from ~180-220 mm2 down to their stable sizes (~ 10 mm2), losing ~90% of their initial sizes. The details of rhodolith breakdown for both individual rhodoliths and rhodolith groups were further investigated by looking at the sizes of each fragment resulting from the mechanical breakdown in each time interval (Figs. 4, 5). In overall, rhodoliths breaking pattern had a “trimodal” pattern. The trimodal referred to the three groups of rhodalgal fragments: “cores”, “branches”, and “crumbs”. The cores were the larger fragments that broke down slower and decreased in size over time. The branches were the smaller pieces that made up the bulk of the fragments produced during the experiment with similar sizes (~10-40 mm2) and their amount increased over time. The crumbs were the finer fragments that were mostly too small to be measured using image analysis technique. The sizes (area 13 and length) of cores and branches were determined by looking at the natural division between cores and branches seen in area and length measurements of all fragments from individual rhodoliths used in the experiment. This natural division occurred at ~10 mm of length (~40 mm2 of area). There were minimal size measurements for crumb that were not enough to show the natural size division that might be present between branches and crumbs. Thus, after consulting the commonly used grain size classes –the Wentworth scale (Wentworth, 1922)– and considering the limitation of the techniques used to measure sediment sizes in this study, 2 mm in length (area ~10 mm2) was decided as an appropriate break to separate the branches from the crumbs. Therefore, the cores were classified as fragments having length >10 mm (area >40 mm2), the branches as fragments with 2-10 mm in length (area 10-40 mm2), and the crumbs as fragments with length <2 mm (area <10 mm2). These sediment groups – cores, branches, and crumbs – had different breaking behaviors as detailed in Fig. 4. Fig. 4A shows the changes in area for cores and branches and the changes in weight for crumbs over time of an individual rhodolith with initial length ~2 cm. Although this graph illustrated the breaking behavior of cores, branches, and crumbs of only a rhodolith with initial length ~2 cm, the same breaking behaviors were observed for rhodoliths with different initial length. In general, there was a clear separation between the sizes of the cores and the branches. The area of the cores decreased as time increased meanwhile the area of the branches stayed at the same range. The production of the crumbs increased over time indicated by the increasing weight. The pictures show all the fragments at 1 second (Fig. 4B) and 1000 seconds (Fig. 4C). The cores, branches, and a portion of the crumbs were easily recognized in these pictures (the orange circles indicated the core fragments in the pictures). 14

The breaking behaviors of these sediment groups for the rhodolith groups also followed similar trend to those in individual rhodoliths. Fig. 5 shows a clear size separation between the cores and the branches for the rhodolith group. The area of the cores for this rhodolith group also decreased over time, the branches stayed at the same range, and the weight of the crumbs increased over time (Fig. 5A). The only difference was the production of more cores over time. Although the area of cores continued to decrease over time, the number of cores produced generally increased over time. The pictures show easily recognized cores fragments at 1-second (Fig. 5B) and at 1000-second (Fig. 5C). The different breaking behaviors of cores and branches were also reflected in their changes of mean sizes over time (Fig. 6). The mean area of the cores showed more variability than the mean area of the branches which had a narrow range of size between ~5 and ~20 mm2. This pattern was consistent for all individual rhodoliths and rhodolith groups. The major changes in the mean size for cores (Fig. 6A) and branches (Fig. 6B) happened at the first 10-second of the breakdown. The mean area of the cores for the individual rhodoliths with initial length ~1 cm did not change much (from ~70 mm2 to ~40 mm2). Meanwhile the mean area of the cores for individual rhodoliths with initial length ~3 cm changed the most (from ~430 and ~540 mm2 to ~80 mm2) with the major decrease occurring at the first 2-second. For the individuals with initial length ~2 cm, the mean size decreased from ~220 mm2 to ~125 mm2. Compared to this, the mean size of the rhodolith groups (with also ~2 cm initial length) greatly decreased – from ~200 mm2 to ~55 mm2. However, the branches from the rhodolith groups fell within the size range of the branches from the individual rhodoliths. 15 Development of Rhodalgal Carbonate Sedimentologic Classification Five sedimentary textural assemblages were identified by visually comparing the sediment texture found in the rhodalgal sediments and their similarity to the texture described in the classic and commonly used Dunham- Embry-Klovan carbonate classification (Dunham, 1962; Embry and Klovan, 1971). These assemblages were classified as “Rhodo-Corestone,” “Grainstone,” “Packstone,” “Wackestone,” and Finer-Wackestone” and established as the rhodalgal carbonate sedimentologic classification (Table 2). The names were adapted from their classification due to the similarity of the rhodalgal sediment texture to the description of texture in Dunham-Embry-Klovan classification. The rhodalgal sediment textures were much coarser than the sediment texture described in Dunham-Embry-Klovan classification. However, there were recognizable major constituent groups in the rhodalgal sediments that took place of the main constituents in their classification. Three constituents were the same rhodalgal sediment groups seen in the mechanical breakdown experiment: “cores,” “branches,” and “crumbs.” “Dusts” was added as the last constituent. The production of dusts was also observed during the mechanical experiment, but the amount was miniscule and their weight could not be detected by the scale. The dusts fragments were finer (<0.5 mm) than the crumbs and were important textural indicators of calmer depositional environment. After recognizing these different constituents, analogy with the constituents in the Dunham- Embry-Klovan classification were made: cores to their >2 mm particles, branches to their grains, crumbs to their coarse mud, and dusts to their fine mud. Table 2 summarizes the comparison between the Dunham-Embry-Klovan classification and the rhodalgal carbonate classification developed in this study. 16

During the process of developing this classification, initially, the sedimentary textures of sediment samples from the cores were roughly matched to the five identified textural assemblages. Then using the sediment size distribution data (Table 3), the percentages of all of the constituents in each assemblage were critically examined to establish percent limit for the presence of these constituents in each category. Since the cores were mostly found in rhodo-corestone and almost none in the other assemblages, the percent limit was only established for the other four categories. The percent limits for branches, crumbs, and dusts were in different units due to the two techniques used for measuring grain sizes: image analysis for particles above 2 mm and laser particle size analyzer for those below 2 mm. The branches and crumbs were the fraction above and below 2 mm measured in percent weight, while the dusts were a portion of only the grains below 2 mm which were measured in percent volume by the laser particle size analyzer. Thus, the percentage limit for the dust components had to be set up in percent volume of below 2 mm percent weight. Table 3 shows the percent limit of these constituent in the classification and the application of these criteria to all the sediment core samples. The development of this classification aided the construction of rhodolith bed framework and the interpretation of its past to present depositional environment. The raw data of all rhodalgal fragments measured in this study are included in an additional compact disc (Appendix E).

Rhodolith Bed Framework The rhodolith bed framework was successfully constructed (Figs. 7, 8) from the sediment cores based on the rhodalgal carbonate sedimentologic classification (Table 2) developed in this study and various sediment analyses (Table 3, 4). In 17 general, all sediment cores showed a vertical trend of coarse-fine-coarse (Figs. 7, 8). This trend was reflected in the classification of the sediment textures in the framework. Rhodo-corestone was only present on the top ~2 cm layer. In transect S3 the sediment textures were mostly classified as “branch-supported” (from grainstone to packstone) while the textures in transect S4 were mostly classified as “crumb-supported” (from wackestone to finer-wackestone). The branch-supported layers occurred mostly at the top of the sediment cores, extending laterally from nearshore (- ~5 m) to offshore (~60 m) in transect S3 and only out to ~10 m offshore in transect S4. The crumb-supported layers tended to concentrate in the bottom layer of the sediment cores, closer onshore (starting at ~0 m) in transect S4, and farther offshore (starting at ~55 m) in transect S3. Grainstone was present in the sediment cores closer to shore and the majority were found in transect S4. Percent of living rhodolith from the upper 7.5 cm of the cores was estimated based on the red pigmentation of the sediments. Higher percentage (5- 100%) of living rhodoliths resided in transect S3 compared to the lower percentage (2-50%) of them found in transect S4 (Table 4). In both transects, a higher percentage of living rhodoliths was found at the top 2-3 cm of the sediment cores. These high percentage (90-100% for transect S3 and 20-50% for transect S4) top layers coincided with rhodo-corestone layer (Figs. 7, 8). Core S4-A in transect S4 –located outside the bed margin – had no living rhodoliths. In general, the percentage of particles >2 mm (cores and branches) originated from rhodolith were much higher in transect S3 than transect S4 and correlated with the rhodalgal carbonate sedimentologic classification (Table 3, Fig. 7, 8). The conspicuous contrast was between the outside of the bed cores, core S3-D had over 40% rhodolith cores and branches while core S4-A had less than 18

20%. Visual observation of cores S4-A showed a very different composition of sediments with few rhodolith originated coarse sediments.

