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Shell abnormalities in Archaias angulatus (foraminifera) from the Florida Keys

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Title:
Shell abnormalities in Archaias angulatus (foraminifera) from the Florida Keys an indication of increasing environmental stress?
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Language:
English
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Souder, Heidi Lynne
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Calcification
Carbonate
Coral reef
Environmental change
Ultra structure
Dissertations, Academic -- Marine Science -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Summary:
ABSTRACT: Historically, Archaias angulatus has been a major contributor to foraminiferal assemblages and sediments in coral-reef environments throughout the Caribbean and tropical Atlantic. A variety of anomalous features were observed in the tests of A. angulatus individuals collected live from the Florida reef tract in 2004 and 2005. Six types of anomalies were documented using scanning electron microscopy: microborings, microbial biofilm, pitted surfaces, dissolution, calcification abnormalities, and growth abnormalities. Calcification abnormalities included mineralogical projections, lacy crusts, and repair marks. These abnormalities were found among both juvenile and adult A. angulatus, and similar features were also found among Cyclorbiculina compressa and Laevipeneroplis proteus specimens collected live in the same samples. In 2006, a comprehensive study was undertaken to see if the occurrence and types of morphological abnormalities have changed in A. angulatus from the Florida Keys over the past 2.5 decades.Archived samples of A. angulatus collected in 1982-83 from John Pennekamp Coral Reef State Park were compared to recent samples. Seven different types of morphological abnormalities and 5 different surface texture anomalies were documented. Eighty-six combinations of abnormalities and surface textures were observed. Physical abnormalities included profoundly deformed, curled, asymmetrical, and uncoiled tests, irregular suture lines, surface "blips," and breakage and repair. Surface texture anomalies included surface pits, dissolution, microborings, microbial biofilm, and epibiont growth. Epibiont growth included bryzoans, cyanobacteria and foraminifers. The archived samples were not obviously more pristine than the recent samples indicating stress was well underway in the early 1980s. Test strength was compromised in deformed specimens.Crushing strength of abnormal individuals was much more variable compared to individuals with irregular sutures and normal specimens. Deformed individuals also exhibited abnormal test wall structure including dissolution and infilling. Mg/Ca ratios for normal and deformed specimens were within normal parameters (12-15 μmol/mol). Implications of these observations are at least twofold. First, in studies of fossil assemblages, damage to tests and changes in test-surface textures should not be assumed to have occurred postmortem, and may provide evidence of environmental stressors acting upon living populations. In addition, we speculate that test dissolution in larger miliolid foraminifers when alive can indicate declining carbonate saturation in seawater, which can result locally from salinity changes or increasing benthic respiration rates, as well as globally from rising concentration of atmospheric CO₂.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
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Includes bibliographical references.
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by Heidi Lynne Souder.
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Title from PDF of title page.
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Document formatted into pages; contains 117 pages.
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Includes vita.

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aleph - 002028812
oclc - 436452378
usfldc doi - E14-SFE0002813
usfldc handle - e14.2813
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Shell Abnormalities in Archaias Angulatus (Foraminifera) from the Florida Keys: An Indication of Increasing Environmental Stress? by Heidi Lynne Souder A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Co-Major Professor: Pamela Hallock Muller, Ph.D. Co-Major Professor: Robert Byrne, Ph.D. Edward Van Vleet, Ph.D. Norman Blake, Ph.D. Lisa Robbins, Ph.D. Date of Approval March 23, 2009 Key Words: calcification, car bonate, coral reef, environmen tal change, ultra structure March 23, 2009, Heidi Lynne Souder

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ACKNOWLEDGMENTS Specimens examined for this research were collected under permits FKNMS-2003-002 and FKNMS-2005-002 from the Florida Keys National Marine Sanctuary with funding from National Oceanic and Atmospheric Ad ministration/National Undersea Research Center UNCW Subcontract 2003-24A, NOAA through the Florida Hurricane Alliance (funding for sampling ~2004-2007) and the U.S. Environmental Protection Agency Gulf Ecology Division Grant No. X7-96465607-0 (20 08). I acknowledge the following for their financial support: University of South Florida College of Marine Science, NSF GK12 Fellowship Program, Center for Ocean Technology, The Society for Underwater Technology, and the Cushman Foundation. I would like to thank Michael Souder and Sherryl Gilbert for their financ ial, emotional, and logistic al support. I thank Pamela Hallock Muller and Robert By rne for acting as my co-major professors, and Edward Van Vleet, Lisa Robbins, and Norman Blake for acting on my committee. Additional thanks go to the following people who help me comp lete this dissertation: Bryan McCloskey, Chad Lembke, Joe Kolesar, Graham T ilbury, Randy Russell, Julie Richey, Ethan Goddard, Chris Simoniello, Lori Adornat o, Michele Winowitch, Alexa Ramirez, Vembu Subramanian, Laura Sherry, Eli Gilbert, Te resa Greely, Angela Lodge, Jim Patten, and Alexa Ramirez.

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv LIST OF PLATES vi ABSTRACT vii 1. INTRODUCTION 1 1.1 Reef Decline in the Florida Keys 1 1.2 Biomineralization 3 1.3 Foraminifera 7 1.4 Morphologic Abnormalities in Porcelaneous Foraminifera 12 1.5 Background on the Foraminifer Archaias angulatus 15 1.6 Objectives 16 2. ANOMALOUS FEATURES OBSERVED ON SHELLS OF LIVE ARCHIASINE FORAMINIFERS FROM THE FLORIDA KE YS, USA 18 2.1 Introduction 18 2.2 Materials and Methods 21 2.3 Results 22 2.4 Discussion 28 2.5 Conclusions 37 3. MORPHOLOGICAL ABNORMALITIES IN A POPULATION OF ARCHAIAS ANGULATUS (FORAMINIFERA) FROM THE FLORIDA KEYS (USA) SAMPLED IN 1982-83 AND 2006-07 39 3.1 Introduction 39 3.2 Materials and Methods 43 3.3 Results 45 3.4 Discussion 68 3.5 Conclusions 73 4. SHELL STRENGTH AND ULTRA ST RUCTURE IN DEFORMED ARCHIAS ANGULATUS FROM THE FLORIDA KEYS (USA): IMPLICATIONS FOR SURVIVAL AND COASTAL SEDIMENTATION 75

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ii 4.1 Introduction 75 4.2 Materials and Methods 79 4.3 Results 83 4.4 Discussion 91 4.5 Conclusions 98 5. CONCLUSIONS 99 6. REFERENCES 103 APPENDIX A 115 ABOUT THE AUTHOR End page

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iii LIST OF TABLES Table 2.1. Types of abnormalities found at each sampling site 23 Table 3.1. Sampling date, number of specimens analyzed per sample, mean diameter, standard deviation 47 Table 3.2. Percentages of specimens exhibiting physical abnormalities (number of specimens examined per 50 Table 3.3. Percentages of specimens exhibi ting surface anomalies (number of specimens examined per 50 Table 3.4. Summary of percent normal and top five most abundant anomalies or combinations of 51 Table 4.1. Results of crushing strength experiments for normal specimens listing maximum shell diameter 86 Table 4.2. Results of crushing experiments for abnormal individuals listing maximum shell diameter (mm) 8 9 Table 4.3. Results of the ANOVA for Mg/Ca ratios 90

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iv LIST OF FIGURES Figure 1.1. A. Light micr ograph of a living adult Archaias angulatus ; B. SEM of adult individuals 15 Figure 2.1. Map of Florida Keys showi ng the location off Key Largo, New Found Harbor, Hawk Channel and John Pennekamp Cora l Reef State Park 22 Figure 3.1. Percentage of individuals with irregular sutures vs diameter (1982-83) 52 Figure 3.2. Percentage of individuals with irregular sutures vs diameter (2006-07) 52 Figure 3.3. Percentage of individuals with curling vs diameter (1982-83) 53 Figure 3.4. Percentage of individuals with curling vs diameter (2006-07) 53 Figure 3.5. Percentage of individuals with asymmetry vs diameter (1982-83) 54 Figure 3.6. Percentage of individuals with asymmetry vs diameter (2006-07 54 Figure 3.7. Percentage of profoundl y deformed individuals vs diameter (1982-83) 55 Figure 3.8. Percentage of profoundl y deformed individuals vs diam eter (2006-07) 55 Figure 3.9. Percentage of break age and repair vs diameter (1982-83) 56 Figure 3.10. Percentage of breakage and repair vs diameter (2006-07) 56 Figure 3.11. Percentage of individua ls with surface blips vs diameter (1982-83) 57 Figure 3.12. Percentage of individua ls with surface blips vs diameter (2006-07) 57 Figure 3.13. Percentage of unco iled individuals vs diameter (1982-83) 58 Figure 3.14. Percentage of unco iled individuals vs diameter (2006-07) 58 Figure 3.15. Percentage of indivi duals with dissolution vs diamet er (1982-83) 59 Figure 3.16. Percentage of indivi duals with dissolution vs diamet er (2006-07) 59

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v Figure 3.17. Percentage of individua ls with surface pitting vs diameter (1982-83) 60 Figure 3.18. Percentage of individua ls with surface pitting vs diameter (2006-07) 60 Figure 3.19. Percentage of individuals with microborings vs di ameter (1982-83) 61 Figure 3.20. Percentage of individuals with microborings vs di ameter (2006-07) 61 Figure 3.21. Percentage of indivi duals with microbial biofilm vs diameter (1982-83) 62 Figure 3.22. Percentage of indivi duals with microbial biofilm vs diameter (2006-07) 62 Figure 3.23. Percentage of indivi duals with epibionts vs diameter (1982-83) 63 Figure 3.24. Percentage of indivi duals with epibionts vs diameter (2006-07) 63 Figure 3.25. MDS plot of archived samples 65 Figure 3.26. Results of cluster analysis on a group-averaged Bray-Curtis similarity matrix for the 66 Figure 3.27. Results of cluster analysis on a group-averaged Bray-Curtis similarity matrix for the recent 67 Figure 4.1. Set up of crushing apparatus; A inverted load cell and B movable platform 81 Figure 4.2. Results of crushing experiments fo r normal shells with shell strength in Newtons plotted 87 Figure 4.3. Results of crushing experiments fo r abnormal shells with shell strength in Newtons plotted 87 Figure. 4.4. Results of crushing experiment s of shell strength (N) plotted against maximum shell diameter 88 Figure 4.5. Mg/Ca ratios for normal and deformed shel ls 90

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vi LIST OF PLATES Plate 2.1. Archaias angulatus : 1-3 normal shells, 1 normal pseudopores and suture lines, 2 juvenile aperture 25 Plate 2.2. Archaias angulatus : 1 2 from same individual, bacteria on adult from Tennessee Reef; 3 26 Plate 2.3. 1 dissolution of previously pitted surface from Tennessee Reef; 2 deformed juvenile Archaias 27 Plate 3.1. Archaias angulatus ; 1 normal adult, 2 normal juvenile aperture, 3-4 profoundly deformed, 5 48 Plate 3.2. Archaias angulatus ; 1 normal adult sutures, 2 dissolution, 3 dissolution and surface pits, 4 49 Plate 4.1. Archaias angulatus ; 1 normal adult, 2 normal juvenile aperture, 3-4 profoundly deformed, 5 85 Plate 4.2. Archaias angulatus ; 1 Normal shell wall, 2-3 individual with breakage and repair exhibited welded 91

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vii SHELL ABNORMALITIES IN ARCHAIAS ANGULATUS (FORAMINIFERA) FROM THE FLORIDA KEYS: AN INDICATION OF INCREASING ENVIRONMENTAL STRESS? Heidi Lynne Souder ABSTRACT Historically, Archaias angulatus has been a major contributor to foraminiferal assemblages and sediments in coral-reef environments throughout the Caribbean and tropical Atlantic. A variety of anomalous features were observed in the tests of A. angulatus individuals collected live from the Florida reef tract in 2004 and 2005. Six types of anomalies were documented using scanning electron microscopy: microborings, microbial biofilm, pitted surfaces, dissoluti on, calcification abnor malities, and growth abnormalities. Calcification abnormalities in cluded mineralogical projections, lacy crusts, and repair marks. These abnorma lities were found among both juvenile and adult A. angulatus and similar features were also found among Cyclorbiculina compressa and Laevipeneroplis proteus specimens collected live in the same samples. In 2006, a comprehensive study was undertaken to see if the occurrence and types of morphological abnormalities have changed in A. angulatus from the Florida Keys over the past 2.5 decades. Archived samples of A. angulatus collected in 1982-83 from John Pennekamp Coral Reef State Park were compar ed to recent samples. Seven different types of morphological abnormalities and 5 di fferent surface texture anomalies were

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viii documented. Eighty-six combinations of abnormalities and surface textures were observed. Physical abnormalities included profoundly deformed, curled, asymmetrical, and uncoiled tests, irregular suture lines, surface “blips,” and breakage and repair. Surface texture anomalies included surface pits, dissolution, microborings, microbial biofilm, and epibiont growth. Epibiont gr owth included bryzoans, cyanobacteria and foraminifers. The archived samples were not obviously more pristine than the recent samples indicating stress was we ll underway in the early 1980s. Test strength was compromised in deformed specimens. Crushing strength of abnormal individuals was much more variable compared to indivi duals with irregular sutures and normal specimens. Deformed indi viduals also exhibited abnormal test wall structure including dissolution and infilli ng. Mg/Ca ratios for normal and deformed specimens were within normal parameters (12-15 mol/mol). Implications of these observations are at least twofold. First, in studies of fossil assemblages, damage to tests and changes in test-surface textures should not be assumed to have occurred postmortem, and may provi de evidence of environmental stressors acting upon living populations. In addition, we sp eculate that test dissolution in larger miliolid foraminifers when alive can indicate declining carbonate saturation in seawater, which can result locally from salinity changes or increasing benthic re spiration rates, as well as globally from rising concentration of atmospheric CO2.