Discussion

Mechanical Breakdown Experiment This study is the first examination of mechanical breakdown in rhodoliths and builds on previous studies of other marine calcifiers including corals, bryozoans, sea stars, mollusks, foraminifera, coralline algae, and calcareous green algae (Chave, 1960; Chave, 1964; Folk and Robles, 1964; Swinchatt, 1965; Force, 1969; Stoddart, 1969; Kotler et al., 1992; Scoffin, 1992; Bone and James, 1993; Smith and Nelson, 1994; Greenstein et al., 1995). The mechanical breakdown experiment demonstrated the consistent breaking patterns of individual 1-3 cm diameter rhodoliths and rhodolith groups into rhodalgal fragments. In general, there were two phases of rhodolith breakdown: the initial phase followed by the stable phase. During the initial phase, rhodoliths broke down rapidly and their sizes were rapidly reduced. The stable phase described the breakdown condition when rhodalgal fragments broke down gradually and their sizes decreased slightly. However, the extreme changes during the initial phase, occurring during the first few seconds of the experiment, demonstrated that these first few seconds breaking are critical moments when a lot of changes in sizes and breaking occurs. Live rhodoliths can range in size from a few mm to over 15 cm in diameter (Foster, 2001). However populations of rhodoliths are generally dominated by smaller individuals (Steller et al., 2003). The starting sizes of rhodoliths in these experiments, ranging from 1-3 cm in diameter only affected the breaking pattern during the initial phase. The starting sizes of rhodoliths only affected the breaking pattern during the initial phase. Once they reach the stable phase, regardless of 19 their initial sizes, rhodoliths fragments were maintained at consistent sizes (~5-20 mm2). That rhodolith with larger starting sizes broke faster and experienced larger size reduction compared to those with smaller initial sizes suggests that in general, larger rhodoliths are more affected by disturbance. The larger rhodoliths (initial length ~2 and ~3 cm) had to break much more in order to reach the stable sizes, which fell within a narrow range, around 10 mm2, and were much smaller than the sizes of the initial rhodoliths. Smaller rhodoliths (initial length ~1 cm) were already closer to these stable sizes, hence, only a small amount of breaking already decreased their sizes to reach the stable condition, suggesting that smaller rhodoliths were more stable than larger rhodoliths in resisting mechanical breakdown. The abundance of smaller sizes rhodoliths (~1 cm) in the El Requesón rhodolith bed (Steller et al., 2003) may be controlled by similar breaking processes. The consistent sizes (~5-20 mm2) at the stable condition suggests that there were inherent characteristics in rhodolith shape influencing the way they break and the sizes of the fragments they produced. The breakdown characteristics of some species of corals, bryozoans, sea stars, mollusks, and green calcareous algae have also produced fragments of specific sizes due to the skeletal structure or shape and the micro-architecture of the contributing organisms (Folk and Robles, 1964; Swinchatt, 1965; Force, 1969; Bone and James, 1993; Smith and Nelson, 1994; Greenstein et al., 1995). This relationship between shape and size of the resulting grains and the morphology and micro-structure of the organism is known as the Sorby Principle (Sorby, 1879). Organisms with similar morphology like branching corals and bryozoans, but different mineralogy (aragonite and calcite), had similar breaking pattern of producing “sticks” from the branches broken off at the joints (Folk and Robles, 1964; Bone and James, 1993). There may be similar 20 factors in shallow-water natural processes (e.g. physical processes like waves and storms) that control the morphology of organisms and the breaking pattern found in different carbonate systems. This study provides the first report of a “trimodal” breaking pattern of spherical branching rhodoliths breaking into core, branch, and crumb fragments. These different breaking behaviors indicate that rhodolith morphology and microstructure play important roles in how they breakdown. The morphology of rhodoliths used in this study, round and ball-like structure with branches radiating out of the center, likely contributed to the way rhodoliths broke and created the trimodal pattern. When impacted, the first fracturing of rhodoliths happened at the juncture points of the branches, leaving behind the more resistant structure of the cores. Depending on the starting size, the resulting cores – still having the complex ball-like structures with branches – were just smaller rhodoliths. As seen in the pictures (Figs. 4, 5), the branches were much simpler in shape: simpler branching form fragments at the beginning of the breakdown to mostly elongated rod-like sediments at the end of the experiment. Other studies showed branching bryozoans and staghorn corals breaking down into sticks at the nodes (Folk and Robles, 1964; Stoddart, 1969; Bone and James, 1993). These sticks were analogous to the rhodalgal branches. The process of initial rhodolith fragmentation created two grain sizes: coarse pebble size cores (30-600 mm2 in area) and fine pebble size branches (6-19 mm2 in area) with less intermediary size fragments. The cores were the main driver that controlled the rapid decrease in overall mean size at the first few seconds. After the rapid breakdown, the more resistant cores decreased in size slower, maintaining the integrity of the initial conditions. However, facing continuous breaking force, the cores continued to break down, and became much 21 smaller, and eventually turned into branches and crumbs. The branches were the main consequence of the rhodoliths breaking down. Therefore branches made the bulk of the rhodalgal sediments produced during the mechanical breakage. These branches were more stable than the cores. Once the fragments reach the branches sizes, they broke more slowly compared to the cores. These branches were the main players in determining the sediment size at the stable condition, between ~5 and ~20 mm2. The crumbs, produced later after the cores and branches were created, were the product of abrasion between the cores and the branches, chipping away finer pieces from the structures of the cores and branches. The poly-modal skeletal grains produced through the mechanical breakdown have also been observed in other organisms like corals, mollusks, and green calcareous algae Halimeda sp. (Folk and Robles, 1964; Swinchatt, 1965). Mechanical breakage and abrasion were the proposed mechanisms for this poly- mode. Mechanical breakage produced pebble size and sand size grains in corals (Folk and Robles, 1964; Swinchatt, 1965), halimeda (Folk and Robles, 1964; Swinchatt, 1965) and mollusks (Swinchatt, 1965). Abrasion cracked and chipped these pebble and sand grains, reducing their sizes into silts. It’s likely that the morphology of original organisms affected the sizes (pebbles and sands) and shape of the grains produced by mechanical breakage, while the microstructure controlled the sediment sizes produced by abrasion as predicted by the Sorby principle. The pebble and sand size of the rhodalgal fragments in this study and the breakage and abrasion mechanisms were consistent with observations and proposed mechanisms of other studies (Folk and Robles, 1964; Swinchatt, 1965; Force, 1969; Bone and James, 1993; Smith and Nelson, 1994; Greenstein et al., 1995). Mechanical breakage drove the initial sediment production and rhodolith 22 branching morphology influenced how broken fragments created cores and branches. After the cores and branches were produced, they abraded each other and produced crumbs. The products of this abrasion process were controlled by the micro architecture of the rhodalgal carbonate. However, in rhodalgal sediments, the pebble size grains comprised of two types of fragments, the cores and the branches. Staghorn corals studied by Folk and Robles (1964) had similar branching morphology to rhodoliths. Unlike free-living rhodoliths, these corals were attached to substrate, hence, the “trunks” of these corals were still attached to the substrate when all their branching “sticks” broke off and became a part of the skeletal debris. The “cores” of rhodalgal sediments were non-attached “trunks” that were also a part of the sediments causing the bimodality in rhodalgal pebbles. The breakdown pattern of the individual rhodoliths was the basic pattern of how one rhodolith breaks. The trimodal breaking pattern was consistent in all individual rhodoliths regardless of their initial sizes. Including rhodolith groups in the experiment, – each group consisting of five individuals – was a step closer to studying a more complex mechanical breakdown of rhodolith community in nature. Since the initial sizes of the rhodoliths in the rhodolith groups were ~2 cm in length, their breaking patterns were compared to those of individual rhodoliths with also ~2 cm initial length. The rhodolith groups followed the trimodal breaking pattern with additional core to core interaction. This interaction caused more breakdown as indicated by the higher amount of decrease in core sizes which was about half of the sizes of the cores of the individual rhodoliths. This suggest that cores were not stable and this phase may be short lived in the natural world where there is a higher number of rhodoliths and grains, thus allowing greater interactions, hence breaking to happen. 23

Understanding the mechanical breaking processes and how the produced sediment groups interact –cores, branches, and crumbs– are the building blocks to understanding the rhodalgal sediment assemblages and carbonate framework of the rhodolith bed. The cores, branches, and crumbs were the main products of the mechanical breaking. Mixtures of these were found in the rhodalgal assemblages in the bed. The different composition of sediment groups in the rhodalgal assemblages could indicate the relative maturity of these assemblages. The mechanical breaking process that produced cores and branches was a fast production and happened at the early breaking processes. While the abrasion process that produced crumbs was a slower process and required more time. Presence of more cores and branches with fewer crumbs in a rhodalgal mixture may imply a short accumulation time with little chance of crumbs building up and could indicate recent formation of the assemblages. On the contrary, the presence of high abundance of crumbs implies longer accumulation time or older creation of the assemblages. The experimental breakdown of whole rhodoliths into cores and branches, and ultimately crumb in this study can inform interpretations of sedimentologic source material. In a rhodolith bed, if a high concentration of whole rhodoliths is considered to be a sediment factory, then the presence of a high abundance of cores and branches in the assemblages could indicate close proximity. Therefore, coarse assemblages with high concentrations of cores and branches may be relatively less mature and/or closer to the sediment factory compared to the finer assemblages with high abundance of crumbs. 24 Rhodalgal Carbonate Sedimentologic Classification This study established a rhodalgal carbonate sedimentary classification. This classification contributes to the interpretation of sediment cores and the depositional environment of the rhodolith beds. Terminology is provided for the texture of rhodalgal sediments in sediment cores, and an interpretation of the bed framework. Fortunately, classification of carbonate rocks such as limestones was already developed (Dunham, 1962; Embry and Klovan, 1971; Folk, 1959). The Dunham carbonate classification was widely known, commonly used, and described similar textural conditions. Therefore, the classification from this study with Embry and Klovan (1971) modification, from rudstone to wackstone, was a good starting point for the development of a classification that would specifically fit the rhodalgal sediments. The rhodalgal sediments have a much coarser texture than the texture of limestones in the Dunham-Embry-Klovan classification. Hence, their classification did not apply well to the rhodalgal sediments due to the differences in size class particles and percentage of these particles described in their classification. Nonetheless, analogies between the rhodalgal sediment texture and limestones can be established. From the results of the mechanical breakdown experiments, three constituents of the rhodalgal sediments were already established: “cores,” “branches,” and “crumbs.” Visual observation of the sedimentary textures indicated the need to add another major constituent, “dusts,” to include the full spectrum of grains sizes and sedimentary texture assemblages. Dusts were smaller to finer fragments than crumbs and a necessary addition since they indicated calmer deposition environment. The rhodalgal cores, branches, crumbs, and dusts were similar to their >2 mm particles, grains, coarse mud, and fine mud respectively. The comparison between the Dunham-Embry-Klovan 25 classification and the rhodalgal carbonate classification was summarized in Table 2. Using this analogy, the application of their rudstone-wackstone classification was adjusted accordingly to better match the texture of the rhodalgal sediments in the sediment cores and five classifications were established for the rhodalgal sediments. The sediment size distribution data from the sediment core samples were used to set-up the percentage limit of the constituents present in each category. Since the Dunham classification have been used widely, adapting similar name to their classification will give a familiar sense of sediment texture that will help those in the future who want to use this new rhodalgal carbonate sedimentologic classification. The five classifications were named as “Rhodo- Corestone,” “Grainstone,” “Packstone,” “Wackestone,” and “Fine-Wackestone.” Similar to the Dunham classification, from the rhodo-corestone (coarser or branch supported texture) to the fine-wackestone (finer or crumb supported texture), the classification generally indicated the trend of higher energy depositional environment to the lower energy depositional environment.