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1 1. INTRODUCTION 1.1 Reef Decline in the Florida Keys Decline of coral reefs has been occurring globally at unprecedented rates over the past few decades (e.g., Santavy et al., 2005; Fran cini et al., 2008; Palandro et al., 2008). Since natural and anth ropogenic pressures exis t in the coastal envi ronment, coral reef decline is a complex and multifaceted dilemma. The plethora of ecological stresses affecting coral reefs range from local to globa l scales and the last thirty years of the 20th century were marked by escalating severity of coral reef perturbations including disease and coral bleaching. Starti ng very early in the 1970s, Cari bbean and Atlantic acroporids were devastated by white band disease (Gladfelter, 1982). Since then, many other diseases such as black band disease, white pox and rapid wasting disease have decimated many types of corals. Santavy et al. ( 2005) assessed the condition of coral reefs throughout South Florida and f ound that coral disease was pr evalent over a large portion of their sampling area, i.e., at least one co ral colony with active di sease was present in about 85% of the sample area. Coral dis ease was extensively di spersed throughout the Florida reef tract and did not appear confined to a ny particular sites. Coral bleaching has also played a signifi cant role in the decline of these diverse ecosystems (Hoegh-Guldberg 1999, 2004). Blea ching is a common stress response of corals to various natural and human-indu ced disturbances including temperature

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2 extremes, increased sedimentation, inorganic nu trients and solar radi ation and is defined as the temporary or permanent loss of sy mbiotic, photosynthetic microalgae or their pigments. Bleaching episodes can be local phenomena or large-scale events occurring over large regions. Since bleaching is a general response to stress, it can be induced by many factors, individually or in combin ation (e.g., Glynn, 1996). The first reported widespread mass coral bleaching events occurred in 1983 (Glynn, 1984) and again in 1987 (Williams and Bunkley-Williams, 1990). Ma ss bleaching events, which can cover thousands of square kilometers, can be initiated by small increases (+1-3O C) in water temperature. These events have increased in frequency, duration and magnitude over the past 25 years (Hoegh-Guldberg, 2004). Many other stresses are contributing to coral reef decline, a list which is extensive, serious and not easily mitigated. Although cora l reefs are among the most ecologically diverse eco systems on Earth, they thrive in oligotrophic (nutrient poor) waters. However, in perturbed coastal ar eas, increased sedimentation, which is often accompanied by nutrient loading, is a significan t problem. In many coastal areas nutrient loading is the combined result of run off fr om land and disposal of human sewage. For example, the Florida Keys have over 600 inje ction wells in operation (Griffin et al., 1999). Although sewage can contain consider able amounts of toxic materials such as pesticides, herbicides, chlorine, and heavy me tals, most reported sewage-related effects on coral reefs have been on the stimulatory rath er than the toxic natu re of sewage (Grigg and Dollar, 1990). Generally speaking, increased sediment and nutrient loading favor growth of macroalgae over hermatypic corals (Grigg and Dollar, 1990; Hallock et al., 1993; Dustan,

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3 1999). Nutrient loading, whether from runoff or sewage dispos al, can have a wide range of cascading effects on a coral-reef ecosyst em ultimately resulting in macroalgal dominance. Suspended sediments and plankt on blooms reduce light levels available to the corals. Algae grow much faster than co rals when nutrients ar e replete due to rapid uptake from the water column (Dustan, 1999). Bare coral skeleton le ft as a result of bleaching or disease can be colonized quickly by algae and sponges, resulting in a shift from coral to algal or sponge-algal communities. Examples of replacement of coraldominated communities by algae, as a result of sewage, can be found all over the globe. A very well known case in point is the overg rowth on the 1960s and early 1970s of mid bay reefs in Kanehoe Bay, Hawaii, by Dictyospheria cavernosa a green bubble algae (Smith et al., 1976; Hallock et al., 1993). Furthermore, algal overgrowth can be further accelerated when levels of herbivory are redu ced or altered (Dusta n, 1999; Miller et al., 1999). Bioerosion, subaereal exposure, epizootics, xenobiotics, freshwater dilution, and solar radiation are other serious problems facing coral reefs (Dustan, 1999). Further, increasing atmospheric CO2 has emerged as a global threat not just to coral reefs, but to many marine calcifiers (Kleypas et al., 1999). 1.2 Biomineralization Biomineralization refers to the pr ocesses by which living organisms form minerals for their shells, skeletons or othe r mineralized structures. Biominerals are typically a composite of inorganic crystals and organic components. Since they are formed under controlled conditions, biominer al properties are often characterized by

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4 particular shapes, sizes, cr ystal structure, and isotopic and trace element composition. Calcium carbonate minerals are the most a bundant biogenic minerals both in terms of their distribution among many different taxa and quantities produced (Weiner and Dove, 2003). The implications of decreased calcium carbonate saturation in marine waters due to increased p CO2 in the atmosphere are profound becau se the energy required to secrete and maintain a calcium carbonate skeleton is a function of how saturated seawater is with respect to calcium carbonate (Morse a nd MacKenzie, 1990; Toler et al., 2001). Furthermore, the solubility product of hi gh magnesian calcite (> 8 mol % MgCaCO3) is even higher than aragonite (Weyl, 1967; Pl ummer and Mackenzie, 1974). Therefore, organisms that produce high Mg-calcite shells may be particularly sensitive to reduced CaCO3 saturation. Calcification refers to the processes that result in the build up of calciumcontaining minerals (not just calcium carbonate) and includ es geochemical precipitation, biologically enhanced geochemi cal precipitation, animal calci fication, algal calcification, and calcification involved in symbioses. Geochemical precipitati on is not biologically mediated and occurs in warm shallow waters with elevated salinities that are supersaturated with respect to CaCO3. Biologically enhanced geochemical precipitation takes place when the biological functions of organisms cause local changes in seawater chemistry that increase carbonate saturation so that calcium carbonate precipitates from seawater around the organism. This type of calcification occurs in stromatolites and whitings. Moreover, a crucial factor controlling carbonate mineralogy is the magnesium/calcium ratio in seawater, which is largely influenced by ion exchange at mid ocean ridges (Hardie, 1996). The alteration of basalt removes Mg2+ from seawater and

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5 releases Ca2+, with the rate of this exchange depe ndent upon the rate of new oceanic crust formation. Therefore, times of high mid-ocean ridge activity not only result in elevated atmospheric CO2 concentrations and HCO3 in seawater, but also higher Ca2+ concentrations in seawater. These conditi ons are energetically more favorable for organisms that produce calcite over those that produce aragonite On the contrary, when seafloor spreading rates slow down, rates of Mg2+ removal from and Ca2+ release into seawater decline. This results in a hi gher Mg/Ca ratio in seawater, which favors aragonite precipitation (Hardie, 1996) or pr ecipitation of variable Mg calcite. Biologically controlled mi neralization processes are much more complex than geochemical precipitation. Organisms use cellu lar processes to dire ct the nucleation, growth, morphology, and final location of the mineral that is deposited. Although the degree of control varies among species, almost all controlled mineralization processes occur in an isolated environment with sophi sticated, species-specific results (Weiner and Dove, 2003). The association between calcification a nd photosynthesis appears strong at the organismal level because calca reous plants and symbioses te nd to calcify faster in the light, calcify faster than mo st non-photosynthetic organisms, and often approach a 1:1 molar ratio of calcificati on to photosynthesis (McConnaughe y, 1994). In coral reef environments scleractinian corals and sy mbiont-bearing foraminifers are prolific calcifiers and conventional wisdom has long he ld that photosynthesis by the symbionts promotes calcification by the splitting of bicarbonate (ter Kuile, 1991, Hallock, 2001) and removing CO2 (Equation 7).

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6 7) Ca2+ + 2HCO3 CO2 (to photosynthesis) + CaCO3 (calcification) + H2O However, McConnaughey and Whelan (1997) ha ve proposed the reverse interpretation where the lack of CO2 limits photosynthesis in warm, sh allow, alkaline environments. Meaning, calcification provide s protons that make CO2 readily available from the much more abundant bicarbonate ions (Equations 8 and 9). In essence, calcification promotes photosynthesis. 8) Ca2+ + HCO3 CaCO3 + H+ 9) HCO3 + H+ CH2O + O2 According to this hypothesis, the electron capture phase of photosynthesis provides ATP for active transport of Ca2+ and H+ ions, promoting calcification and making bicarbonate ions a viable source of CO2 for the organic carbon-synthe sis phase of photosynthesis. Further supporting this hypothesis, Erez (1983) found essentially normal calcification rates in symbiont-bearing foraminifers that had been treated with an herbicide that blocks photosystem II, the carbon fixation step in phot osynthesis. However, photosystem I, the initial step in which solar energy in capt ured and fixed into ATP, was unaffected, indicating direct en ergetic control. The photosynthetic uses of calcifica tion are easily appreciated because bicarbonate, the most abundant carbon source in alkaline wate rs, is inaccessible without a source of protons. It is po ssible that diffusion from ambient waters can supply these protons. But the photosynthetic organism is then bathed in an alkaline, CO2 depleted

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7 micro-environment, which actually inhibits photosynthesis. Ca lcareous plant and symbioses discharge protons from calcificati on into their boundary layers and maintain CO2 concentrations despite photosynthetic CO2 uptake. Combining Equations 8 and 9, a 1:1 ratio of calcification to photosyn thesis is obtained (Equation 10). 10) Ca2+ + 2HCO3 CaCO3 + CH2O + O2 Equation 10 does not consume or produce H+ or CO2, so this affects solution pH and p CO2 less than calcification and photosynt hesis individually (McConnaughey and Whelan, 1997). 1.3 Foraminifera Members of the class Foraminifera ar e shelled protists whose higher-level taxonomy has traditionally been based on shel l mineralogy. Extant forms are generally categorized into four major groups: a) taxa which produce organic she lls, b) agglutinated taxa, c) calcareous perforate ta xa, and d) calcareous imperfor ate (porcelaneous) taxa (Sen Gupta, 1999; Erez, 2003). Hallock (2000) pr oposed that reef-dwelling foraminifers, especially larger taxa that hos t algal symbionts, have substa ntial promise as indicators of coral reef vitality because physiological an alogies between zooxanthellate corals and foraminifers with algal symbionts result in similar environmenta l requirements. Furthermore, similar types of stress sympto ms have been observed in foraminiferal populations as those reported fo r corals themselves. Cockey et al. (1996) reported that considerable changes in the foraminiferal assemblages of the Fl orida reef tract had

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8 occurred even before the onset of fora miniferal bleaching in 1991. Comparisons of surface sediment samples collected in 1982, 1991, and 1992 with samples collected in 1960 revealed there was a shift in dominance from symbiont-bearing foraminifers, such as Amphistegina gibbosa and Archaias angulatus to smaller detritus-consuming taxa. This shift in foraminifersl assemblages occu rred at the same time coral cover in the Florida Keys decreased while algal and sponge cover increased (Dustan and Halas, 1987; Hallock et al., 1993; Dustan 1999). Perforate foraminifers dominate in to day’s oceans. They can have simple morphologies constructed of one or a few cham bers or can be very complex composed of many chambers arranged in various threedimensional configurations. Perforate foraminifers may produce either low magnesium or variable calcite shells, many of which exhibit coiling with planispiral or trochospira l geometries. All perforate foraminifers are covered in microscopic pores sealed by organic caps or plugs which prevent the cytoplasm from flowing out of the shell (Erez, 2003). Another distinguishing feat ure found in these foraminife rs is the presence of laminations in the fabric of the shell wall. Laminations are formed when individuals cover their pre-existing shell with a new laye r of calcite, sandwiched between layers of organic matrix, as they add new chambers. Therefore, the shell is composed of many layers of alternating organic matrix and radi al calcite, the number of layers depending upon the number of chambers per whorl. A lthough little is known a bout the calcification mechanism itself, the bulk of the shell is composed of secondary laminations (Erez, 2003). In short, calcification takes place in situ (Angell, 1980).