Rhodolith Bed Framework Rhodolith bed framework was constructed based on the sediment cores collected from the middle section and southern edge of El Requesón bed. The rhodalgal carbonate sedimentologic classification applied to these sediment cores sample showed the construction of various sediment assemblages that made up the rhodolith framework. This construction based on the classification assisted in recognizing relative depositional environment with relative high or low energy. Percentage of living rhodolith and percentage of >2 mm particles originated from rhodoliths were additional data used to create a more complete construction of the 26 bed framework. The percentage if living rhodolith data were used to identify the living layer of the bed and the percentage data of sediment origin from rhodolith were used indicated relative high or low productivity of sediment factory and proximity to the factory. There was no time frame correlated with the sediment cores which were mostly about 20 cm deep. However, considering that the rhodoliths in El Requesón grow from about 1-5 mm per year (Steller et al., 2007), the sediment cores collected were assumed to represent processes occurred in the scale of hundred years. Most of the building materials of this framework were non-living carbonate sediments. The living layer of the rhodolith bed occupied only the first top centimeters, indicated by the Rhodo-corestone texture and the high percentage of living rhodoliths (30-100%). This living rhodolith community was composed of mostly whole living rhodoliths mixed with core fragments. It was extended farther offshore in the middle part of the bed, ~60 m, and closer onshore, ~20 m, at the southern edge of the bed. Living rhodoliths were highly concentrated in the middle of the bed and decreased in half or less in the southern part of the bed. Since the percentage of living rhodoliths were estimated based on the pigmentation seen in the sediment cores, this percentage may be overestimated due to preservation of pigment in some sediments even after they were long dead as seen in the stored sediment cores. This overestimation of living rhodoliths could indicate that the extent of the living part of the bed was also overestimated laterally and vertically. The living portion of the bed is mainly concentrated over the branch- supported framework. The coarse texture of branch-supported framework with over 60% branches and mostly over 40% of >2 mm sediments coming from rhodoliths indicated that these sediments were closer to the sediment source. As seen in the progression of mechanical breakage of rhodoliths during the 27 experiment, the coarser textures of the sediments were present in the earlier phase of breaking and the finer textures were found in the later. Therefore, if the coarser texture had not gone through much breaking processes, it could indicate close proximity to the sediment source. In this case, the source would be the whole rhodoliths. Thus, it would be appropriate to label the living layer, the whole living rhodolith population found on the surface, as the rhodalgal sediment source or factory. The contrast between the two cores collected above the upper edge of the bed (S3-D and S4-A) suggests that the middle part of the bed was the more productive sediment factory. Core S3-D had branch-supported texture (packstone and grainstone) with high percentage of living rhodoliths and over 40% of the >2 mm sediments coming from rhodolith, while core S4-A had crumb-supported texture (finer-wackestone to wackestone) with no living rhodoliths and low percentage (<20%) of >2 mm sediments coming from rhodoliths. Core S3-D was closer to the more productive sediment factory in the middle of the bed and perhaps its textures were made up of the spillover rhodoliths materials, meanwhile core S4-A was isolated enough from the spillover of rhodolith fragments produced in the less productive sediment factory in the southern edge of the bed. Core S4-A was also in the transitional zone where higher mixing of other sediment sources such as corals and Amphiroa sp. (articulated corlline algae) may be more prominent. To further understand the construction of the rhodolith bed framework, the mechanisms of how the framework formed needed to be explored. There were three mechanisms to considered: ‘in-situ’ sediment production, sediment transport and deposition, and sorting. Two factors, physical processes and biological actions, play important roles in these mechanisms. The physical processes that 28 occurred in the El Requesón rhodolith bed were those that operated during the normal everyday conditions produced by waves and currents (Merrifield et al., 1987; Marrack, 1999) and those that operated during short-term extreme conditions produced by storms and hurricanes (Schalanger and Johnson, 1969; Gutiérrez-Sánchez, 1987; Galli-Oliver et al., 1995). Biological actions that effect the formation of rhodolith bed framework would be induced by activities of living organisms like movement, bioturbation, boring, and ingestion (Marrack, 1999; James, 2000). Physical processes and biological actions would affect the in-situ sediment production by initiating breaking, chipping, and erosion of the sediment source. Daily physical processes like waves and currents may not be a major player in the initial fragmentation of rhodoliths, however storms and hurricanes have enough force to break these rhodoliths. Waves and currents may be important in inducing abrasion between grains to produce finer particles. During calmer conditions, bioturbation and movement of organisms living in the rhodolith bed might be more important than water motions and could cause the initial breaking of rhodoliths. Rhodolith ingestion and excretion by urchins could also add to sediment production (James, 2000). Boring organisms like polychaeta (Steller, 1993) could weaken the structure of rhodoliths and aided in sediment production. Some boring organisms had been shown to cause some dissolution of the coral reef framework that weaken the reef structure and cause disintegration (Aller, 1982; Scoffin, 1992). Physical processes and biological actions would affect sediment transport and deposition at different scales. Water motion is the major transport mechanism for surface sediments. These processes transported grains laterally and covered more distance. Storms and hurricanes could rework, mix, and turn over sediments 29 in a short period. While bioturbation works more on a local scale and may be important in the vertical mixing of sediments (Scoffin, 1992; Marrack, 1999). A larger scale effect of biological activities would be the effect of growing macroalgae on the rhodalgal sediments. These algae stabilized the sediments and dampen lower energy hydraulic transport. Both physical and biological processes can affect sediment sorting. Generally, sorting increases with increasing hydraulic energy. Higher energy waves and storms can pick up, rework, and deposit coarse sediment, while winnowing the finer grains away. Meanwhile, low energy currents could only transport and deposit finer grains. Hence, well-sorted sediment in conjunction with the presence of coarser sediments indicates higher energy environment and poorly sorted sediments with the presence of finer grains indicate a low energy environment. Movement of organisms in the rhodolith bed would increase mixing between layers, hence decreasing sorting. In addition, this movement would also shake off the finer particles to fall into the interstitial spaces of the framework, hence increasing mixing and decreasing sorting. Most likely, these physical and biological processes are working together in forming the framework of the rhodolith bed, though some processes may be more dominant than the others at different part of the bed. Considering only the influence of physical processes, the rhodalgal carbonate classification can be used to interpret as depositional environment. Simplifying the classes into two depositional environments, the branch-supported classification (grainstone and packstone) would indicate higher energy environment and the crumb-supported classification (wackestone and Finer-wackestone) would indicate lower energy environment. 30

The rhodolith bed framework showed that the branch-supported framework occurs directly under the living part of the bed, indicated that this part of the bed experienced higher energy physical processes (Fig. 9). In general, the crumb- supported framework was located towards the onshore and offshore edge of the bed and below the branch-supported framework. The location of this crumb- supported framework towards offshore and the deeper edge of the bed was at the depth where energy of the waves might dissipate. On the southern edge of the bed, a big portion of the framework was crumb-supported which might be the characteristics of sediment framework of bed edges. The crumb-supported framework found below the branch-supported layer, however, might indicate a past environment with lower energy succeeding to the present environment with high energy. The vertical trend of coarse-fine-coarse generally found in all the sediment cores, from both the middle and southern transects ~350 m apart, was in agreement with the evolution higher energy environment to lower energy environment and then back to higher energy environment again. The branch-supported framework contained a high percentage of branches and this may be an indicator of active sediment production. Thus, the branch- supported framework and the living layer could be considered as the active part of the bed and the crumb-supported framework as the passive part of the bed where there was little to no sediment production. In the present condition, the onshore location of the active part of the bed was correlated with higher energy environment and the offshore location of the passive part of the bed was correlated with lower energy environment. The current active part of the bed sitting on top of the passive part of past bed suggests that in the past, the active part of the bed might have been further north then the present condition. This is corroborated by longterm observations (D. Steller and M. Foster, personal communications, 2009). 31

The vertical trend coarse-fine-coarse could then be interpreted as the succession of the active bed migrating or shrinking and expanding in the south-north-south direction over the scale of hundred years. In other words, the southern edge of the bed is more dynamic and the middle part of the bed is more stable.

Implication of the Framework as a Unique Habitat The framework of the rhodolith bed not only informed the depositional environment but also the habitat type, specifically for infaunal organisms like polychaeta, mollusks, and crustraceans. Other studies had previously shown correlation between infauna and sediment type (e.g. McNulty et al., 1962; Perkins and Halsey, 1971; Schroeder, 1972; Hines and Comtois, 1985). Burrowing behavior, abiotic requirement, and size of organisms might be correlated with the texture type. Branch-supported framework had more interstitial spaces than crumb-supported framework and might be better suited for certain species. The abundance and diversity of organisms living in a rhodolith bed might also be correlated with sediment textures (Grall et al., 2006). Further investigation of species and habitat/framework type correlation is needed to confirm this idea. The unique carbonate framework of the rhodolith bed was a product of the unique free-living nature of rhodoliths. Free-living rhodoliths are both living photosynthetic organisms and also living sediments. The formation of rhodalgal sediment assemblages depends on the depositional energy at the time. This creates an adaptable habitat; rhodolith reefs rock and roll to adapt to changing environments which could be advantageous for the longevity of the habitat and its inhabitants. As living sediments with branching morphology and vegetative reproduction capability (Bosence, 1983a; Bosence, 1983b), free-living rhodoliths can survive physical processes better, hence increasing their viability. The 32 branching morphology allows rhodoliths to be more efficient sediments. The branches act as a tripod, allowing larger rhodoliths –with more branching– to stay on top of smaller sediments produced and deposited at the same area. The branches also act like a sieve allowing smaller sediments to fall in between, keeping them from being buried. This branching morphology also allows light to penetrate deeper into the substrate so neighboring rhodoliths could photosynthesize and survive. Interlocking their branches with one another increases stability of these living rhodoliths, forming a network of rhodoliths, so together, they are more resistant to breaking caused by physical processes, except for extreme events like storms and hurricanes. In the laboratory, the network of interconnecting rhodoliths was much harder to break in a rotating tumbler than when they were separated (personal observation). In the field, if they do break, some of the broken pieces can keep growing, and with hydraulic transport, these pieces can extend the range of the bed or populate a new depositional area. Presumably, larger broken pieces have more likelihood to survive. These pieces tend to be sorted towards the surface of the substrate, allowing maximum access to light for . Most likely, there is a size limit for fragments to be successful in vegetative reproduction. The crumbs are less likely to succeed, hence not important for the survival of the community. These unfit crumbs would be sieved through and swept below the carpet of living rhodoliths and coarse fragments. The migrating framework of the rhodolith bed reflects this survival strategy and the advantage of being free-living organisms/sediments.

Conclusions • Rhodoliths followed a “trimodal” breakdown pattern with three main groups representing the modes: “cores,” “branches,” and “crumbs.” 33

• Each group had different breaking behavior but over the time of the experiment, they reached a stable range of mean sizes. • The first few seconds of the breakdown process were critical since the initial breaking happened, which caused rapid size reduction and an increase in production of smaller fragments. • Understanding the basic breakdown mechanism allowed identification of sediment group players in the rhodalgal assemblages and the correlated breakdown phases, in which assisted in the development and refinement of their textural classification. • The rhodalgal carbonate sedimentologic classification was essential for interpreting the present and past depositional environment of the rhodolith bed and the dynamic of rhodolith beds. • The framework showed the living layer of rhodoliths and rhodalgal fragments occurring at the top few centimeters of the bed, which was supported by “branch-supported” framework beneath. • The general vertical trend of coarse-fine-coarse texture found in the sediment cores, hence reflected in the framework, might suggest succession of the active part of the bed migrating in a south-north-south direction; and the present location of the active bed was southern of the past bed. • Understanding the supporting structure of the living rhodolith community provide a more complete picture of the rhodolith bed habitat and its dynamic as reefs that rock and roll • This framework construction might also be useful for further interpretation of habitat structure for infaunal organisms. 34 References

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Abstract

Declining seawater pH due to increasing dissolved atmospheric pCO2 is a global threat to marine calcifiers and their carbonate systems, including coralline red algal rhodoliths and their associated rhodolith beds. Because rhodoliths are made of high magnesium calcite, the most soluble type of calcium carbonate, they are at high risk to dissolution in acidic waters. Due to their global distribution, the effect of dissolution on this system might be seen worldwide. In this study, rhodoliths were subjected to a treatments with pCO2 adjusted to a variety of IPCC climate scenarios. Experimental results showed dissolution of rhodoliths rapidly increased with the decreasing seawater pH. Total inorganic carbon and derived total alkalinity data show detectable dissolution, starting at pH ~7.8 based on

TCO2 or between pH ~7.8 and ~7.5 based on TA. At 30% dissolution, the rhodoliths are more susceptible to mechanical breakage and increase production of smaller fragments that might alter the rhodolith bed framework. Projected estimates of rhodolith bed longevity indicate that these effects could occur within 30 years after dissolution started.