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9 Biomineralization in porcelaneous foramini fers is quite different. In general, the walls of porcelaneous shells include a thic k layer of high magnesi um calcite needles arranged randomly in three dimensions in an organic matrix and coated with a thin layer of regularly arranged high magnesium calcite rhombohedral plates (Lipps, 1973; MacIntyre and Reid, 1998; Debena y et al., 2000). The crystals composing the bulk of a porcelaneous shell wall are formed in the cyto plasm and transported to the newly forming chamber wall. The random orientation of cal cite rods and the presence of rhombohedral plates block light causing the shell to a ppear opaque. The opaque appearance coupled with the veneer formed by the rhombohedral plates creates a porcelan eous finish to the shell, hence the name (Erez, 2003). Porcel aneous foraminifers are not perforated although some, such as Archaias angulatus are covered with pseudopores which allow for gas exchange and promote light penetration to symbiotic algae. Biomineralization in symbiont-bearing foraminifers is further complicated by host-symbiont interactions influencing uptak e of inorganic carbon and internal carbon cycling. Ter Kuile and Erez (1987) investigated the incorporation of inorganic carbon in Amphistegina lobifera which is perforate, and Amphisorus hemprichii which is porcelaneous. They concluded that perforate species appear to have a large internal inorganic carbon pool, which serves mainly fo r calcification. Due to this large pool of inorganic carbon, A. lobifera showed a time lag for incorpor ation of inorganic carbon into its shell. Conversely, imperfor ate taxa have little or no internal inorganic carbon pool and may take up carbon for calcification dir ectly from seawater. Consequently, photosynthesis and calcification appeared to be simultaneous. Ter Kuile et al. (1989a,b) further investigated decoupling of photosynt hesis-calcification processes in symbiont-

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10 bearing foraminifers and revealed compe tition between photosynthesis and calcification for inorganic carbon in A. lobifera The classification of Foraminifers ha s largely been based on morphology and wall structure (Loeblich and Tappan, 1987), both of which are governed by the processes involved in chamber formation (Wetmore, 1999). Although chamber formation has been described for only a few foraminiferal species, in A. angulatus has been well documented by still photography, cytological work and vi deo recording (Marszalek, 1969; Wetmore, 1999). New chamber formation starts with the creation of a protective cyst that encloses the area where the new chamber will form. Within the cyst, the reticulopodia form a dense network and are in contact with the cy st. The reticulopodia retract from the cyst once it is complete and are ve ry uniform in length and closely spaced. Next, an anlage, or template, forms. This large mass of vesi cular cytoplasm assumes the general shape of the new chamber and probably serves as the substrate for the secretion of the outer organic membrane. Initial thickening of the cytoplasm occurs out near the growth cyst but then retracts along with the mass of retic ulopodia to the final position for the new chamber. The organic membrane forms within the anlage as relatively thick structure that appears as thick as the calcified wa ll, and displays the final surface morphology including the pseudopores. After formation of the outer membra ne, clear cytoplasm enters the new chamber prior to influx of co lored cytoplasm. Fina lly, calcification begins after cytoplasm enters the new chamber (Marszelak, 1969; Wetmore, 1999). The functional morphology of the shell in benthic foraminifers is not well understood, although it has been proposed the she ll serves primarily as a physical barrier against a changing external environment, as protection against predators, and as support

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11 for the cell. However, the shell must remain intact in order to function (Wetmore, 1987). Shell strength is a key element in the surviv al and distribution of benthic foraminifers, and in the post-mortem distribution of their shells as sedimentary particles. Braiser (1975) reported that agglutinated shells tended to be weaker than calcareous shells based on measurements of survival times of two speci es in agitated glass b eads. Shells of the calcareous species Cibicides lobatulus remained intact longer than the shells of the agglutinated species Reophax atlantica Wetmore (1989) and Wetmore and Plotnick (1992) experimentally determined shell stre ngth of smaller benthic foraminifers and found that shell strength was significantly gr eater in those from more physically stressed habitats. Further, a correlation between shell strength and habitat has also been suggested for larger symbiont-bearing foraminifers ba sed on correlations between shell morphology and habitat. Overall shape, wall thickne ss, chamber size and arrangement, shell composition, and strength of connections be tween chambers could all affect shell strength. Biconvex, thick-walled shells without spin es or other ornamentation tend to be associated with high energy environments. Wetmore and Plotnick (1992) specifically looked at correlations between shell morphology, crushing stre ngth, and habitat of three biconvex species, Amphistegina gibbosa, Archaias angulatus and Laevipeneroplis proteus from Bermuda. They compared individu als from a shallow-water (1 m depth) protected embayment to indivi duals collected from the reef (10 m depth) and reported that, in specimens from both habitats, a resi stance to crushing gene rally increased with increasing size. Within the reef habitat al l three species had e qually robust shells. However, Archaias angulatus from the reef loca lity were significantly more robust than

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12 similar-sized individuals from the embayment (site to site co mparisons could not be made with A. gibbosa and L. proteus because there were not enough individuals in the embayment). They repo rted that shells of A. angulatus from the reef were on average slightly heavier than similar-sized shells of A. gibbosa from the same location. Dramatic differences in the inner organic lining of select ed shells were also evident. The organic lining of A. gibbosa did not maintain its shape when the shell wall was dissolved. In contrast, the organic lining of A. angulatus was more self-supporti ng and appeared to be more robust in specimens from the reef versus the embayment. This may indicate that individuals from the reef are mechanically stronger. 1.4 Morphologic Abnormal ities in Foraminifera Shell abnormalities in foraminifers due to natural variation and anthropogenic influences have been well documented. I ndustrial and domestic po llution (Yanko et al., 1994; Alve, 1995; Yanko et al., 1998; Yanko et al., 1999; Stouff et al., 1999a; Stouff et al., 1999b; Samir, 2000; Samir and El Din, 2001; Geslin et al., 2002; Saraswat et al., 2004;), heavy metals (Banerji, 1990; Yanko et al., 1994; Alve a nd Olsgard, 1999), low pH (Geslin et al., 2002; Le Ca dre et al., 2003), and salinity (S touff et al., 1999a; Geslin et al., 2002) have been investigated in field a nd laboratory investigations. Miliolids in particular have exhibited numerous a bnormalities in response to anthropogenic influences. Samir and El-Din (2001) conducte d a study comparing two bays in Egypt: El Mex Bay, one of the most metal-polluted areas along the Alexandria Coast, and Miami Bay which is subject to domestic waste but not metals. They found that deformities were restricted mainly to miliolids including the families Hauerinidae, Peneroplidae,

PAGE 23

13 Soritidae, and one rotaliid family, Cibicididae. They noted one type of deformation in Amphisorus hemprichii (family Soritidae) from Miami Bay which was similar to the wing found on Cycorbiculina compressa from New Found Harbor in the Florida Keys (Crevison and Hallock, 2007). Other sy mbiont-bearing miliolids, such as Peneroplis pertusus and P. planatus exhibited an uncoiled chambe r arrangement, reduction in the size of the last chamber, and protuberances. Smaller miliolids from El-Mex Bay, such as Quinqueloculina seminulum and Quinqueloculina disparilis, possessed multiple apertures, displayed a change in the direction of the axis of coiling, and lateral asymmetry of apertural position. Yanko et al. (1998) documented mo rphological deformities in benthic foraminifers along the Mediterran ean coast north of Israel. Th eir study area is subject to heavy metal pollution from industrial waste. Larger miliolids including P. pertuses and P. planatus exhibited twinning of two individu als as well as double apertures and additional chambers. Smaller miliolids, including Miliolinella subrotunda Triloculina earlandi and Trilocilina schreiberiana, were marked by wrong direction of coiling, double apertures, and aberrant chamber shape. Furthermore, Adelosina pulchella and Quinqueloculina phoenicia exhibited twisted chambers, wrong dire ction of coiling, and double apertures. Yanko et al. (1999) also reviewed the effects of marine pollution, such as municipal sewers, fertilizer, aquacultur es, paper mills, dredging, and hydrocarbons on benthic foraminifers in and around Haifa Bay near Israel. Examples of affected taxa and specific deformities were similar to those prev iously mentioned in Yanko et al. (1998). Moreover, additional deformities in larger miliolids from Haifa Bay were noted.

PAGE 24

14 Other studies looking speci fically at heavy metal contamination noted stunted foraminifersl shells (Banerji, 1990; Yanko et al., 1994) and low abundance and diversity (Yanko et al., 1994). Geslin et al. (1998) de scribed abnormal wall structures and shell deformation in Ammonia due to heavy metal contamination. The “crystal disorganization” they described may have been the result of alien elements, such as Cu and Zn, being introduced into the crystalline framework (Sharifi et al., 1991). Environmental factors unrelated to pollution also produce morphologic abnormalities in benthic foraminifers. Le Cadr e et al. (2003) showed low pH resulted in decalcification in culture experiments using Ammonia Morphological anomalies were also evident when these individuals started to recalcify after being returned to normal environmental conditions. Stouff et al. (1999) investigated the infl uence of hypersalinity on cultured specimens of Ammonia Five categories of shell malformations were identified in juveniles cultur ed in hypersaline conditions (s alinity 50): a) abnormal size or shape of the proloculus or first chambers; b) modifications of coiling plane of the first chamber; c) development of two different whorls; d) fusion of young and development of complex abnormal forms; and e) excrescences (unusual growths) on chambers. Adults which were placed in hypersaline conditi ons also exhibited malformations. One individual produced chambers of greater si ze than those chambers constructed under normal saline conditions (salinity 37). Another individual exhibited complex development of many chambers with a perturbed arrangement. Debenay et al. (2001) in vestigated foraminiferal assemblages in a hypesaline lagoon in Brazil. Triloculina oblonga a smaller miliolid, and Ammonia tepida a rotaliid, were the dominant taxa in their samples. They observed a high percentage of aberrant

PAGE 25

15 shells. They concluded anthropogenic stre ss was not responsible for the morphological abnormalities, but rather high salinity condi tions and changes in salinity were. 1.5 Background on the Foraminifer Archaias angulatus Archaias angulatus are porcelaneous foraminifers w ith planispiral involute shells covered with pseudopores (Fig.1.1) (Fichtel and Moll, 1798; Cottey and Hallock, 1988). Their shells are compressed and characteri zed by numerous chamberlets and pronounced flaring in the outermost whorls of mature individuals (Loeblich a nd Tappan, 1988). They host chlorophyte endosymbionts of the genus Chlamydomonus (Lee and Bock, 1976). Studies of modern shallow-water marine carbonate platforms have underscored the role of scleractinian corals and calcareous algae as contributors to reefal sediments. However, foraminifers are frequently a ma jor component of the sedimentary record (Muller, 1976; Hallock, 1981; Hallock et al., 1986; Harney et al., 1999). Historically, Archaias angulatus has been considered a major contri butor to foraminifersl assemblages and sediments in coral reef environments throughout th e Caribbean and Atlantic (Marshall, 1976; Martin, 1986; Cottey a nd Hallock, 1988), specifically Florida Bay (Bock, 1971), Florida Keys (Wright and Hay, 1971), and the Florida-Bahamas carbonate province (Rose and Lidz, 1977; Lidz and Rose 1989) because shells are thick-walled, robust, and are structurally reinforced by internal pillars (M artin, 1986).Cottey and Hallock (1988) investigated post-mortem surface degradation of A. angulatus in sediment samples collected from Key Largo, Florida a nd La Parguera, Puerto Rico. Laboratory and field-conducted experiments produced degr aded shells from pa rtial removal of the outer tile-roof layer to complete loss of the outer shell wall resulting in exposure

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16 Figure 1.1. A. Light micrograph of a living adult Archaias angulatus ; B. SEM of adult individual of the underlying septa and chamberlets. An alysis of field samples revealed several different types of degradational features in cluding dissolution, breakage, impact features, pitted surfaces, scratches and microborings. No ne of these characteri stics are out of the ordinary for biological sedimentary constituents such as foraminiferal shells, since many biological, physical, chemical, and geological processes immediately act on the shell after the individual dies. Although the literature provi des a comprehensive overview of foraminiferal shell abnormalities and the causes of morphologica l and textural anomalies (Alve, 1995; Yanko et al., 1999; Samir and El-Din, 2001), th ere is little documentation concerning A. angulatus MacIntyre and Reid (1998) ex amined recrystallization in living A. angulatus and, although they found textural changes wit hout mineralogical alte ration, their study focused on ultrastructure rather than surf ace texture or morphologic abnormalities. 1.6 Objectives The objectives of my research are to a) document textural and morphological anomalies in archaiasine foraminifers, b) de termine if such anomalies have changed in A B

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17 prevalence in a population of Archaias angulatus previously studied in 1981-82, and c) experimentally determine if shell strength is compromised in deformed versus normal individuals. Question 1. What types of textur al and morphologic anomalie s can be identified using light microscopy and SEM analysis of A. angulatus collected live from the Florida Keys? Question 2 Are the textural anomalies structural or caused by a secondary agent such as microorganisms? Question 3. Are similar textural or morphologic anomalies evident in archived samples of A. angulatus collected live from the Florida reef tract? Question 4. Are there visible differences in test wall fabric of normal versus deformed individuals? Question 5. Are Mg/Ca ratios of normal and deformed A. angulatus specimens consistent with ratios previous ly reported for this species?

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18 2. ANOMALOUS FEATURES OBSERVED ON SHELLS OF LIVE ARCHAIASINE FORAMINIFERS FROM THE FLORIDA KEYS, USA 2.1 Introduction Archaias angulatus (Fichtel and Moll), Cyclorbiculina compressa (d’Orbigny), and Laevepeneroplis proteus (d’Orbigny) are porcelaneous foraminifers with planispiral involute shells covered with pseudopores (Fichtel and Moll, 1798; Cottey and Hallock, 1988). Archaias angulatus and C. compressa are further charact erized by numerous chamberlets and pronounced flaring in the out ermost whorls of mature individuals (Loeblich and Tappan, 1987). These protists host chlorophyte endosymbionts of the genus Chlamydomonus (Lee and Bock, 1976; Pawlowski et al., 2001; Pocock et al., 2004). The walls of porcelaneous foraminiferal sh ells (Order Miliolida) characteristically include a thick layer of magnesian-calcite n eedles arranged randomly in three dimensions and coated with a thin layer of regularly a rranged rhombohedral plates, also composed of magnesian calcite (Lipps, 1973; MacIntyre and Reid, 1998; Debenay et al., 2000). Soritaceans have a third smooth inner layer th at coats the interior surface of chambers and forms internal structures such as pillar s and walls. The calcite needles are produced within the Golgi apparatus and transported via vesicles to the location of chamber formation, where they are laid into place by the granulose reticulopodia (Angell, 1980; Ter Kuile, 1991: Erez, 2003).

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19 Studies of modern shallow-water, ma rine-carbonate platforms have underscored the role of foraminifers that host algal endos ymbionts as contributors to reefal sediments (Muller, 1976; Hallock, 1981; Hallock et al., 1986; Langer et al., 1997 ). Historically, Archaias angulatus has been considered a major contri butor to foraminiferal assemblages and sediments in coral-reef environments throughout th e Caribbean Sea and western North Atlantic Ocean (Marshall, 1976; Ma rtin, 1986; Cottey and Hallock, 1988), Florida Bay (Bock, 1971), Florida Keys (Wright and Hay, 1971), and the Florida-Bahamas carbonate province (Rose and Lidz 1977; Lidz and Rose, 1989). Cockey et al. (1996) reported dramatic changes in the foraminiferal assemblages of the Florida reef tract over the past 50 y ears. Comparisons of surface sediment samples collected in 1982, 1991 and 1992 with samples co llected in 1960 revealed a shift in dominance from symbiont -bearing taxa, such as Amphistegina gibbosa d’Orbigny and Archaias angulatus to smaller detritus-consuming taxa, such as Discorbis, Quinqueloculina, Rosalina and Triloculina Consistent with this shift in foraminiferal assemblages, coral cover in the Florida Keys has declined while algal and sponge cover has increased (Dustan and Halas, 1987; Porter and Meier, 1992; Dustan, 1999). Cottey and Hallock (1988) investigated post-mortem (taphonomic) surface degradation of Archaias angulatus specimens in sediment samples collected from Key Largo, Florida, and La Parguera, Puerto Ric o, in the early and mid 1980s. Analysis of field samples revealed several different types of degradationa l features including dissolution, breakage, impact features, pitt ed surfaces, scratches and microborings. Laboratory and field-conducted experiments produced a range of taphonomic features, from partial removal of the outer layer to comp lete loss of the outer shell wall resulting in

PAGE 30

20 exposure of underlying septa and chamberlets. None of these features were considered unusual taphonomic alterations. Coral bleaching, which results from either the loss of symbiotic algae or reduction of photosynthetic pigments within the alg ae, was considered an unusual phenomenon prior to 1980 (Glynn, 1996; Hoegh-Guldberg, 1999). Two widespread coral-bleaching events, the first in 1982-83 and the second in 1987-88, were key events in the recognition of worldwide decline in co ral reefs. Bleaching was di scovered in populations of Amphistegina gibbosa in the Florida reef tract in 1991 (Hallock et al., 1993), and subsequently documented in Amphistegina spp. worldwide (Hallock, 2000). Along with bleaching, Hallock and co-workers also documented unusually high incidences of developmental deformities, microborings and infe station, and structural damage in shells of live Amphistegina (Hallock and Talge, 1994; Hallock et al., 1995; Toler and Hallock, 1998). Shell anomalies in co-occurring Arch aiasinae foraminifers were occasionally noted but not routinely documented (Williams et al., 1997). In a sample collected from New Found Harbor in the Florida Keys in May 2004, surface texture anomalies appeared to be unusually common among live Archaias angulatus individuals. Because the anomalies were so common, the sample was saved for later examination. Under light micr oscopy, many specimens of symbiont-bearing porcelaneous taxa, including also Laevipeneroplis proteus and Cyclorbiculina compressa appeared to have a rough, etched finish to their shells. Some of these individuals exhibited a range of physical abnormalities in cluding rows of mangled-looking chambers, ragged suture lines, and complete shell malformations.

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21 Although the literature provi des a comprehensive overview of foraminiferal shell abnormalities and the causes of morphological a nd textural anomalies (Alve, 1995; Samir and El-Din, 2001; Yanko et al., 1999), there is little documentation of abnormalities in Archaias angulatus MacIntyre and Reid (1998) exam ined recrystallization in living A. angulatus and, although they found textural chan ges without mineralogical alteration, their study focused on ultrastr ucture rather than surf ace texture or morphologic abnormalities. The purpose of this paper is to document anomalous shell-surface textures and morphological abnormalities in A. angulatus collected live along the Fl orida reef tract. 2.2 Materials and Methods I examined samples collected from severa l sites and depths along the Florida reef tract: New Found Harbor (3 m water de pth) behind Looe Key in May 2004, and Molasses Reef (15 m depth) off Key Largo and Tennessee Re ef (10 and 30 m depth) off Long Key in July 2005 (Fig. 2.1). Specimens were determined to be living when collected by their algal-symbiont coloration a nd the presence of granulose reticulopodia. Juvenile and adult specimens were examin ed using light microscopy for any surfacetexture or morphological abnormalities. Affected individuals were air dried and stored. Prior to examination using scanning el ectron microscopy (SEM), specimens were rinsed in deionized water and air dried on paleontological slides. They were mounted onto aluminum SEM stubs using doublesided adhesive tabs and sputter coated with gold-palladium (to approximately 10-nm thickness) using a Hummer 6.2 Sputtering

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22 System. Samples were then examined using a Hitachi S-3500N scanning electron microscope. Figure 2.1. Map of Florida Keys showing the location of Key Largo, New Found Harbor, Hawk Channel and John Pennekamp Coral Reef State Park 2.3 Results Under light microscopy, examples of sh ell-surface anomalies and morphological abnormalities were observed among both juvenile and adult specimens of Archaias angulatus, Cyclorbiculina compressa and Laevipeneroplis proteus All indivi duals that appeared unusual under a stereomicroscope we re examined using SEM. Six basic types of features were observed: microborings, pi tted surfaces, microbial biofilm, calcification (structural) anomalies, dissolution, and sh ell deformation. Table 2.1 summarizes which abnormalities were found at each site.

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23Table 2.1. Types of abnormalities found at each sampling site Sampling Location and Depth Microborings Pitted Surfaces Microbial Biofilm Calcification (Structural) Anomalies Dissolution Shell Deformation Newfound Habor 3m Tennessee Reef 10 m Tennessee Reef 30 m Molasses Reef 15 m Normal Archaias angulatus possess clearly define d, round pseudopores; crisp concentric suture lines; and smooth surface texture (Pl. 2.1, Fi gs.1-3). Microborings (Pl. 2.1, Fig. 4) were present on bot h juvenile and adult individuals from New Found Harbor and adult specimens from 30 m depth at Te nnessee Reef. In general, microborings appeared straight, had fairly smooth edges, and avoided contact with pseudopores. Some individuals were completely covered with mi croborings while others exhibited sporadic smaller patches. Pitted surfaces were observed on juvenile and young adult Archaias angulatus from New Found Harbor (Pl. 2.1, Fig. 5), 30 m depth at Tennessee Reef (Pl. 2.1, Fig. 6), and Molasses Reef Some pits were as sm all as 25 m, and circular in shape with ragged edges. In some individuals, pits co alesced into large pockmarks (100 m) giving a crumbly appearance to shell surfaces (Pl. 2.1, Fig. 6).