Introduction Ocean acidification is a threat to carbonate systems worldwide (e.g. The Royal Society, 2005; Kleypas et al., 2006) and is caused by increasing atmospheric carbon dioxide that dissolves into surface seawater, thus lowering ocean pH (Feely et al., 2004; Kleypas et al., 2006; Orr et al., 2005). Ocean acidification models predict that by the next century, the continuing increase of

CO2 will cause a significant decrease in ocean pH, as much as 0.7 pH units (pH 42

~7.3-7.5; Caldeira and Wickett, 2003; Orr et al., 2005). Acidified seawater corrodes and dissolves the calcium carbonate structures of marine calcifiers (Orr et al., 2005; Kleypas et al., 2006), including coralline algae rhodoliths (Jokiel et al., 2008). Rhodoliths are also known as ‘maerl’ and their carbonate skeletons are made of high-magnesium calcite (Kamenos et al., 2008), which is the most soluble type of calcium carbonate when compared to other biogenic carbonates, such as aragonite in corals and calcite in shellfish (Kleypas et al., 1999). Rhodoliths break down into smaller fragments through a combination of mechanical and chemical processes. Rhodoliths and their rhodalgal sediments accumulate in depositional environments and form rhodolith beds. The rhodalgal skeletal remains are the primary constituents of the carbonate framework of the beds, supporting the living rhodolith communities that cover only the top few centimeters of the deposits (Steller and Foster, 1995; Foster, 2001). Rhodolith beds are unique habitats with high biodiversity comparable to coral reefs and kelp forests (Barbera, 2003; Steller et al., 2003; Hinojosa-Arango and Riosmena- Rodríguez, 2004; Foster et al., 2007). They harbor many endemic species (Scoffin et al., 1985; Ballesteros, 1988; Freiwald et al., 1991; Reyes-Bonilla et al., 1997; DeGrave, 1999; Clark, 2000; James et al., 2006) and are also important nursery grounds for juvenile fishes and invertebrates (Scoffin et al., 1985; Barbera, 2003; Steller et al., 2003; Kamenos et al., 2004; Steller and Cáceres-Martínez, 2009). The effect of declining pH on these underwater ‘tumbleweeds’ and their beds will affect the organisms that depend on this particular habitat. Due to their worldwide distribution, extending from the intertidal zone to depths of over 200 m (Bosence, 1976; Scoffin et al., 1985; Littler et al., 1991; Freiwald, 1993; Foster, 2001), these effects could be widespread. 43

The predicted increase of carbonate dissolution during high CO2 conditions will enhance the role of chemical weathering in rhodolith beds. As seen in corals, dissolution could weaken the structural integrity of carbonate structures (Kleypas et al., 1999). Dissolution might cause the carbonate structure of rhodoliths to become more fragile, increasing their susceptibility to breaking. Hence, the effect of dissolution on rhodoliths may alter the sediment production of rhodalgal sediments and consequently change the framework and the habitat quality of the rhodolith beds. Therefore, the threat of ocean acidification on rhodoliths is two fold: 1) the dissolution of rhodoliths and their bed habitat and 2) the increase of mechanical breakdown due to the weakening of corroded calcium carbonate structures. Understanding the dissolution of rhodoliths due to declining seawater pH will provide information regarding how fast rhodoliths may dissolve and how robust rhodolith beds may be in a high CO2 ocean condition. Understanding the effect of dissolution on the mechanical breakdown of rhodoliths into rhodalgal fragments will provide insight into how the production of rhodalgal sediment is changed by the dissolution process. By investigating these dynamics in sediment production, possible consequences due to this change on the framework of the rhodolith bed can be predicted. This study investigated the dissolution of rhodoliths under decreased pH conditions and the effect of dissolution on the mechanical production of rhodalgal sediment. Implications of these effects on the framework of the bed are discussed.

Study Settings El Requesón is one of many rhodolith beds found throughout the Gulf of California coast in Baja California, Mexico (Steller and Foster, 1995; Foster et al., 1997; Riosmena-Rodriguez et al., 2010). Compared to the other beds in the Gulf, 44 more is known about this bed due to numerous studies previously performed on rhodolith distribution and morphology (Steller & Foster, 1995; Foster et al., 1997) physical processes (Marrack, 1999), associated species (Steller et al., 2003; Hinojosa-Arango and Riosmena-Rodríguez, 2004) and settlement and recruitment (Steller et al., 2003; Steller et al., 2009). This bed was also sampled due to its easy accessibility for sample collection and that it is a good representative of other beds in the gulf. Located in Bahia Concepción, on the windward (east side) of Isla Requesón , the bed is about 0.5 km by1 km, at about 3-12 m depth (Marrack, 1999; Steller et al., 2003). The tombolo that connects the island to the mainland and the surrounding beaches nearby is composed of carbonate sediments primarily from rhodoliths (Hayes et al., 1993; McFall, 1968). Due to the high energy environment and the influence of stronger hydraulic action (e.g. waves and storms) particularly in winter months, El Requesón bed is classified as a “wave” bed (Foster et al., 1997, Marrack, 1999) and is dominated by Lithophyllum margaritae rhodoliths (Steller et al., 2003).

Methods

Rhodoliths Collection, Storage, and Preparation Whole rhodoliths were collected using SCUBA from the bed at 7 m depth on 10 October 2008 and brought back to the laboratory. They were immediately desiccated in air for a week and stored. Before exposing them to experimental treatment, rhodoliths were sonicated for 30 minutes in seawater and 20 minutes in

3% H2O2 to clean off adhering sediment debris and organic matter that might interfere with the measurement of dissolving CaCO3 from rhodoliths in the dissolution experiment. 45 Chemical Dissolution Experiment Five groups of twenty rhodoliths were used in this experiment and incubated at five different pH levels (between 6.5 and 8.0). This range of pH values included different Intergovernmental Panel on Climate Change (IPCC) scenarios (Bindoff et al., 2007) and lower pH extrapolation of severe acidic environment. From the pool of rhodoliths with roughly 2 cm in length (longest axis), rhodoliths were randomly placed in groups while maintaining similar group ending weight of ~20 grams. Each group was incubated for 90 hours in a 1-gallon glass container filled with seawater. Control groups –containing no rhodoliths– were set up to match each pH treatment. Incubation glass containers were then placed in a water bath maintained at 20o C. Seawater used for this experiment was filtered twice using UV light and Millipore-Stericap (0.22 μm) filters to reduce the amount of particulates and respiring organisms in the seawater. This water was then bubbled with CO2 gas to make five batches of water at five pH levels. The salinity of each water batch was measured using a Bausch and Lomb refractometer. Ampicillin antibiotic solution (25 mg/L) was used to limit the amount of exogenous CO2 contributed by respiring bacteria still present in the water after filtration (Mickel and Chilldress, 1978). Dow Corning silicone based anti- foaming agent (22 μL per 22 mL of sample) was added to the seawater sample prior to the TCO2 measurement to reduce foaming caused by Ampicillin (E. Pane, personal communication, 2009). To test the contribution of bacterial respiration on rhodolith dissolution, another glass container containing a rhodolith group treatment (without antibiotic) was added at pH ~7.5. Roughly 25 mL of seawater were sampled from both control jars and rhodolith group jars before and after incubation for triplicate TCO2 and pH 46 measurements. Three 3.5 mL sub-samples were used for pH measurements using Shimadzu spectrophometer (model UV-1601) following methods developed by Dickson (Dickson et al. (Eds.), 2007). Another three replicates of 3.75 mL sub- samples were used for TCO2 measurement using non-dispersive infrared analysis technique (Friederich et al., 2002) with Li-Cor CO2/H2O (model 6262). Standard reference seawater (Dickson) was used at the start and the end of TCO2 sample measurement to standardize readings into micromoles per kilogram of seawater

(μmol/kgSW). Total alkalinity (TA) was calculated using CO2SYS program

(Pierrot et al., 2006) based on the pH, TCO2, and salinity measurements. Since non-living rhodoliths were used in this experiment, no calcification would occur, hence TA would only be affected by dissolution. Therefore, TA was used as a direct indicator for dissolution. If TCO2 and TA were due to dissolution alone,

1 mol of TCO2 would equal to 2 mol of TA, with a 1:2 ratio.

Since there were three replicate measurements of TCO2 before and after combination, there were nine pairs of TCO2 and TA to compare for every pH treatment. These were used in regression to examine the linear relationships between TCO2 or TA and hydrogen ion concentration for both control and treatment groups. Concentration of hydrogen ion was used instead of the pH since unlike pH that is logarithmic, hydrogen ion concentration is linear. T-test were then used to test for any significant difference in the slope of linear regression between control and rhodolith groups. The dissolution rate of rhodoliths was then calculated based on changes

TCO2 or TA. Using the molecular weight of CaCO3 (100.09 g/mol) and the assumption that 1 mol of TA is equal to 0.5 mol of dissolved calcium carbonate

(CaCO3) or 1 mol of TCO2 to 1 mol of CaCO3, the dissolution rate was extrapolated to determine the percent dissolution per year. The initial pH (ranging 47 from 7.82 to 6.62) from the rhodolith groups represented the pH of ocean condition in high CO2. Finally, the residence time () of a rhodolith bed at different ocean scenarios (based on initial pH used in the experiment) was derived from this dissolution rate extrapolation.

Chemical Dissolution-Mechanical Breakdown Experiment Rhodoliths used in this experiment were dissolved for 3-4 days in 0.5% HCl in a 1-gallon glass container to reach a 30% of initial weight loss. The 30% weight loss was chosen based on preliminary trials showing a visible change of breaking pattern at this percentage on individual rhodoliths. All rhodoliths selected were roughly 2 cm in length (longest axis). The goal of this experiment was to investigate the effect of 30% dissolution on rhodalgal sediment production of an individual rhodolith and a group of five rhodoliths. They were shaken in the mechanical shaker over time (0-1000 seconds, Appendix E). The sizes of fragments larger than 1 mm were tracked using image analysis methods (described below). Dry weights were measured for those smaller than 1 mm. Comparisons of the breaking patterns (i.e. their sediment size distribution) between these 30% dissolved rhodoliths were made to those of the non-dissolved rhodoliths previously acquired in the mechanical breakdown experiment (see chapter 1).

Image Analysis Samples from the chemical dissolution-mechanical breakdown experiment contained coarse fragments that could not be adequately measured by sediment sieve. The size of every particle in the sample was measured at once by taking digital images of each sample that include every particle. ImageJ (Rasband, 1997- 48

2009), an image analysis software, measured the area and length (longest axis) of each particle in an image. All particles in each sample were poured randomly on a black paper background and arranged so that not one particle was touching another and a millimeter scale was placed as size reference for the image taken. Digital images of this sediment arrangement were taken by Leica® Z6 APO zoom macroscope with attached Leica® DFC 320 camera. The sizes of these 3-dimensional particles were not fully represented in the two dimension plane of these images. Therefore, four different arrangements were set up and their average was calculated to get a better representation of these rhodalgal sediments. When necessary, several photo frames were taken to include all particles in the arrangement and then stitched later in Photoshop® to make up one photomontage representing one arrangement. This allowed measurement of sizes of all the particles in the arrangement at once in ImageJ so that the final results for every sample would be four set of sediment measurement corresponding to the four arrangements. Each photo or photomontage per arrangement was converted into a binary image of black particles on white background in ImageJ. Then, each particle in the binary image was detected, outlined, and measured using the “Analyze Particles” feature. The measurement results per photo was listed and saved as a text file. Program scripts were written and run as macros in ImageJ to process batches of photos from many samples to speed up the process. Further processing of this high volume of sediment size data –sorting, analyzing, and graphing– was performed in Matlab. 49 Results

Chemical Dissolution Experiment During the chemical dissolution experiments, rhodolith dissolution was successfully measured by examining the changes in total inorganic carbon (TCO2) and seawater pH (Table 1). In general, after 90 hours of incubation at pH range between 6.5 and 8.0, dissolution was evident as changes in total inorganic carbon and total alkalinity in the rhodolith groups: as pH decreased, TCO2 and TA increased rapidly. After incubation, the pH for the rhodolith groups generally increased but little change was observed in the controls. Change of pH observed in rhodolith groups increased as the pH of treatments decreased. Regression analysis was performed to examine the linear relationships between TCO2 and TA and initial hydrogen ion concentration. However pH could be used interchangeably to interpret the results. Fig. 1A showed the

2 relationship between TCO2 and pH for both rhodolith groups (r = 0.991, p = 2.61E-45) and controls (r2 = 0.984, p = 2.77E-40). Total alkalinity was derived from

TCO2, pH, and salinity (35 psu). Fig. 1B showed that as initial pH increased, TA was also increasing for rhodolith groups (r2 = 0.993, p = 9.63E-48) and controls (r2 = 0.527, p =1.71E-8). There were significant differences between the controls and

-82 -78 the rhodolith groups for both TCO2 (p = 1.37E ) and TA (p = 3.68E ) relationships with hydrogen ion concentration. These were clear indicators that the presence of rhodoliths affected the TCO2 and TA.

The change of TCO2 in rhodolith treatment ranged from 22.13 μmol/kgSW (CI. 0.17) to 453.66 μmol/kgSW (CI. 1.01) indicating that dissolution of rhodolith increased rapidly as pH decreased. The TCO2 showed an increase at initial pH of 7.82. However, there was no difference between the rhodolith group and control at pH 7.82 for TA as indicated by their overlapping confidence intervals. Hence 50 based on TA, dissolution could not be detected at pH 7.82. The TA change in rhodolith groups showed definite dissolution of 46.78 μmol/kgSW (CI. 13.74) starting at initial pH 7.52 that continued to increase up to 772.28 μmol/kgSW (CI. 8.04) with decreased pH down to 6.62. At pH 7.46, treatment without antibiotic showed TCO2 of 177.08 μmol/kgSW and TA of 156.64 μmol/kgSW. The observed TCO2 and TA did not exactly match the 1:2 ratio. The TCO2 was slightly higher than expected based on TA which indicated the presence of other contributing factors to TCO2 in addition to CaCO3 dissolution.

Chemical Dissolution-Mechanical Breakdown Experiment The rhodoliths used in this chemical dissolution-mechanical breakdown experiment were dissolved to reach 30% weight loss prior to subjecting them to mechanical force. One individual and a group of five rhodoliths were used in this experiment to study the effect of dissolution on individual rhodolith as well as rhodolith groups. Dissolved rhodoliths were brownish and some of their branches were shorter and slightly thinner with more delicate ends. The results from this experiment were compared to the results from the previous mechanical experiments (see chapter 1) since these rhodoliths were not dissolved prior to their mechanical trials. During mechanical trials, the first few seconds were critical moments when major changes occurred. The results presented in this section focus on mostly the changes occurring during first few seconds by comparing the 30% dissolved rhodoliths to the 0% dissolved rhodoliths for both individual rhodoliths and rhodolith groups. The effect of 30% dissolution on individual rhodoliths was shown in the rapid breakdown of the cores after the first 2-second time interval and the higher number of finer sediments production throughout the breakdown process (0-1000 51 seconds) in the experiment. The cores of the 30% dissolved individual rhodolith broke down more (from ~200 mm2 to ~40 mm2) than the non-dissolved cores (from ~220 mm2 to ~125 mm2) (Fig. 2A). The rate fracture of dissolved rhodoliths was about double the rate of the non-dissolved cores. The stable size (40 mm2) of the 30% dissolved rhodolith cores was much lower than the stable size (~125 mm2) of the non-dissolved cores. The dissolved rhodolith cores reached their stable size earlier (after 2-second) than the non-dissolved rhodolith cores (after 10- second). The production of small size fragments between 0-10 mm2 was quicker and occurred at the first 2-second of breakdown in the 30% dissolved rhodolith when compared to the non-dissolved rhodolith (Fig. 3). The series of pictures in Fig. 3 confirmed this pattern. For the non-dissolved rhodolith, the cores were easily identified even after 1000-second. Meanwhile the cores for the 30% dissolved rhodolith broke down to much smaller fragments and were harder to distinguish after 2-seconds. There were a greater amount of fine fragments in the 30% dissolved rhodolith at 1-2 seconds as shown in Fig. 3. Unlike apparent changes seen in the breaking pattern of the individual rhodolith, the effect of 30% dissolution on the rhodolith group was not as obvious. Early and rapid breaking of cores was the most noticeable effect, however increased production of finer sediments was only seen during the first 2 seconds. The cores of the 30% dissolved rhodolith group rapidly broke down during the first 2 seconds, reaching their stable size earlier than the cores of the non-dissolved rhodolith groups (Fig. 2B), but their stable sizes (~74 mm2) were larger than those cores (~55 mm2) of the non-dissolved rhodolith group. At this first 2-second time interval, the production of finer fragments was much higher compared with the 0% dissolved group, but eventually the finer-fragment production of the 0% dissolved 52 group increased, reaching similar quantity as those in the dissolved rhodoliths (Fig. 4).

From the two dissolution scenarios based on TCO2 and TA, the rate of dissolution (Fig. 5A) and residence time (Fig. 5B) of this rhodolith bed was calculated. Based on TCO2 extrapolation, at pH ~7.8, rhodolith beds will be dissolving at 1.1% per year, disappearing completely in 90 years (Fig. 5A). At this pH, rhodolith beds will reach 30% dissolution within 27 years, a time at which the effect on mechanical breakdown and sediment production were noticed. Both

TCO2 and TA scenarios showed dissolution at pH ~7.5, yet, the amount of dissolution differed. Based on the TCO2 projections made here, rhodolith beds will dissolve at 2.9% per year. This implies a residence time equal to 35 years, the time it takes to dissolve the entire bed, or only 10 years to get to 30% dissolution (Fig. 5A). Under the TA scenario, rhodolith bed will dissolve at 1.2% per year, equaling to 85 years to dissolve the entire bed or 26 years to reach 30% dissolution (Fig. 5B). The raw data of all rhodalgal fragments measured in this study are included in an additional compact disc (Appendix E).

Discussion

Chemical Dissolution Experiment

The increase in total inorganic carbon (TCO2) and total alkalinity (TA) in rhodolith groups after a 90-hour incubation indicated that the dissolution of rhodoliths increased rapidly as seawater pH decreased. Although both TCO2 and

TA showed sign of rhodolith dissolution, TCO2 might indicate that dissolution occurred at a higher pH than TA. The confidence intervals for TA between the control and the rhodolith treatment overlapped at higher pH level (~7.8) – 53 indicating no TA difference between them– hence there was not enough evidence by this parameter that dissolution occurred at this pH. On the other hand, at the same pH, there was enough difference in TCO2 between control and treatment. These differences resulted in two scenarios of rhodolith dissolution, one according to TCO2 and another relating to TA. In both scenarios, dissolution became obvious at pH ~7.5 and below.

If TCO2 and TA were caused only by dissolution of rhodoliths, then the ratio of TCO2 to TA will be 1:2. The results from this experiment did not follow this ratio, hence there were other factors, besides dissolution, that contributed to this discrepancy. Since only non-living rhodoliths were used in this experiment, calcification – another factor that could influence TA – was absent. Thus, the dissolution of rhodoliths was assumed to be the only factor that contributed to TA. More factors could influence the changes in TCO2 in addition to dissolution such as CO2 leakage and bacterial respiration. If these factors did not have much affect during the experiment, then the controls would show insignificant change of TCO2 with changing pH. Yet, there was a significant

2 -40 relationship between TCO2 and pH in the controls (r = 0.984, p = 2.77E ).

TCO2 was declining with lowering pH and this increased as pH decreased.

At a lower pH, CO2 might have leaked out of the jars since the concentration of CO2 was much higher at lower pH, for example the marked decrease of TCO2 in the control at pH ~6.6. The effect of bacterial respiration might have been variable in relation to pH. Treatment at pH ~7.5 without the use of antibiotic showed a dissolution rate that was more than double the rate of the treatment with antibiotic. It is possible that even after the use of antibiotics in the experiment, bacterial respiration was still present. If TA was assumed to be the true measure of dissolution, the discrepancy between TCO2 and TA at pH ~7.8 54 might indicate that the TCO2 was caused by bacterial respiration because TA dissolution was not present to contribute to this TCO2.

The discrepancy between observed TCO2 and expected TCO2 might be caused by the factors mentioned above. However, these factors could not fully explain the excess TCO2, which also correlated with pH. Generally, excess

TCO2 increased with decreased pH. Therefore, there must be other contributors to this excess TCO2 that could explain the correlation with pH. The existence of particulate carbonate in the seawater due to dissolution may have contributed to excess TCO2. More particulates would be released into the water as dissolution increased with decreasing pH. Since the pH was measured using a spectrophotometer, most likely, the carbonate particulates would not affect the pH measurements. The technique used to measure TCO2 required acidification of sample prior to infrared analysis. If present, these particulates would be acidified during this process and would contribute to the overestimation of TCO2. No investigation was conducted to confirm the presence of these particulates in the samples, so this idea remained untested and consequently, the different rhodolith dissolution scenarios due to the discrepancy between the TCO2 and TA remained unresolved. Yet, the only practical differences were the amount of dissolution and at what pH level rhodoliths dissolved. Based on TCO2, dissolution already occurred at pH ~7.8 while it happened somewhere between pH ~7.5 and ~7.8 according to TA.

As rhodoliths dissolved, the calcium carbonate (CaCO3) in their skeletons was released into the water. This dissolved CaCO3 acted as a buffer, making the water less acidic (increase of pH) after the incubation. Since rhodoliths dissolved more as pH decreased, this pH buffering also increased as pH decreased, slowing down the rate of dissolution. The rate of dissolution in this experiment was only 55 for non-living rhodoliths and presumably overestimated for living-rhodoliths since the living ones with their metabolic activities would be more resistant to dissolution. Data collected from the El Requesón rhodolith bed suggested pH values drop at night (R.Sanders, personal communication, 2006). Moreover, in the summer, the strong thermocline stratification in Bahia Concepción caused a hypoxic – anoxic (low pH) deep water layer (Lechuga-Devéze et. al., 2001). Tidal and wind motion could induce mixing of this deep water layer with the surface water, advecting the acidic bottom water onshore (Félix-Pico, 1987; Lechuga- Devéze et. al., 2001). The combination of night time pH drop, seasonal low-pH water mass intrusion, and low-pH ocean acidification condition might enhance dissolution and accelerate the dissolution rate of rhodoliths and the bed.

Chemical Dissolution-Mechanical Breakdown Experiment Rhodoliths used in this experiment were dissolved to reach 30% weight loss prior to breaking them down in the mechanical shaker. In the preliminary trials, rhodoliths with 30% weight loss started to show noticeable physical degradation through brownish coloration and shorter delicate tips. Thus, the 30% weight loss level was chosen for this experiment. Changes in breaking patterns and sediment size distribution were discerned by comparing these 30% dissolved rhodoliths to those non-dissolved rhodoliths from the previous mechanical experiment. The sample size for this experiment is low, one individual rhodolith and one rhodolith group, but the trends suggested by the changes in breaking behavior during the initial phase were obvious. The pronounced decrease in fragment sizes and increased production of fine sediments were additional evidence of dissolution effects seen in the individual rhodolith. 56

The rapid decrease in size at the first 2-second interval in the cores of the 30% dissolved individual rhodolith suggested that the 30% dissolution prior to the mechanical breakdown increased the fragility of the rhodolith resulting in earlier and faster breakdown. Increasing structural fragility also created more chipping points in rhodoliths. In addition, the shorten branches due to dissolution might also be the source of fine fragments which would add to the increase of fine sediment production seen in the 30% dissolved individual rhodolith. Production of fine sediments will also constitute a loss in strong hydrodynamic regimes. The effects of 30% dissolution on the rhodolith group were not as striking as those on the individual rhodolith. Yet, the obvious evidence of rapid size decrease at the first 2-second suggested that these rhodoliths were also more fragile. It is possible that, stronger and more resilient rhodoliths could have been randomly picked for this group. Hence, dissolution did not affect these rhodoliths as much and perhaps only weakened the natural breaking points in these rhodoliths for rapid breaking to occur at the first few seconds. There were no apparent size changes or detectable shifts towards smaller size fragments (except for the first 2- second) seen in the size distribution of the rhodolith group. Dissolution may be more obvious in the microstructural scale; hence, if there was any change in sizes due to dissolution, the scale of the rhodalgal fragments were measured at might not capture minor changes. Breaking is a one-way process. Earlier and faster breaking increased production of sediments that would have not occurred otherwise. Although eventually the size distribution after the experiment showed no different between the dissolved and non-dissolved rhodoliths, in the field, this initial fragmentation might affect rhodoliths’ fitness. Once they lost their branches, they would lose the ability to interlock with the neighboring rhodoliths and could easily be picked up 57 and transported away by water motion. Since these Lithophyllum margaritae rhodoliths were slow-growing about 1-5 mm a year (Steller et al., 2007), a community of mostly small rhodoliths may not be able to provide enough structure to maintain an existence of a living rhodolith bed. This study is one of the first to examine the effect of dissolution on the breakdown and production of carbonate sediments. Kotler et al. (1992) however reported an increase in destruction by abrasion after dissolution in some foraminifera. This helped inform the effect of dissolution on the abrasion mechanism but not the mechanical breakage of coarse grains. The increase in rhodalgal crumbs production found in the 30% dissolved individual rhodoliths were likely accelerated by abrasion. If dissolution accelerated abrasion, it might also accelerate breaking, perhaps by the weakening of the joints. In addition, the production of fine particles would also constitute a loss of calcium carbonate from systems characterized by strong currents.