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24 A microbial biofilm was found on one adult individual from the 10-m-depth site at Tennessee Reef (Pl. 2.2, Figs. 1-2). The bacteria were capsule-shaped and approximately 1 m in length. In some areas, bacteria looked melted to the shell surface (Pl. 2.2, Fig. 2). Elsewhere, bacteria were di screte entities that cl ung to the shell surface or nestled in the pseudopores. Calcification anomalies were structurally very different. One juvenile individual from 10 m depth at Tennessee Reef was cove red with projections, which protruded from the pseudopores (Pl. 2.2, Fig. 3). These prot rusions varied in length (about 5-25 m) and girth, and some appeared slightly curved while others were straight. Crystal faces were visible on nearly all of the projections. A juvenile from 30 m depth at Tennessee Reef was covered in a lacy-looking crust (Pl. 2.2, Fi g. 4). The crust appeared thick in some areas, completely obscuring the pseudopores. However, in other areas, pseudopores could be discerned through the lacy outer layer. Finally, multiple adult individuals from New Found Harbor exhibited repair marks in areas th at were previously pi tted (Pl. 2.2 Fig. 5). In one individual, a large pit within a single row of chamberlets was repaired by regrowth from a different row of chamberlets. C onsequently, areas of this individual look smeared. This same individual also exhibited irregular suture lines. Dissolution was evident on multiple indi viduals from both depths at Tennessee Reef. Some specimens from the 10-m dept h exhibited extremely shallow pseudopores, so shallow that the bottoms of the pseudopor es were visible (Pl. 2.2, Fig. 6). These individuals were also cove red in bacteria. Other indi viduals from the 30 m depth appeared to have pitted surfaces, which were partially dissolved (Pl. 2.3, Fig. 1). These

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25 Plate 2.1. Archaias angulatus : 1-3 normal shells, 1 normal pseudopores and suture lines, 2 juvenile aperture, 3 normal adult; 4 microborings on juvenile from New Found Harbor; 5 pitted surfaces on juvenile from New Found Harbor; 6 pitted surface on adult from Tennessee Reef.

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26 Plate 2.2. Archaias angulatus : 1 2 from same individual, bacteria on adult from Tennessee Reef; 3 mineralogical projections on juvenile surface from Tennessee Reef; 5 lacy crust on juvenile surface from Tennessee Reef; 6 repair marks on adult from New Found Harbor; 6 shallow pores as a result of dissolution from Tennessee Reef

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27 Plate 2.3. 1 dissolution of previously pitted surface from Tennessee Reef; 2 deformed juvenile Archaias angulatus from New Found Harbor; 3 deformed A. angulatus from Tennessee Reef; 4 deformed adult Cyclorbiculina compressa from New Found Harbor; 5 microborings on adult C. compressa from New Found Harbor; 6 microborings and pitted surface in Laevipeneroplis proteus from New Found Harbor

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28 individuals not only had large coalescing pits (nearly 75 m along the longest axis), but the pits looked very smooth and somewhat polished. The last type of abnormality documented was shell deformation (Pl. 2.3, Fig. 2-4). In one juvenile individual fr om New Found Harbor, the planispi ral nature of the shell was completely obscured and the shell looked lumpy. No clearly defined cluster of apertures could be seen. This individual also e xhibited microborings on its surface. An intermediate-size individual from the same site also exhibited chamber malformation. Similar anomalies were also found in Cyclorbiculina compressa and Laevipeneroplis proteus from the same samples. Several specimens of C. compressa from New Found Harbor were marked by mo rphological abnormalities or shell-surface anomalies. Instead of a flat disc-shaped shell, one individual ha d a “wing,” which was perpendicular to the shell su rface (Pl. 2.3, Fig. 4). Aper tures were present along the margin of the wing. Other C. compressa individuals from New Found Harbor were affected by microborings (Pl. 2.3, Fig. 5). Th ese microborings appeared dendritic instead of the short, strai ght burrows seen on Archaias angulatus Microborings were also observed on some Laevipeneroplis proteus individuals. One L. proteus specimen was affected by multiple abnormalities including microborings, pitted surface, and chamber malformation (Pl. 2.3, Fig. 6). Again, the micr oborings seemed to a void contact with the pseudopores. 2.4 Discussion Shell abnormalities in foraminifers, which can be associated with either natural variation or anthropogenic pol lutants, have been widely reported. Industrial and

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29 domestic pollution (Yanko et al., 1994; Alve 1995; Yanko et al., 1998; Yanko et al., 1999; Stouff et al., 1999a; Stouff et al., 1999b; Samir, 2000; Samir and El Din, 2001; Geslin et al., 2002; Saraswat et al., 2004; ), heavy metals (Banerji, 1992; Yanko et al., 1994; Alve and Olsgard, 1999), low pH (Geslin et al., 2002; Le Ca dre et al., 2003), and salinity (Stouff et al., 1999a; Geslin et al., 2002) have b een implicated as causes for abnormalities in field and laboratory investigat ions. Shell construction in the Miliolida appears to be particularly sensitive to envi ronmental influences. Samir and El Din (2001) noted twinning in Amphisorus hemprichii Ehrenberg (family Soritidae) from a polluted bay in Egypt that was similar to what we saw on a C. compressa specimen from New Found Harbor (see Pl. 3, Fig. 4). Othe r symbiont-bearing miliolids, such as Peneroplis pertusus (Forsskl) and P. planatus (Fichtel and Moll), exhi bited an uncoiled chamber arrangement, reduction in the size of the last chamber, and protuberances (Samir and El Din, 2001). Smaller miliolids from their study si te exhibited multiple apertures, a change in the direction of the axis of coiling, and la teral asymmetry of apertural position. Yanko et al. (1998) also documented a similar variety of morphological deformities among miliolids, including P. pertusus and P. planatus. Other studies looking speci fically at heavy metal contamination noted stunted foraminiferal shells (Yanko et al., 1994), as well as low a bundance and diversity (Yanko et al., 1994). Geslin et al. (1998) desc ribed abnormal wall structures and shell deformation in Ammonia due to heavy metal contamination. The “crystal disorganization” they described may have been the result of alien elements, such as Cu and Zn, being introduced into the crystalline framework (Sharifi et al., 1991).

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30 Environmental factors unrelated to pollution also can produce morphologic abnormalities in benthic foraminifera. Le Ca dre et al. (2003) found that low pH resulted in decalcification in cu lture experiments using Ammonia Debenay et al. (2001) reported a high percentage of aberrant shells from a hypersaline lagoon, which they concluded were associated with high and variable salinity. Many of the morphological abnormalit ies we observed in the Archaiasine foraminifers from the Florida Keys are similar to abnormalities reported among Amphistegina gibbosa during the 1990s, following the onset of bleaching in Amphistegina gibbosa populations in the summer of 1991 (Hallock et al., 1995). Hallock and co-workers documented frequent incide nces of broken shells and calcification anomalies, including surface electron-densi ty anomalies observed by SEM (Toler and Hallock, 1998); reproductive dysfunction, including development of profoundly deformed offspring in broods produced by multip le fission (Hallock et al., 1995; Harney et al., 1998); and predation and microborings (Talge and Hallock, 1995). Williams et al. (1997) and Williams and Hallock (2004) conclude d that environmental factors that affect the spectral quality and quantity of solar radiation reaching the seafloor (e.g., ozone depletion and/or local changes in wate r transparency) can induce bleaching and associated symptoms. Williams et al. (1997) also noted some analogous symptoms in Cyclorbiculina compressa and Heterostegina depressa and suggested that studies of other symbiont-bearing larger forami nifers should be undertaken. One of the most significant destructive processes affecting carbonate grains in modern marine environments is biochemical dissolution by endol ithic microorganisms such as cyanobacteria, chlorophytes, r hodophytes, and fungi (Perry, 1998). In 1998,

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31 Perry investigated grain susceptibility to the effects of micr oborings on carbonate sediments from Discovery Bay, Jamaica. He described 16 different species of microborers, including six speci es of cyanobacteria, four species of chlorophytes, one species of rhodophyte, two species of sponge s, two species of fungi, and one unknown borer. The microbores present in A. angulatus are very similar to those produced by cyanobacteria. A more definitive assessm ent of microborer taxonomy would require impregnation of the foraminifera with resin, which would cast the microbores in three dimensions to identify distinctive character istics (Perry, personal communication, 2006). Although microbioerosion is a natural pr ocess on coral reefs, its prevalence can be associated with nutrient pollution. Chazotte s et al. (2002) and Silv a et al. (2005) found higher bioerosion rates by microborers in r eefs subject to eutrophication compared to reefs in nutrient-poor areas. The Florida reef tract has experienced a shift from coral-algal dominated reef communities to algal-sponge dominated hard-bottom communities over the past several decades (Dustan, 1999; Porter et al., 2002), a shift th at is reflected in changes in foraminiferal assemblages (Cockey et al., 1996; Hallock et al., 2003). If this shift has occurred in response to increased nut rient flux, as some have suggested (Hallock et al., 1993), bioerosion should have increased comparably, both on the reefs and within the sedimentary constituents such as forami niferal shells. Szmant and Forester (1996) and Szmant (2002) argue that eutrophication has only occurred in inshore waters, and not at the offshore reefs. However, Lapoin te et al. (2004) found that regional-scale agricultural runoff from the mainland Everglad es watersheds, as well as local sewage discharges from the Florida Keys, were significant nitrogen sources supporting

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32 eutrophication and algal blooms in sea gra ss and coral reef communities in the Lower Florida Keys. The patterns of microborings present on our foraminifers may provide clues to their defense mechanisms. The microborin gs do not come in contact with the pseudopores, suggesting the pseudopores are und esirable targets for the microborers. Chemical defenses in unicellu lar algae have been well documented (Turner and Sheller, 1997; Wolfe, 2000; Hay and Kubanek, 2002; Pohnert 2005). It is important to recognize that phytotoxins are not only produced by harmful algal blooms, but also by many unicellular algae, such as diatoms, which ar e generally regarded as a primary food source for zooplankton (Hay and Kubanek, 2002; Pohnert 2005). Phytotoxins are also produced by cyanobacteria (Hay and Kubanek, 2002). Pohnert (2005) reviewed diatom-copepod interactions and indirect chemical defens es in diatoms. Oxylipins (unsaturated aldehydes), which are produced by wounded diat oms, appeared to greatly decrease copepod egg production and egg-ha tching success. It is not unreasonable to speculate that symbiotic algae might produce chemical defenses advantageous to the symbiotic relationship. In a healthy foraminifer, phytotoxins produced by symbiotic algae may be one mechanism by which the hosts keep th emselves clean of epibionts, including predatory foraminifers and microborers. Chemical interactions between protoctists and procaryotes are a fertile realm for future research. Kearns and Hunter (2000) demonstrated that toxin production by a freeliving freshwater cyanobacterium was regulated in part by the presence of extracellular products of Chlamydomonas reinhardtii At high concentrations of extracellular products of C. reinhardtii microcystin accumulation was comp letely inhibited. Microboring was

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33 a common secondary indicator of stress in partly bleached Amphistegina (Hallock, 2000) Talge and Hallock ( 1995) reported that Amphistegina in early stages of bleaching were most susceptible to predati on by the microboring foraminifer Floresina amphiphaga No studies have yet investigated whether th e degree of microboring and encrustation by epibonts differs between miliolid and rotaliid foraminiferals. The pitted surfaces found on Archaias angulatus resembled ‘karst topography’, as the pits appear to have collapsed from th e shell surface much like sinkholes. Although dissolution is evident on the outsi de of the shells, the actual formation of pits may be due to dissolution of the interior smooth layer rather than disso lution of the outer layer of rhombohedral plates. Many of the anom alies we have described have profound implications for the fossil record in terms of taphonomy a nd interpretation of paleoenvironmental conditions. Researcher s examining the taphonomy of foraminiferal shells should be aware that such modification ca n occur to the shells while the protists are still alive. Toler and Hallock (1998) describe d how to distinguish ev idence of stress in the living populations from postmortem processes in Amphistegina To their list, we can add when examining Archaiasines, the pr esence of microborings that avoid the pseudopores indicates that the boring likely occurred while the foraminifer was alive. South Florida environments, both terre strial and marine, have experienced dramatic changes in the past several decad es. The human population has increased to over 75,000 in Monroe County and almost 2.3 mi llion in the greater Miami area (U.S. census, 2005). Nevertheless, neither acute pollution sources nor hypersaline conditions are likely explanations for the morphologi cal abnormalities we have observed. Chronic nutrient (Szmant and Forrester, 1996) and pesticide (Pierce et al., 2005) pollution

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34 certainly exists inshore along the Florida Ke ys. But evidence for acute pollution is lacking as, ironically, the inshor e patch reefs, which are closer to sources of pollution and salinity variations, have experi enced less decline in their co ral populations than have the offshore reefs (Callahan, 2005). The Florida reef tract is also impacted by global-change factors, including higher ultraviolet radiation reaching the sea surface as a consequence of stratospheric ozone depletion, global-climate change and increasing atmospheric CO2 (Hallock, 2005; Precht and Miller, in press). However, larger foraminifers appear to be more tolerant of temperature stresses than corals, and bleaching prevalence in Amphistegina is clearly not related to temperature (Hallo ck et al., 1995; Talge and Hallock, 2003). As noted above, Hallock (2000) and Williams and Hallock (2004) concluded that changes in the quality and quantity of light reaching the seafloor pl ays an important role in inducing bleaching and related shell anomalies in Amphistegina There are several reasons why Archaias angulatus populations should be less susceptible to changes in solar irradiance than Amphistegina spp. The miliolid shell is naturally more opaque than the hyalline shell of Amphistegin a. Furthermore, Archaias angulatus tolerates very high and vari able light regimes. Thes e foraminifers thrive in shallow water, where they can be exposed to ne arly full sunlight at low tide and very low light as rising tides bring turbid wate rs over their habitats. In 2003, Gorton and Vogelmann reported that the snow alga, Chlamydomonas nivallis was able to withstand high levels of UV radiation because it contained extrachloroplastic UV-absorbing cytoplasmic compounds known as astaxanthins. Future research is required to determine if Chlamydomonas that live symbiotically with A. angulatus also produce astaxanthins.

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35 One global-change condition that may be impacting the miliolid larger foraminifers is the increasing acidity of the oceans. Small changes in CO2 concentrations in surface waters can have significant negati ve impacts on marine calcifiers and oceanic biogeochemical cycles (Kleypas et al., 1999; Pecheux, 1999; Langdon et al., 2003, Langdon and Atkinson, 2005). Severa l laboratory studies reported calcification rates of reef-building corals and algae declined by 10-50% under doubled CO2 conditions (Gattuso et al., 1998; Langdon et al., 2000; Langdon et al., 2003; Langdon and Atkinson, 2005). Laboratory and field experiments on coccolithophorids reported diminished calcification, malformed coccoliths, incomple te coccospheres (Reibesell, 2004; Reibesell et al., 2000), and a decrease in the average coccolith and coccosphere size as p CO2 increased (Engel et al., 2005). Magnesian-cal cite shells, such as those produced by miliolid foraminifers, have an even higher solubility product than aragonite and should be particularly sensitive to th e declining saturation of CaCO3 in seawaters, (Weyl, 1967; Plummer and Mackenzie, 1974), which is a consequence of rising atmospheric CO2 (Kleypas et al., 1999). Carbonate saturation in aquatic envir onments is a consequence not only of atmospheric CO2 concentration, but also of local changes in p CO2 associated with temperature, salinity, or diur nal and seasonal cycles of phot osynthesis and respiration. As noted above, temperature and salinity were probably not variables contributing to the shell dissolution we observed. However, with increasing algal domi nance of the benthos as noted previously (LaPointe et al., 2004) more organic substrate is available for microbial communities. Yates and Halley (2003) measured coral-reef community metabolism using a submersible habitat in the Florida Keys and Hawaii, documenting

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36 that dissolution exceeded calcification duri ng darkness on many types of coral reef substrate types. Increasing atmospheric CO2 can also influence rates of photosynthesis by the algal symbionts. Many studi es have reported that CO2 enrichment associated with pH decline results in an incr ease of primary production by ma rine phytoplankton (Hein and Sand-Jansen, 1997; Riebesell, 2004) and incr eased growth of freshwater microalgae (Yang and Gao, 2003). Yang and Gao (2003) in vestigated the eff ects of increased CO2 on Chlamydomonas reinhardtii Chlorella pyrenoidosa and Scenedesmus obliquus They reported that increased concentrations of CO2 significantly enhanced the growth rate of all three taxa. They also reported that C. reihhardtii had enhanced photoinhibition under elevated CO2. The response of chlorophyte endosymbionts to elevated levels of CO2 is unknown, so predicting the response of the forami niferal-symbiont system to increasing p CO2 in seawater is not possible. To mainta in the symbiotic relationship, the host must retain control over the amount of nitrogen reaching the symbionts (Hallock, 2000). Additional research is required to determine if the host also must maintain control over the inorganic carbon reaching the symbionts. Symbiont-bearing foraminifera are pro lific calcifiers and conventional wisdom has long held that photosynt hesis by the symbionts prom otes calcification by splitting bicarbonate ions a nd removal of CO2 (ter Kuile, 1991). However, McConnaughey (1989) and McConnaughey and Whelan (1997) ha ve proposed a revers e interpretation, suggesting the lack of CO2 limits photosynthesis in warm, shallow, alkaline environments so that calcification promotes photosynthes is. If this hypothesis is valid, rising CO2 concentrations may render calcificat ion less important as a source of CO2 for

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37 photosynthesis, thereby reducing the compe titive advantage of algal symbiosis for calcifying protists and metazoans (Hallock, 2000). Kleypas et al. (2006) proposed the need for additional research on the effects of increasing atmospheric CO2 on marine systems. We propose that larger miliolid taxa should be included in such efforts for severa l obvious reasons. First, as noted above, magnesian calcite is the least stable form of calcium carbonate commonly secreted by organisms and, therefore, is potentially mo st sensitive to declining oceanic carbonate saturation. Second, among the larger miliolids ar e families or subfamilies that specifically host chlorophyte, rhodophyte, diatom and dinoflagellate symbi onts (Hallock, 1999). Thus, comparative research on calcification of these different taxa may elucidate if and how photosynthetic rates of different sy mbiont taxa respond to changes in p CO2. 2.5 Conclusions Deformed shells and unusual shell-surface features were observed in juvenile and adult Archaias angulatus and other miliolids with algal endosymbionts collected live along the Florida reef tract. Calcification anomalies includ ed mineralogical projections and lacy crusts. Features typically consid ered taphonomic included microborings, pitted surfaces, bacterial infestation, and dissoluti on; evidence of shell repair was also documented. Prevalence of such features may indicate that these foraminifers experienced environmental stress. Given th e inherent solubility of their magnesiancalcite shell mineralogy, these fo raminifers are anticipated to be sensitive indicators of declining carbonate saturation in seawater, wh ich can result locally from low temperature

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38 or salinity, or increasing benthi c respiration rates associated with coastal nutrification, as well as globally with rising c oncentration of atmospheric CO2.

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39 3. MORPHOLOGICAL ABNORMALITIES IN A POPULATION OF ARCHAIAS ANGULATUS (FORAMINIFERA) FROM THE FLORIDA KEYS (USA) SAMPLED IN 1982-83 AND 2006-07 3.1 Introduction Many papers have documented the occurrence of shell abnormalities in Foraminifera. Abnormalities associated with heavy metals (Banerji, 1990; Yanko et al., 1994; Alve and Olsgard, 1999, LeCadre and Debenay, 2006), industrial and domestic pollution (Alve, 1995; Yanko et al., 1998; Ya nko et al., 1999; Stouff et al., 1999a; Stouff et al., 1999b; Geslin et al., 2002; Saraswat et al., 2004), low pH (Geslin et al., 2002; Le Cadre et al., 2003), and salinity (Stouff et al., 1999a; Geslin et al., 2002) have been investigated in field and la boratory studies. Miliolids in particular have exhibited numerous abnormalities in response to anth ropogenic influences (Yanko et al., 1998; Samir and El-Din, 2001). A study compari ng two bays in Egypt, Samir and El-Din found that deformities were found primarily in miliolids including the families Hauerinidae, Peneroplidae, Soritidae, and one rotaliid family, Cibicididae. Symbiontbearing miliolids, such as Peneroplis pertusus and P. planatus exhibited uncoiled chamber arrangements, reductions in the size of the last chamber, and protuberances. Studies looking specific ally at heavy metal contamination noted stunted foraminiferal shells (Banerji, 1990; Yanko et al., 1994) and low abundance and diversity (Yanko et al., 1994). Geslin et al. (1998) de scribed abnormal wall structures and shell deformation in Ammonia due to heavy metal contamination. The “crystal

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40 disorganization” they described may have been the result of alien elements, such as Cu and Zn, being introduced into the crystalline framework (Sharifi et al., 1991). Natural environmental variability can also induce morphologic abnormalities in benthic foraminifera. Le Cadre et al. (2003) showed that low pH resulted in decalcification in culture experiments using Ammonia Morphological anomalies were also evident when these individuals started to recalcify after being returned to normal environmental conditions. Stouff et al. (1999b) investigated the influence of hypersalinity on cultured specimens of Ammonia Shell malformations were identified in juveniles grown in culture under hypersaline co nditions (salinity 50), as well as in adults that were placed in hypersaline conditions. Debenay et al. (2001) observed high percentages of aberrant shells in forami niferal assemblages from a hypesaline lagoon in Brazil. They concluded that anthropoge nic stress was not responsible for the morphological abnormalities, but rather high salinity conditions and changes in salinity were. Changes in foraminiferal assemblages of the Florida Keys reef tract have been well documented. Cockey et al. (1996) compar ed surface sediment samples collected in 1982, 1991, and 1992 with samples collected in 1960 and reported significant changes in foraminiferal assemblages along two traverse s off Key Largo, Florida. The 1960 samples were dominated by larger symbiont-beari ng foraminiferal taxa (LBF), including Archaias angulatus (Lidz and Rose, 1989). The 1991-92 samples were dominated by smaller rotaliid and miliolid taxa indi cative of more abundant food sources. The foraminiferal assemblages in the 1982 samples were interm ediate between these two extremes.

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41 Further influencing the larger forami niferal populations, bleaching (anomalous loss of algal endosymbionts) began to impact Amphistegina gibbosa in the summer of 1991 (Hallock et al., 1993; 1995). In Se ptember of 1991, more than 50% of A. gibbosa specimens collected at several reefs in the Fl orida Keys showed anomalous loss of color, from slight mottling to complete bleaching (Hallock et al., 1995). By November 1991, A. gibbosa densities had declined by 95% a nd remained low through 1992, rebounding to densities comparable to those found pre-bl eaching (Hallock et al., 2005). Affected individuals also exhibited br oken shells, microborings, and reproductive dysfunction that resulted in either reproductive failure or broods exhibiting she ll abnormalities that included twinned, twisted, and encrusting mo rphologies (Toler and Hallock, 1997). Archaias angulatus are symbiont-bearing porcela neous foraminifers with planispiral involute shells covered with ps eudopores (Fichtel and Moll, 1798). Their robust shells are composed of high magnesian-c alcite needles arranged randomly in three dimensions coated with a thin layer of re gularly arranged rhombohe dral plates (Cottey and Hallock, 1988). Historically, A. angulatus has been considered a major contributor to foraminiferal assemblages and sediments in coral-reef environments throughout the Caribbean Sea and western North Atlantic Ocean (Marshall, 1976; Martin, 1986). However, comparisons of multiple data sets have shown a significant reduction of A. angulatus in sediments during the last several d ecades. Lidz and Rose (1989) reported on Foraminifera in surficial sediments from th e backreef of Molasses Reef collected in 1959-1961. Total assemblages were 60-80% from the family Soritidae, mainly the species A. angulatus and Peneroplis proteus Martin (1986) found that A. angulatus shells made up approximately 15-20% of the total assemblage from sediments collected

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42 in 1974. Samples collected in 1982, reported by Cockey et al. (1996), revealed that percentages of the family Soritidae had dropped to 5-27%, and A. angulatus to about 5%. They also reported that relative abundances of the family Soritidae were around 10% and A. angulatus about 5% for samples collected in 1991-1992. Chapter 2 (see also Crevison and Hall ock, 2007) reported anomalous features found in living A. angulatus from the Florida Keys. In samples collected from New Found Harbor, surface texture anomalies a ppeared to be unusually common among specimens collected live. Under light micros copy, many specimens appeared to have a rough, etched finish to their shells. Under SEM, these individuals were affected by a variety of surface anomalies including micr oborings, microbial biofilm, mineralogical projections, dissolution and lacy crusts. Other individuals exhibited a range of physical abnormalities including rows of mangled-l ooking chambers, ragged suture lines, and complete shell malformations. Given the documented decline in LBF densities in Florida reef-tract sediments in the past few decades (Cockey et al., 1 996) and the prevalence of morphological abnormalities in very recently collected sa mples, a logical question is: “Has the prevalence or types of abnormalities increased as populations have declined?" Archived samples of A. angulatus collected live in 1982-83 from John Pennekamp Coral Reef State Park on Key Largo, Florida (Hallock et al., 1986), were available for study. In 2006 a study was performed to determine if th e occurrence and type s of morphological abnormalities have changed in A. angulatus over the past 2.5 decades at this site. The objectives of this study were to document mo rphological and textural anomalies in these two sample sets and to compare archived to recent samples to determine if the types of