Implication of Dissolution Estimates

Based on TCO2 and TA, two dissolution scenarios were proposed showing the rate of dissolution and residence time of this rhodolith bed. Most of initial pH used in the experiment was based on future IPCC acidification scenarios and one initial pH of a more severe condition. This was the first estimate of the effect of ocean acidification on rhodolith beds and allows exploration into further questions, such as “How quickly does a rhodolith bed dissolve?” and “How robust will a rhodolith bed be under various future ocean scenarios?” Although TCO2 extrapolation is more extreme compared to the TA extrapolation, this worse case scenario might be better used for resource management purposes, which will result in a more conservative protection policy. As seen in the example from the 58 northeastern Pacific Ocean, current ocean acidification condition is already more intense (pH <7.75) than what had been previously predicted (Feely et al., 2008). If the TA were used instead, the doubling rate of loss in about half of the time (e.g. at pH ~7.5, the rate based on TA will be 1.2% per year instead of 2.9% per year based on TCO2) would be the consequence when TCO2 extrapolation was later found to be more accurate. However, more importantly, both TCO2 and TA extrapolations suggest significant dissolution over relatively short time scales.

These results demonstrate that rhodolith dissolution is inevitable under high CO2 conditions, suggesting very few mitigation measures (other than a reversed of atmospheric CO2) that would have a positive affect on this problem. Conservative actions based on the TCO2 estimate are more appropriate to better protect this system. The effect of dissolution due to ocean acidification on the rhodalgal sediment production would also affect the framework of the bed and their survival strategy. Early and rapid initial fragmentation seen in the chemical dissolution- mechanical breakdown experiment resulting from 30% dissolution is a one-way process. This increase susceptibility to breaking could increase sediment production and lower the ability for rhodoliths to interlock with one another, creating a less stable rhodolith network. Although not consistent, increased production of small grain size sediments was observed in one rhodolith as an effect of 30% dissolution. This potential increase in crumb production might lead to a shift in sediment textures, perhaps from branch-supported to crumb-supported, making the framework more compacted with less interstitial spaces leading to a compromised quality of this habitat especially for the infaunal organisms. Increase production of rhodalgal fragments could also negatively impact the bed quality as a nursery habitat for and other invertebrates. Steller and Cáceres-Martínez 59

(2009) showed significantly smaller larval settlement and post-settlement growth for scallops that settled onto rhodalgal sediments compared to settlers on intact rhodoliths. According to the estimates in this study, the substantial changes in rhodalgal sediment production and framework alteration could be seen within the span of a human lifetime. Other studies showed evidence of dissolution on rhodoliths and other coralline algae. Jokiel et al. (2008) observed occurrence of substantial rhodolith dissolution at pH ~7.9 relative to the ambient pH ~8.2. Kuffner et al. (2008) also observed a drop in recruitment rate and percent cover of coralline algae at pH 7.9. In the field, Martin et al. (2008) noticed a more than 50% decrease in percent cover and calcium carbonate mass at pH 8.0. These studies provide independent support for the results found here and indicate that the estimate based on TCO2 (showing 1.1% per year dissolution at pH ~7.8) is robust. The results of this study suggest ocean acidification may pose significant threats to rhodolith communities and rhodolith bed habitats but further study is needed to validate the trends. A direct measurement of total alkalinity and total inorganic carbon in the dissolution experiment would address the discrepancy between TCO2 and TA. Increasing the sample size of dissolved rhodoliths would further assess the effects of dissolution on rhodolith breaking observed in this study. Investigating the sizes of fine sediments and their resistance to dissolution would define the range of sediment sizes that could be preserved in the rhodolith bed framework. Setting up an experimental manipulation directly in the rhodolith bed would fine-tune the dissolution rate estimates, which could better inform resource managers about the effect of acidic seawater directly on rhodolith bed communities. 60 Conclusions • The dissolution experiments show that with decreasing pH, rhodolith dissolution increased rapidly. This dissolution buffered the pH of seawater, causing the pH increase seen after incubation.

• Dissolution was seen earlier at pH ~7.8 based on TCO2 data and later at pH ~7.5 according to TA data.

• Earlier and higher estimates of dissolution based on TCO2 imply better protection guidelines to the rhodolith bed and might be a better estimate to use for conservation efforts since there are very few mitigation options

other than a reversed in atmospheric CO2 that could positively affect this problem. • At 30% dissolution, rhodoliths were more susceptible to breaking. Faster and earlier breakdown and increased production of fine fragments during the initial breaking moments were observed after 30% dissolution. • These effects of dissolution as seen in the laboratory experiments might cause a shift in the rhodolith bed framework, resulting in more compaction with less pore spaces. According to the estimate from the dissolution experiment, this shift could happen in less than 30 years. 61

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Orr, J.C., Fabry, V.J., Aumont, O., Bopp, L., Doney, S.C., Feely, R.A., Gnanadesikan, A., Gruber, N., Ishida, A., Joos, F., Key, R.M., Lindsay, K., Maier-Reimer, E., Matear, R., Monfray, P., Mouchet, A., Najjar, R.G., Plattner, G.-K., Rodgers, K.B., Sabine, C.L., Sarmiento, J.L., Schlitzer, R., Slater, R.D., Totterdell, I.J., Weirig, M.-F., Yamanaka, Y., Yool, A., 2005. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437, 681-686.

Pierrot, D., Lewis, E., Wallace, D.W.R., 2006. MS Excel Program Developed for CO2 System Calculations., ORNL/CDIAC-105a. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee.

Rasband, W.S., 1997-2009. ImageJ. U. S. National Institutes of Health, Bethesda, Maryland, USA. http://rsb.info.nih.gov/ij/.

Reyes-Bonilla, H., Riosmena-Rodriguez, R., Foster, M.S., 1997. Hermatypic corals associated with rhodolith beds in the Gulf of California, México. Pacific Science 51, 328-337. 65 Riosmena-Rodriguez, R., Medina-López, M.A., 2010. The Role of Rhodolith Beds in the Recruitment of Invertebrate Species from the Southwestern Gulf of California, México. In: Israel, A., Seckbach, J., Einav, R. (Eds.), Seaweeds and their Role in Globally Changing Environments. Cellular Origin, Life in Extreme Habitats and Astrobiology. Springer, Netherlands, pp. 127-138.

Scoffin, T.P., Stoddart, D.R., Tudhope, A.W., Woodroffe, C., 1985. Rhodoliths and coralliths of Muri Lagoon, Rarotonga, Cook Islands. Coral Reefs 4, 71- 80.

The Royal Society, 2005. Ocean acidification due to increasing atmospheric carbon dioxide, Policy document 12/05. The Royal Society, London.

Steller, D.S., Foster, M.S., 1995. Environmental factors influencing distribution and morphology of rhodoliths in Bahia Concepcion, B.C.S., Mexico. Journal of Experimental Marine Biology and Ecology 194, 201-212.

Steller, D.L., Cáceres-Martínez, C., 2009. Coralline algal rhodoliths enhance larval settlement and early growth of the Pacific calico scallop Argopecten ventricosus. Marine Ecology Progress Series 396, 49-60.

Steller, D.L., Riosmena-Rodriguez, R., Foster, M.S., Roberts, C.A., 2003. Rhodolith bed diversity in the Gulf of California: the importance of rhodolith structure and consequences of disturbance. Aquatic Conservation: Marine and Freshwater Ecosystems 13, S5-S20.

Steller, D.L., Hernández-Ayón, J.M., Riosmena-Rodríguez, R., Cabello-Pasini, A., 2007. Effect of temperature on photosynthesis, growth and calcification rates of the free-living coralline alga Lithophyllum margaritae. Ciencias Marinas 33, 441-456. CHAPTER 3: THE OVERALL CONCLUSIONS OF THIS STUDY

In conclusion, there were two parts that this study accomplished: the methodology contributions and research findings. The methodology contributions showed methods successfully developed in this study in order to conduct the experiments to answer the questions posed by this study. The research findings showed the results that answered the posed questions and contributed to new insights about rhodalgal sediment production and rhodolith framework.

Methodology Contributions • The image analysis method was successfully developed so that pictures of rhodalgal sediments could be easily processed and each particle in the picture could be measured in batches efficiently. This method was used to measure the size of coarse sediments above 1mm in two out three experiments in this study, and as one of the analyses performed on sediment core samples. • Dissolution of rhodoliths was effectively detected by measuring the difference in the chemistry (i.e. pH and total inorganic carbon) of the seawater used in the incubation. Total inorganic carbon and pH measurements allowed easy calculation of total alkalinity as a measure of dissolution. • The development of sedimentologic classification scheme for rhodalgal sediments was successful. The sediment cores were categorized based on this classification, which had helped with the interpretation of the depositional environment in the rhodolith bed. 67 Research Findings • Rhodoliths followed a “trimodal” breakdown pattern with three main groups representing the modes: “cores”, “branches”, and “crumbs”. Each group had different breaking behavior but over the time of the experiment, they reached a stable range of mean sizes. • The first few seconds of the breakdown process were critical since the initial breaking happened, which caused rapid size reduction and an increase in production of smaller fragments. • The dissolution experiment showed that with decreasing pH, dissolution increased rapidly. In a closed system, this dissolution buffered the pH of seawater, causing the pH increase seen after the experiment. Open Ocean systems may not experience such buffering thus the dissolution rates measured here are conservative. • From the dissolution data, extrapolation was made to provide an estimate of rhodolith bed dissolution. The dissolution rate of 1.2% per year at initial pH 7.52 condition suggested that a rhodolith bed could last only for ~85 years. • Due to 30% dissolution, rhodoliths were more susceptible to breaking. Early breakdown and increased production of fragments during the critical moments were the consequence seen after 30% dissolution. • The construction of rhodolith bed framework, based on various sediment analyses, and experimental results was successful. This framework model showed relative high and low depositional energy environments occur in the rhodolith bed. This could be useful for further interpretation of habitat structure for infaunal organisms. 68

• The framework showed that the living layer of rhodoliths and rhodalgal fragments occurring at the top few centimeters of the bed, supported by “branch-supported” framework underneath. • The general vertical trend of coarse-fine-coarse texture found in the sediment cores, hence reflected in the framework, might suggest succession of the active part of the bed migrating in a south-north-south direction; and the present location of the active bed was southern of the past bed. • The effects of dissolution as seen in the laboratory experiments (i.e. increased initial breakage and early phase sediment production) might cause a shift in the framework from “branch-supported” to “crumb- supported”. This shift, according to the estimate from the dissolution experiment, could happen in ~25 years under a pH 7.52 ocean acidification scenario. APPENDICES APPENDIX A: CHAPTER 1 TABLES Table 1. Sediments cores collected on 29 March 2007 from the El Requeson bed, Baja California Sur, Mexico. Relative distance was to the beginning live margin (point 0 m) of the bed. Two cores (S3-D and S4-A) were collected outside the upper bed margin.

TransectsSediment Vertical Water depth (m) Relative transect core ID core length (cm) distance (m)

Transect S3 S3-D 22 -4.3 -5 S3-29 17 -4.9 0 26o 38.572' N S3-28 19 -7.9 30 111o 49.823' W S3-27 21 -8.5 55 S3-26 22.5 -9.1 56 S3-25 21 -10.7 59

Transect S4 S4-A 11 -3.4 -5 S4-18 19.5 -5.5 0 26o 38.456' N S4-19 17 -6.1 3 111o 49.659' W S4-20 19 -8.8 16 S4-21 18.5 -8.8 20 S4-23 19.5 -12.2 45 S4-24 17.5 -12.2 50 71 72 Table 2. Summary showing similarities and differences of Dunham-Embry-Klovan carbonate classification (1961, 1971) and rhodalgal carbonate classification (this study).

Dunham-Embry-Klovan Rhodalgal carbonate classification carbonate classification Name Criteria Name Criteria Picture

Rudstone ->2 mm Rhodo- -High percentage of particles - Coresto whole rhodoliths supported ne mixed with cores ->10% of >2 and very few mm branches components -Whole rhodoliths and cores form the framework

Grainstone -"Rocks in Grainst -Branch-supported which grains one -Branches are so are so abundant abundant as to as to support support one another one another" -Lacking dust - Lacking mud - <10% of >2 mm components

Packstone - "Grain Packsto -Branch-supported supported ne with some crumbs muddy -60%-90% of carbonate branches rocks" - <10% of >2 mm components - Contains mud

73 Table 2. (continued).

Dunham-Embry-Klovan Rhodalgal carbonate classification carbonate classification Name Criteria Name Criteria Picture

Wackesto -"Mud- Wackes -Crumb-supported ne supported tone with some carbonate "branches" rocks" - <60% of branches -<10% of >2 mm components ->10% of grains -Contains mud

Finer- - Crumb-supported Wackst with some branches one and a lot of smaller dust particles -<60% of branches - 25% of dust

74 Table 3. The percentage of rhodalgal sediment constituents (“branches,” “crumbs,” and “dusts”), sediment origin from rhodolith, and the rhodalgal carbonate sedimentologic classification (G = grainstone, P = packstone, W = wackestone, fW = finer-wackestone) from the sediment core samples. Vertical distance is from the top of each core.

Core Sample Vertical “Branches” “Crumbs” “Dusts” Rhodolith Rhodalgal ID ID distance (%) (%) (%) origin carbonate pot morf pot )%( sedimentologic )mc( noitacifissalc

S3-D A 0 99 1 - 65 G B 3.5 92 8 - 56 G a 6 83 17 6 63 P b1086147 67P c1578221168P d1865351850P

S3-29 A 2 80 20 - 69 P a 5.5 58 42 25 53 W b 8.5 49 51 28 41 fW c 10.5 56 44 17 47 W d1446543738fW e 15.5 58 42 18 50 W

S3-28 A 1.5 88 12 - 73 P a 4 53 47 23 42 W b 6 62 38 19 60 P c 5 60 40 18 45 P d1365351959P e1763371358P 75 Table 3. (continued).

Core Sample Vertical “Branches” “Crumbs” “Dusts” Rhodolith Rhodalgal ID ID distance (%) (%) (%) origin carbonate pot morf pot )%( sedimentologic )mc( noitacifissalc

S3-27 a 3.5 61 39 21 54 P b 6.5 52 48 23 44 W c1062381147P d1360401651P e 16.5 48 52 26 36 fW f2073271060P

S3-26 a 1.5 72 28 11 53 P b 7 72 28 7 43 P c1170301254P d1342582826fW e1855452444W f2164361543P

S3-25 a 2 70 30 11 49 P b 8 53 47 16 38 W c 10.5 48 52 24 37 W d 12.5 54 46 31 41 fW e1554462141W f1957431740W

S4-A a 0 29 71 28 7 fW b 3 42 58 25 16 W c 6 18 82 28 3 fW d 9 20 80 35 7 fW

S4-18 A 1.5 99 1 - 83 G B 4.5 99 1 - 96 G a 9 70 30 11 58 P b1359412047W c1871291029P 76 Table 3. (continued).

Core Sample Vertical “Branches” “Crumbs” “Dusts” Rhodolith Rhodalgal ID ID distance (%) (%) (%) origin carbonate pot morf pot )%( sedimentologic )mc( noitacifissalC

S4-19 A 1.5 99 1 - 94 G B 3 86 14 - 83 P a 5.5 74 26 13 71 P b 7.5 51 49 22 48 W d1546542035W

S4-20 A 1 68 32 - 65 P a 4 45 55 26 40 fW b 7 33 67 30 27 fW c1031692623fW d1443571025W e1641592435W

S4-21 A 2 57 43 - 45 W a 4.5 40 60 20 30 W b 5 23 77 38 17 fW c1351492141W d1636643126fW

S4-23 a 1 37 63 19 25 W b 5 57 43 11 31 W c 8 32 68 30 24 fW d1329712422W e1839611529W

S4-24 a 1 52 48 18 40 W b 5 42 58 19 31 W c1123772610fW d1650511739W 77 Table 4. The percentage of living rhodalgal sediments from the top 7.5 cm of the sediment cores.

Core ID Living rhodalgal sediments (%) 0 to 2.5 cm 2.5 to 5 cm 5 to 7.5 cm

S3-D 50 5 0 S3-29 100 50 0 S3-28 90 10 0 S3-27 95 5 0 S3-26 100 80 0 S3-25 100 80 0

S4-A 0 0 0 S4-18 45 6 12 S4-19 50 10 0 S4-20 20 5 0 S4-21 30 50 0 S4-23 5 0 0 S4-24 2 0 0 APPENDIX B: CHAPTER 1 FIGURES 79 www.maps.com A

Transect S3

Transect S4

B maps.google.com 1 cm 1 cm

C D Fig. 1. (A) Map of Baja California Peninsula showing location of (B) El Requesón rhodolith bed and transects where (C) rhodoliths and cores of (D) rhodalgal sediments were collected. Transect notation was relative to a previous study (Steller, 1993). 1 cm

A C

ID Area Peri X Y Length Width

B D Fig. 2. Image sequence to illustrate sediment size processing in ImageJ of one sediment sample. (A) Initial image taken using Leica® Z6 APO zoom macroscope with attached Leica® DFC 320 camera. (B) Black and white binary image con- verted in ImageJ. (C) Each particle was outlined and measured for size. (D) Result of size measurements saved as a text file. 80 600 1 cm Individual 1 cm Individual 500 2 cm Individual 2 cm Individual 3 cm Individual 400 )

2 3 cm Individual 2 cm Group 300 2 cm Group

Mean area (mm 200

100

0 0123456789101000 Time (s)

Fig. 3. The mechanical breakdown of individual rhodoliths and rhodolith groups (consisting of 5 individuals per group). Starting rhodoliths were roughly 1 cm, 2 cm, and 3 cm for individuals and 2 cm for rhodolith groups. Vertical error bars represent 2 standard error for the analytical uncertainty associated with measuring area. 81 A 240 45 B “core” 40 1 1 “branches” “crumbs” 35 180 30

25 2 ) 120 20 C Area (mm

Weight (mg) 2 15 2 60 10 5 0 0 1 10 100 1000 Time (s)

Fig. 4. Trimodal pattern of rhodolith breakdown in the mechanical experiment of an individual rhodolith with initial length roughly 2 cm. (A) The breakdown process of the “cores” and “branches” are shown as changes of area (mm2) on the left y-axis and the production of the “crumbs” are shown as changes in weight (mg) on the right y-axis plotted against time (second) in a logarithmic scale. The number 1 and 2 (orange) on the graph refer the “cores” at (B) 1 and (C) 1000 seconds indicated by the orange circles in the pictures. The orange bars in the pictures mark a 1-cm scale. 82 A B 1

200 90 “core” 80 1 “branches” “crumbs” 70 150 C 2 60

50 2 ) 100 40 Area (mm Weight (mg) 30 2 50 20

10

0 0 1 10 100 1000 Time (s)

Fig. 5. Trimodal pattern of rhodolith breakdown in the mechanical experiment of a rhodolith group (consisting of 5 individuals) with initial length roughly 2 cm. (A) The breakdown process of the “cores” and “branches” are shown as changes of area (mm2) on the left y-axis and the production of the “crumbs” are shown as changes in weight (mg) on the right y-axis plotted against time (second) in a logarithmic scale. The number 1 and 2 (orange) on the graph refer the “cores” at (B) 1 and (C) 1000 seconds indicated by the orange circles in the pictures. The orange bars in the pictures mark a 1-cm scale. 83 84 A 550 2 cm Group 3 cm Individual 475 2 cm Group 3 cm Individual

) 2 cm Individual 2 400 2 cm Individual 1 cm Individual 325 1 cm Individual 250 Mean area (mm 175

100

25 0 5 10 15 20 25 30 Time (s) B 25 2 cm Group 3 cm Individual 2 cm Group 3 cm Individual 20

) 2 cm Individual 2 2 cm Individual 15 1 cm Individual 1 cm Individual 10 Mean area (mm

5

0 0 5 10 15 20 25 30 Time (s) Fig. 6. A closer look of the changes in area (mm2) of (A) the “cores” and (B) the “branches” of individual rhodoliths and rhodolith groups (consisting of 5 indi- viduals per group) at the first 30 seconds. Initial lengths of rhodoliths were roughly 1 cm, 2 cm, and 3 cm for the individuals and 2 cm for the rhodolith groups. Vertical error bars represent 2 standard error for the analytical uncer- tainty associated with measuring area. S3-D Rhodhalgal carbonate -5 m, -4.3 m S3-29 0 sedimentologic A ¢ 00 m, -4.9 m T classification (cm) B A l ¢ Rhodo-corestone a S3-28 ¢ a 30 m, -7.9 m Grainstone l 0 S3-27 T 55 m, -8.5 m 0 S3-26 Packstone b b A ¢ T 56 m, -9.1 m l 0 ¢ T S3-25 Wackestone a a c l a l l 59 m, -10.7 m b l 0 d T Finer-wackestone c l b a ¢ ¢ c l l b % Living rhodolith e l l l >80-100% 17 c d d l >60-80% c ¢ >40-60% l d l b ¢ 22 l c >20-40% e e d ¢ ¢ l >0-20% d ¢ l 19 e % Rhodolith origin e l >80-100% f l l l 21 ¢ >60-80%

f l l f >40-60% 22 ¢ ¢ >20-40% 21 l >0-20% Fig. 7. Detailed sediment cores analyses from the middle bed transect S3 showing rhodalgal carbonate sedimentologic classification, percentage of living rhodolith, and percentage of >2 mm particles originated from rhodolith. Relative dis- tance and depth are given respectively below core label. Rulers indicate vertical distance (cm) from the top of cores. 85 S4-A Rhodhalgal carbonate -5 m, -3.4 m 0 sedimentologic a S4-18 l 0 m, -5.5 m S4-19 classification (cm) 0 3 m, -6.1 m T 0 b T Rhodo-corestone l A l A c l S4-20 S4-21 Grainstone

l B 16 m, -8.8 m 20 m, -8.8 m B l l 0 0 T T Packstone d a l ¢ A a ¢ A l 11 Wackestone b l l a a S4-23 S4-24 ¢ ¢ 45 m, -12.2 m 50 m, -12.2 m Finer-wackestone b b 0 0 ¢ a a % Living rhodolith l b ¢ ¢ d c l >80-100% ¢ c ¢ c b b >60-80% 17 ¢ ¢ ¢ l >40-60% 19 d ¢ c >20-40% ¢ d e ¢ ¢ c >0-20%

d l % Rhodolith origin 19 19 l >80-100% ¢ ¢ >60-80% l d ¢ >40-60% 17 e ¢ ¢ >20-40% 19 l >0-20% Fig. 8. Detailed sediment cores analyses from the southern edge transect S4 showing rhodalgal carbonate sedimentologic classification, percentage of living rhodolith, and percentage of >2 mm particles originated from rhodolith. Relative dis- 86 tance and depth are given respectively below core label. Rulers indicate vertical distance (cm) from the top of cores. S3-29