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43 anomalies and the percentages and sizes of individuals affected by them have changed since 1982. 3.2 Materials and Methods Sample Collection A target of at least 150 living specimens per sample of A. angulatus were collected quarterly (March, June, September, and December) from Thalassia beds in the swimming area of John Pennekamp Coral Reef State Park on Key Largo, Florida (Fig. 2.1). These sampling months corresponded to sampling months of archived individuals collected in the early 1980s from the same location. The water was very shallow and samples were collected while snorkeling. Archaias angulatus can live attached to seagrass, therefore thr ee to four handfuls of Thalassia testudinum blades were collected at 3 sites along the swimming area demarcation rope. The Thalassia were picked above the rhizome and placed into resealable pl astic bags filled with seawater. Surface sediments around the seagrass were also collecte d to catch any individu als that fell from the blades as they were gathered. In the fi eld, Foraminifera were carefully scraped from the seagrass blades and rinsed with seawater. Foraminiferal specimens and sediment were transported back to laboratory in seaw ater-filled plastic containers. While in the lab, all samples were transferred to Petri dishes and allowed to settle in an environmental chamber for 24 hours. Live individuals were picked directly into buffered deionized water and rinsed for 5-20 minutes in buffe red deionized water, depending on how much debris was present on their surfaces. Live individuals were determined by presence of

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44 granulose reticulopodia and endosymbiont co lor. Specimens were then placed on paleontological slides to air dry. The archived samples were collected by snorkeling with three samples collected on each sampling date: a rubble sample, a mixed algal-seagrass samples, and a predominantly seagrass sample (Hallock et al., 1986). Samples were preserved in buffered formalin and shipped from Key Largo to the senior author’s laboratory. This treatment preserved the green symbiont color, facilitating identific ation of individuals that were collected live, but exposed the sa mples to foramilin for several days. Upon arrival at the laboratory, samples were washed in freshwater over a 63 m sieve, dried at 40OC, and individuals determined to be a live when collected (based on preserved symbiont color) were picked to micropaleontological slides. These specimens were stored in a wooden cabinet between the time of their original evalua tion and their use in my study. All specimens of A angulatus were viewed under light microscopy to determine the types and numbers of sh ell anomalies present. If an individual had a single abnormality, it was categorized according to that particular anomaly. If an individual had more than one abnormality, it was categorized according to all abnorma lities present. For instance, if an individual had ir regular sutures, it was tallied accordingly. If an individual had irregular sutures and was curled, it was tallied into its own category based on the combination of abnormalities so each individual was counted only once. Individuals that appeared to have a su rface texture anomaly, as well as a random sample of normal individuals, were prep ared for SEM to further identify surface abnormalities. Individuals were mounted onto aluminum SEM stubs using double-sided

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45 adhesive tabs and sputter co ated with gold-palladium (a pproximately 10 nm thickness) using a Hummer 6.2 Sputtering System. Samples were then examined using secondary electron imaging on a Hitachi S-3500 N scanning electron microscope. Analysis of Foraminiferal Anomalies The foraminiferal anomaly data were analyzed using Plymouth Routines in Multivariate Ecological Research (PRIMER) Since the data were not normally distributed and some anomalies or combina tions of anomalies were very common while others were rare, the data set was square -root transformed to achieve a more normal distribution. Cluster analyses and MDS (mu ltidimensional scaling) plots were created based on Bray-Curtis similarity matrices to illustrate how the variables (abnormalities) and samples clustered. Two-dimensional MD S plots were used to show similarity between sampling dates. For an MDS plot, a stress level of < 0.2 was considered to be a useful representation of re lationships of the similari ty among samples (Clarke and Warwick, 2001). The percent of specimens exhibiting each abnormality was calculated for all samples and pooled into recent and ar chived dates to determine trends between each anomaly and shell diameter. 3.3 Results A total of 5,510 Archaias angulatus shells were examined for this study (Table 3.1) and nearly 1,400 were examined under SEM. Seven different t ypes of morphological abnormalities and five different surface texture anomalies were documented in 86 combinations. Physical abnormalities included profoundly deformed, curled,

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46 asymmetrical, and uncoiled shells, irregular su ture lines, surface “blips,” and breakage and repair (Pl. 3.1, Table 3.2). Surface te xture anomalies included surface pits, dissolution, microborings, microbial biofilm, an d epibont growth (Pl. 3.2, Table 3.3). Epibont growth included bryzoans, cyanobacteria and foraminifers. A detail description of all anomalies are in Appendix A. Many individuals exhibited multiple abnormalities. Table 3.4 summarizes the average percent normal and five most abundant anomalies or combin ations of anomalies for each sampling date. For the archived samp les, the percent normal individuals ranged from 14% (September 1982) to 37% (December 1982). The most abundant abnormality or combination of anomalies for each sa mpling month were as follows: June 1982-dissolution, pits, and irregular sutures (23%); September 1982--irregular sutures (14%); December 1982--dissolution, pits, microborings, i rregular sutures, and curled (13%); and March 1983--irregular sutures ( 26%). The largest individuals were in the September 1982 samples. For the recent samples, per cent normal ranged from 77% (June 2006) to 5% (December 2006). The most abundant abnormality for June and September 2006 and March 2007 were irregular sutures at 15%, 40% and 12% respectiv ely. The largest individuals were found in December 2006. Some abnormalities showed obvious trends with increased shell diameter in both recent and archived samples (Figs. 3.1-3.24). Curled tests were very common and the highest percentages of curled tests were f ound in the largest size class (1 mm) for all sampling months except for December 2006 (for this month curled tests were most common in the dominant size class of > 1 mm although the largest size class was > 2 mm) (Figs 3.3 and 3.4). Irregular sutures were also very common. In both archived and

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47 recent samples, percentages of irregular suture s were highest in the largest size class (1 mm) for September and December (Fig. 3.1 and 3.2). For June and March (both archived and recent) irregular sutures were most common in both the >0.5 mm and >1 mm size classes (Fig. 3.1 and 3.2). Dissolution and su rface pitting were very pronounced in the >0.5 mm and > 1 mm size classes for the 1982-83 samples, although in September and December the highest percentages of these tw o surface textures were found on the largest individuals (1 mm) (Figs. 3.15 and 3.17). In 2006-07 dissolution and surface pitting were far less pronounced and seemed to have a fairly even distribution among all size classes except for December, where both textur es were very prominent on individuals in the 1 and 2 mm size classes (F igs. 3.16 and 3.18). Very few specimens in the recent samples exhibited microborings. However, n early 14% of individuals found in the 198283 samples exhibited microborings, the majority of which were found in the largest size class (1 mm) (Fig. 3.19). Table 3.1. Sampling date, number of specimens analy zed per sample, mean diamet er, standard deviation, median diameter, and size range Sampling Date Total Number of Specimens Per Date Number of Specimens Per Size Class .125-<.25 mm .25-<.5 mm 5-<1 mm 1-<2 mm > 2 mm June 1982 408 114 74 267 174 0 Sept 1982 285 4 30 77 174 0 Dec 1982 158 4 25 61 68 0 Mar 1983 224 5 62 101 56 0 June 2006 2830 7 128 1844 851 0 Sept 2006 827 0 25 201 601 0 Dec 2006 353 0 11 32 285 25 Mar 2007 425 34 144 166 81 0

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48 Plate 3.1. Archaias angulatus ; 1 normal adult, 2 normal juvenile aperture, 3-4 profoundly deformed, 5 asymmetry, 6 curled, 7 uncoiled, 8 surface blips and irregular sutures, 9 breakage and repair

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49 Plate 3.2. Archaias angulatus ; 1 normal adult sutures, 2 dissolution, 3 dissolution and surface pits, 4 microborings, 5 (bryzoan), 6 dissolution and microbial biofilm

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50Table 3.2. Percentages of specimens exhibiting physical abnormalities (number of specimens examined per date listed in Table 3.2; percentages exceed 100% because any one specimen can have multiple anomalies) Table 3.3. Percentages of spec imens exhibiting surface anomalies ( number of specimens examined per date listed in Table 3.2; percentages exceed 100% because any one specimen can have multiple anomalies) Sampling Date Normal Irregular Sutures Curled Asymmetrical Un coiled Profound Surface Blip Breakage and Repair June 1982 27.9 57.4 9.58 2.71 1.49 1 0.25 1.23 Sept 1982 13.7 78.6 29.1 9.83 0 1.4 1.05 4.2 Dec 1982 37.3 43 27.8 6.32 0 0.63 0.63 1.26 Mar 1983 24.6 68.8 24.6 3.13 0.45 0 0 0 June 2006 76.5 19.7 3.5 0.82 0.04 0.39 0.15 0.51 Sept 2006 15.4 76.3 19.9 2.06 0.12 1.09 1.21 3.26 Dec 2006 5.1 88.4 68.3 3.11 1.13 1.13 0.28 14.7 Mar 2007 46.4 33 6.13 9.69 2.85 0.24 0.24 2.13 Sampling Date Dissolution Surface Pits Microborings Microbial Biofilm Epibiont June 1982 46.6 43.9 9.35 0 0 Sept 1982 57.9 55.1 21.1 0.35 0 Dec 1982 46.8 41.1 13.9 1.26 0.63 Mar 1983 41.5 40.2 15.2 0 0.89 June 2006 7.31 6.18 0.04 0.04 0 Sept 2006 17.5 15.4 0.6 0.12 0 Dec 2006 46.0 49.0 2.55 1.97 0.56 Mar 2007 22.2 12.1 0.24 1.89 0

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51 Table 3.4. Summary of percent normal and top five most abundant anomalies or combinations of anomalies for all sampling dates Date Percent normal Five most common abnormalit ies or combinations of abnormalities and average relative percent June 1982 28% Dissolution, pits, irregular sutures (23%) Irregular sutures (17%) Dissolution, pits (7%) Irregular sutures, curled (5%) Dissolution, pits, microborings, irregular sutures (3%) September 1982 14% Irregular sutures (14%) Dissolution, pits, irregular sutures, curled (13%) Dissolution, pits, irregular sutures (12%) Irregular sutures, curled (9%) Dissolution, pits, microborings, irregular sutures (11%) December 1982 37% Dissolution, pits, microborings, irregular sutures, curled (13%) Dissolution, pits (8%) Dissolution, pits, microborings, irregular sutures (6%) Irregular sutures, curled (6%) Irregular sutures (4%) March 1983 25% Irregular sutures (26%) Dissolution, pits, irregular sutures (14%) Dissolution, pits, microborings, irregular sutures, curled (10%) Dissolution, pits, irregular sutures, curled (7%) Irregular sutures, curled (5%) June 2006 77% Irregular sutures (12%) Dissolution, pits, irregular sutures (3%) Irregular sutures, curled (2%) Dissolution, pits (2%) Dissolution, irregular sutures (1%) September 2006 14% Irregular sutures (43%) Irregular sutures, curled (14%) Dissolution, pits, irregular sutures, curled (11%) Dissolution, pits, irregular sutures (8%) Dissolution, pits (2%) December 2006 5% Dissolution, pits, irregular sutures, curled (34%) Irregular sutures, curled (24%) Irregular sutures (6%) Dissolution, pits, irregular sutures (5%) Dissolution, pits, irregular sutures, curled, breakage and repair (4%) March 2007 46% Irregular sutures (15%) Dissolution (7%) Dissolution, pits (6%) Irregular sutures, curled (5%) Dissolution, pits, irregular sutures (2%)

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52 Figure 3.1. Percentage of individuals with irregular sutures vs diameter (1982-83) Figure 3.2. Percentage of individuals with irregular sutures vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 20% 40% 60% 80%Percent Irregular Suture s Size Class (mm)Percent Irregular Sutures (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 20% 40% 60%Percent Irregular Suture s Size Class (mm)Percent Irregular Sutures (1982-83)