S3-28 S3-27S3-26 S3-25

S4-18

Nearshore S4-19

S4-20 S4-21

S4-23 S4-24 Offshore Living layer

Branch-supported = high energy Crumb-supported = low energy

Fig. 9. Simplified cross section of transect S3 (top) and S4 (bottom) and interpretation of relative depostional energy (high or low). Transects were ~350 m apart. 87 APPENDIX C: CHAPTER 2 TABLE Δ Δ Table 1. Changes in total inorganic carbon ( TCO2), total alkalinity ( TA), pH values, initial hydrogen concentration, and rhodolith group weight (grams) from the chemical dissolution experiment. Dissolution in rhodolith groups was detected by Δ Δ TCO2 and TA. Error is expressed as standard error (SE) of the TCO2 and pH measurements. Confidence intervals of Δ Δ TCO2 and TA were derived from combination of nine delta pairs.

Hp laitinItnemtaerT Hp Initial [H+] gnidnE Hp TCO2 TA )ruoh-0 ta( )ruoh-0 ta( )ruoh-0 retfa( )ruoh-09 ( mol/kgSW) ( mol/kgSW)

Control 1 7.81 (SE. 0.0013) 1.56E-08 (SE. 4.79E-11) 7.77 (SE. 0.0029) -0.75 (CI. 0.66) -12.31 (CI. 2.97) Control 2 7.53 (SE. 0.0031) 2.96E-08 (SE. 2.11E-10) 7.47 (SE. 0.0032) -7.52 (CI. 0.28) -16.88 (CI. 1.05) Control 3 7.25 (SE. 0.0012) 5.63E-08 (SE. 1.60E-10) 7.28 (SE. 0.0005) -22.65 (CI. 0.74) -8.29 (CI. 0.80) Control 4 7.16 (SE. 0.0015) 6.93E-08 (SE. 2.33E-10) 7.09 (SE. 0.0003) -18.15 (CI. 0.44) -31.03 (CI. 0.72) Control 5 6.58 (SE. 0.0023) 2.61E-07 (SE. 1.40E-09) 6.67 (SE. 0.0096) -78.28 (CI. 1.10) 13.54 (CI. 7.13)

Rhodolith Grp 1 7.82 (SE. 0.0082) 1.53E-08 (SE. 2.91E-10) 7.72 (SE. 0.0015) 22.13 (CI. 0.17) -7.96 (CI. 3.09) (19.45 g) Rhodolith Grp 2 7.52 (SE. 0.0042) 3.05E-08 (SE. 2.93E-10) 7.50 (SE. 0.0024) 57.44 (CI. 2.27) 46.78 (CI. 13.74) (19.35 g) Rhodolith Grp 3 7.29 (SE. 0.0015) 5.08E-08 (SE. 1.73E-10) 7.42 (SE. 0.0025) 130.89 (CI. 0.87) 171.50 (CI. 1.33) (19.41 g) Rhodolith Grp 4 7.17 (SE. 0.0033) 6.76E-08 (SE. 5.14E-10) 7.35 (SE. 0.0038) 120.44 (CI. 0.75) 199.22 (CI. 1.82) (19.33 g) Rhodolith Grp 5 6.62 (SE. 0.0091) 2.39E-07 (SE. 4.97E-09) 7.18 (SE. 0.0033) 453.66 (CI. 1.01) 772.28 (CI. 8.04) (19.55 g) 89 APPENDIX D: CHAPTER 2 FIGURES 91 A 500 Controls Rhodolith groups 400 y = 2E+09x + 6.8081 R2= 0.9907 300 (μmol/kgSW) 2 200

100 Mean Δ TCO

0 6.4 6.8 7.2 7.6 8.0 y = -3E+08x + 0.9204 R2= 0.984 -100 pH B 900 800 700 y = 3E+09x - 39.77 600 R2= 0.9929 500 400 300 -10

TA (μmol/kgSW) Mean Δ TA 200 100 -20 y = 1E+08x - 21.747 R2= 0.5264 0 6.4 6.8 7.2 7.6 8.0 -100 pH

Fig. 1. Mean changes in (A) total inorganic carbon (ΔTCO2) and (B) total alkalin- ity (ΔTA) in relation to pH (graphed in logaritmic scale) for the duration of 90-hour incubation in the chemical dissolution experiments. Vertical bars repre- sent 2 confidence intervals. Overlapping confidence intervals between control and treatment at pH ~7.8 is detailed in the blue box. 92

A 270 0% Dissolved Individual 220 0% Dissolved Individual ) 2 30% Dissolved Individual 170

120

Mean area (mm 70

20 0 5 10 15 20 25 30 Time (s)

B 270 0% Dissolved Group 0% Dissolved Group 220

) 30% Dissolved Group 2

170

120 Mean area (mm

70

20 0 5 10 15 20 25 30 Time (s)

Fig. 2. Changes in mean area (mm2) of the “cores” for 0% and 30% dissolved (A) individual rhodoliths and (B) rhodolith groups at the first 30 seconds. Vertical error bars represent 2 SE for the analytical uncertainty associated with measuring area. 0% Dissolved 30% Dissolved 100 0% dissolved 50

0s 30% dissolved

0 50 100 150 200 250 60 40 1s 20 0 50 100 150 200 250 60 40 2s 20 0

Frequency (%) 50 100 150 200 250 60 40

10s 20 0 50 100 150 60 40

1000s 20 0 50 100 150 Size Bin (mm2)

Fig. 3. Size (area) frequency distribution of rhodalgal fragments produced from 0% and 30% dissolved individual rhodoliths over time (0-1000 seconds). Pictures are showing the fragments produced at each time intervals for both 0% and 30% dissolved rhodoliths. The orange bars in the pictures mark a1-cm scale. 93 94 40 30 0% dissolved 20 0s 30% dissolved 10 0 50 100 150 200 250 40 30 20 1s 10 0 50 100 150 200 250 40 30 20 2s 10 0 50 100 150 200 250 Frequency (%) 60 40

10s 20 0 50 100 150 200 250 60 40

1000s 20 0 50 100 150 200 250 Size Bin (mm2)

Fig. 4. Size (area) frequency distribution of rhodalgal sediment produced from 0% and 30% dissolved rhodolith groups over time (0-1000 seconds). 95 A 25

ΔTCO 20 2 ΔTA

15 (%/year) 3

10 CaCO

5

0 6.50 6.70 6.90 7.10 7.30 7.50 7.70 7.90 Initial pH B 100 Δ TCO2 90 100% Dissolved 80 30% Dissolved ΔTA 70 100% Dissolved 60 30% Dissolved 50 τ (years) 40 30 20 10 0 6.62 7.17 7.29 7.52 7.82 Initial pH

Fig. 5. (A) Rate of CaCO3 dissolution and (B) residence time extrapolated from

ΔTCO2 and ΔTA in the rhodolith groups from the chemical dissolution experi- ment. This plot predicts how quickly a rhodolith bed could dissolve or how long it would last under different ocean pH scenarios based on the initial pH of the rhodolith groups in the experiment. The mark of 30% dissolution indicates early signs of changing rhodalgal sediment production. APPENDIX E: DATA 97 A. List of mechanical shaking time (seconds) for rhodoliths and rhodoliths groups in the mechanical breakdown and the chemical dissolution-mechanical breakdown experiments

1. Mechanical breakdown experiment • Individual rhodoliths o 1 cm initial size  R10: 0s, 2s, 5s, 10s, 21s, 103s, 1000s  R11: 0s, 2s, 10s, 20s, 100s o 2 cm initial size  R3: 0s, 1s, 2s, 5s, 10s, 20s, 100s, 1000s  R4: 0s, 1s, 2s, 5s, 10s, 20s, 100s, 1000s o 3 cm initial size  R8: 0s, 1s, 2s, 5s, 10s, 21s, 101s  R9: 0s, 1s, 2s, 5s, 10s, 20s, 100s • Rhodolith groups o 2 cm initial size  RGa: 0s, 1s, 2s, 5s, 10s, 20s, 100s, 1000s  RGb: 0s, 1s, 2s, 5s, 10s, 20s, 100s, 1000s 2. Chemical dissolution-mechanical breakdown experiment • Individual rhodoliths o 2 cm initial size  Rz: 0s, 2s, 5s, 10s, 20s, 100s, 1000s • Rhodolith groups o 2 cm initial size  HM5: 0s, 1s, 2s, 10s, 100s, 1000s

B. Description of data compact disc included in this thesis (also in READ_ME_FIRST.rtf)

This data CD contains all the raw size data from rhodoliths and rhodalgal sediments in this thesis study.

Thesis title: The Role of Mechanical and Chemical Processes on Rhodalgal Sediment Production and Implication of Ocean Acidification (Baja California Sur, Mexico) Author: Elsie Tanadjaja

Data organization and description: • Data are organized in three different main folders: SedimentCores, MechanicalBreakdownExperiment, and ChemicalDissolution- 98 MechanicalBreakdownExperiment. • In each folder, data are organized by method of acquisitions in folders: "ImageJ_Data" and "LaserParticleSizer_Data" • Data acquired by ImageJ freeware are in text files and those measured by laser particle sizer are in an Excel file, with the results showing frequency vs. volume for 115 size bins between 0.04 and 2000 m. • The text files from ImageJ are organized in columns. Column titles are ParticleID, Area (mm2), Perimeter(mm), Length(mm), and Width (mm) respectively. • Sediment core data are organized based on location where sample was collected in the sediment core ("Top" and "Downcore"), transects ("ERS3" and "ERS4"), and core ID number (25 - 29 and D for transect S3 and 18-24 and A for transect S4) • In the "MechanicalBreakdownExperiment" folder, the data are organized based on sample id as following:  Individual rhodoliths: - R10 and R11 with initial size 1cm - R8 and R9 with initial size 2 cm - R3 and R4 with initial size 3 cm  Rhodolith groups: - RGa and RGb with initial size of 2 cm • In the "ChemicalDissolution-MechanicalBreakdownExperiment" folder, the data are organized based on sample id as following:  Individual rhodoliths: - Rz and R9 with initial size 2 cm  Rhodolith groups: - HM5 with initial size of 2 cm Individual rhodoliths:

• Text files name for both "MechanicalBreakdownExperiment" and "ChemicalDissolution-MechanicalBreakdownExperiment" follow this naming system: • the four numbers preceding "s" indicate the time interval • the number after "s" indicates the replication number • for example: "R110100s3.txt" means data in this file come from individual rhodolith R11 (with initial size 2 cm) at 100 s time interval and these were the third replication. California State University, Fresno

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Elsie Dekawati Tanadjaja

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26 October 2010

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