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53 Figure 3.3. Percentage of individuals with curling vs diameter (1982-83) Figure 3.4. Percentage of individuals with curling vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 20% 40% 60% 80%Percent CurledSize Class (mm)Percent Curled (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 10% 20% 30%Percent CurledSize Class (mm)Percent Curled (1982-83)

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54 Figure 3.5. Percentage of individuals with asymmetry vs diameter (1982-83) Figure 3.6. Percentage of individuals with asymmetry vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r Ma r c h >0.125 >0.250 >0.500 >1 >2 0% 2% 4% 6%Percent AsymmetrySize Class (mm)Percent Asymmetry (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 2% 4% 6%Percent AsymmetrySize Class (mm)Percent Asymmetry (1982-83)

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55 Figure 3.7. Percentage of profoundly deformed individuals vs diameter (1982-83) Figure 3.8. Percentage of profoundly deformed individuals vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 1% 2%Percent Profoundly Deforme d Size Class (mm)Percent Profoundly Deformed (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 1% 2%Percent Profoundly Deforme d Size Class (mm)Percent Profoundly Deformed (1982-83)

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56 Figure 3.9. Percentage of breakage and repair vs diameter (1982-83) Figure 3.10. Percentage of breakag e and repair vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r Ma r c h >0.125 >0.250 >0.500 >1 >2 0% 4% 8% 12% 16%Percent Breakage and Repai r Size Class (mm)Percent Breakage and Repair (2006-07) J u n e S e p t e m b e r D e c e m b e r Ma r c h >0.125 >0.250 >0.500 >1 >2 0% 1% 2% 3%Percent Breakage and Repai r Size Class (mm)Percent Breakage and Repair (1982-83)

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57 Figure 3.11. Percentage of individuals with surface b lips vs diameter (1982-83) Figure 3.12. Percentage of individuals with surface b lips vs diameter (2006-07) June September D ece m ber Ma r ch >0.125 >0.250 >0.500 >1 >2 0.00% 0.40% 0.80% 1.20%Perecent Surface BlipsSize Class (mm)Percent Surface Blips (2006-07) J une Septembe r Dece m b e r March >0.125 >0.250 >0.500 >1 >2 0.00% 0.40% 0.80% 1.20%Perecent Surface BlipsSize Class (mm)Percent Surface Blips (1982-83)

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58 Figure 3.13. Percentage of uncoiled individuals vs diameter (1982-83) Figure 3.14. Percentage of uncoiled individuals vs diameter (2006-07) June Se pte m be r D e cem b er M ar c h >0.125 >0.250 >0.500 >1 >2 0.00% 0.40% 0.80% 1.20%Perecent UncoiledSize Class (mm)Percent Uncoiled (2006-07) June S ep temb e r D ece mb er M a rch >0.125 >0.250 >0.500 >1 >2 0.00% 0.20% 0.40% 0.60% 0.80% 1.00%Perecent UncoiledSize Class (mm)Percent Uncoiled (1982-83)

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59 Figure 3.15. Percentage of individuals with dissolution vs diameter (1982-83) Figure 3.16. Percentage of individuals with dissolution vs diameter (2006-07) June Septembe r D e ce m ber M a r c h >0.125 >0.250 >0.500 >1 >2 0% 10% 20% 30% 40%Percent DissolutionSize Class (mm)Percent Dissolution (2006-07) June S e p tember Dec e mbe r March >0.125 >0.250 >0.500 >1 >2 0% 10% 20% 30% 40% 50%Percent DissolutionSize Class (mm)Percent Dissolution (1982-83)

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60 Figure 3.17. Percentage of individuals with surface p itting vs diameter (1982-83) Figure 3.18. Percentage of individuals with surface p itting vs diameter (2006-07) June S eptembe r December Marc h >0.125 >0.250 >0.500 >1 >2 0% 10% 20% 30% 40% 50%Percent Suface Pittin g Size Class (mm)Percent Surface Pitting (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 10% 20% 30% 40% 50%Percent Surface Pittin g Size Class (mm)Percent Surface Pitting (1982-83)

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61 Figure 3.19. Percentage of individuals with microborings vs diameter (1982-83) Figure 3.20. Percentage of individuals with microborings vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0% 4% 8% 12% 16% 20%Percent MicroboringsSize Class (mm)Percent Microborings (1982-83) June September De c e m b er Ma r ch >0.125 >0.250 >0.500 >1 >2 0% 1% 2% 3%Percent Microboring s Size Class (mm)Percent Microborings (2006-07)

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62 Figure 3.21. Percentage of individuals with microbial biofilm vs diameter (1982-83) Figure 3.22. Percentage of individuals with microbial biofilm vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0.00% 0.20% 0.40% 0.60% 0.80%Percent Micrbial Biofil m Size Class (mm)Percent Microbial Biofilm (1982-83) June S e p t e m b er De c em b e r March >0.125 >0.250 >0.500 >1 >2 0.00% 0.40% 0.80% 1.20% 1.60% 2.00%Percent Microbial Biofil m Size Class (mm)Percent Microbial Biofilm (2006-07)

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63 Figure 3.23. Percentage of individuals with epibionts vs diameter (1982-83) Figure 3.24. Percentage of individuals with epibionts vs diameter (2006-07) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 >2 0.00% 0.20% 0.40% 0.60% 0.80% 1.00%Percent Epibon t Size Class (mm)Percent Epibont (1982-83) J u n e S e p t e m b e r D e c e m b e r M a r c h >0.125 >0.250 >0.500 >1 2 0.00% 0.20% 0.40% 0.60%Percent Epibon t Size Class (mm)Percent Epibont (2006-07)

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64 Irregular sutures and curled shells were the most common physical abnormalities and were found in all samples. The number of individuals with irregular sutures ranged from about 20 to 88%, w ith the highest percen tage found in the December 2006 samples (Table 3.2). Curled and asymmetrical shells were also present in all sampling dates. Surface pits and dissolution were the dominant surface texture anomalies (Table 3.3). The number of indivi duals with surface pits ranged from 6 to 55% and the number of individuals with dissolution ranged from about 7 to 58%. Percentages of shells with surface pits a nd dissolution were highest in September 1982, both over 50%. In addition, microborings were observed for all sampling dates. Among Sample Comparisons An MDS plot of all samples by date produced a stress value of 0.11, indicating a useful representation of the relative si milarities among samples (Fig. 3.25). The 1982-83 samples exhibit relatively high betw een sample variability, which often exceeded between date variability. The 2006-07 samples were much less variable within any sampling date, but ranged from relatively few anomalies in June 2006 (76.5%) to relatively few normal specimens in December 2006 samples (about 5%). Comparisons Among Variables Cluster analyses were performed on group-averaged Bray-Curtis similarity matrices by variables to see which shell anom alies occurred together. In the archived samples five groups were evident (Figs. 3.26). Dissolution and surface pits clustered

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65 Figure 3.25. MDS plot for all sampling dates

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66 Figure 3.26. Results of cluster analysis on a gr oup-averaged Bray-Curtis si milarity matrix for the archived samples Similarity 1 2 3 4 5

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67 Figure 3.27. Results of cluster analysis on a groupaveraged Bray-Curtis similar ity matrix for the recent samples Similarity 5 6 7 1 2 3 4

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68 together at a similarity of about 98. These two anomalies then clus tered with irregular sutures and curled tests with similarities 80. Within the recent samples, seven groups clustered out with similarities 60 (Fig. 3.27). Again, di ssolution and surface pits (similarity of 95) clustered with irregular sutures and curled tests with similarities 75. 3.4 Discussion The basic finding of this study is that shell anomalies were common in the Archaias angulatus population from Pennekamp State Park, Florida, in both the 198283 collections and the 2006-07 collections. Th e sample site was originally chosen in 1982 because these foraminifers were abundant and the location was convenient for repeated sampling. The continued abundance and lack of significan t change in kinds and frequency of morphological anomalies indicates that environmental conditions, including the variability of the geochemical habitat, are still well within the range that A. angulatus can thrive. However, given that thes e are Mg-calcite taxa that are adapted to high carbonate saturati on, I highly recommend mon itoring this population on 10-20 year intervals to determine if and when o cean acidification begins to impact such foraminifers. Morphological abnormalities have long been observed in foraminifers. For example, Brady’s (1884) report of forami nifers collected during the Challenger expeditions (1873-76) included illustrations of twinning, doub le apertures, irregular sutures and asymmetry in planispiral taxa. Moreover, shell anomalies are particularly common in geochemically stressed environmen ts including euryhaline environments (Yanko et al., 1998; Stouff et al., 1999a; Debena y et al., 2001; Geslin et al., 2002) and

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69 environments contaminated by heavy metals (Yanko et al., 1999). Foraminifers of the Order Miliolida seem to be particularly susceptible to morphological abnormalities (Yanko et al., 1998; Samir and El-Din, 2001). A variety of researchers have proposed that morphological anomalies have potential as indicators of anthropogenic contamination (Alve, 1995; Yanko et al., 1998; Yanko et al., 1999; Stouff et al., 1999a; Stouff et al., 1999b; Geslin et al., 2002; Saraswat et al., 2004; many othe rs). To utilize shell anom alies as stress indicators, questions that must be addressed include: “Which anomalies are cause for concern?” and especially, “What frequency of occurrence is cause for concern?” Time series studies such as this are essential to begin to address these questions. The criteria for determination of normal versus anomalous shell morphologies were based on presence or absence of features and not the degree to which an individual was affected. For example, if an individual exhibited one section of an irregular suture line, that sp ecimen was tallied in the irregu lar suture category although most of the suture lines appeared normal While many individuals were affected by multiple anomalies, some actually looked al most normal but were categorized based on the anomaly, no matter how slight. Irregular sutures are likely superficial features analogous to scars. Their presence did not affect shell strength in la boratory experiments (C hapter 4). Generally they were visible on only one side of the shell, indicating the entire chamber wall was not affected. Moreover, irregular suture s commonly were noted on specimens of A. angulatus collected from other locations. Thus, they probably should not be classified as anomalies, except when they occur with great frequency on individual shells.

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70 Dissolution is an indication of decr eased carbonate saturation and possible reasons on the scale of individual foraminife rs can include hyposalinity and oxidation of organic matter. My field-based study provides baseline information for future assessments to determine if and when rising CO2 begins to influence calcification of miliolid Foraminifera. Epiphytic Foraminifera can exhibit a great deal of mor phological variation. Langer (1993) investigated epiphytic Fora minifera in the Me diterranean Sea and reported that foraminiferal morphology can va ry greatly within one species depending on type of phytal substrate and on whether i ndividuals lived on blades, rhizomes, or holdfasts. Permanently and tempor arily attached species including Planorbulina and Rosalina can exhibit curling, asymmetry, and other unusual forms because they possess multiple apertures and adaptive attachment surfaces that mold to the surface on which they adhere. Thus, some feat ures that were observed in A. angulatus such as curled or asymmetrical shells, might be related to th e phytal substrate sampled. Most individuals for this study were scraped from Thalassia blades while others were picked out of the rhizomes and off algal blades and holdfasts. Certainly the discoid soritids can mold to phytal substrates, but whethe r the same is true for A. angulatus requires further study. Profoundly deformed and uncoiled shells, as well as surface blips, epibionts, and microbial biofilm, were uncommon to ra re, generally occurring in fewer than 2% of the shells examined. Percentages of she lls exhibiting breakage and repair were more variable, though also low except for December 2006 where the percentage was nearly 15%. Samples that month had the highest per cent of large individua ls and that larger surface area means higher potential for breakage. Studies of Amphistegina spp.

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71 collected prior to worldwide documentation of bleaching beginning in 1991-92, found that about 5% of shells collected live exhi bited breakage and repair (Toler and Hallock, 1998), and that deformities were seldom observed. After bleaching began in Florida populations in 1991, breakage and breakage and repair became much more common, sometimes occurred in more than 40% of specimens, which was clearly anomalous. Further, breakage and repair and surface pitting are part of life in an energetic environment where individual gr ains, including foraminiferal shells, interact with each other causing damage. Given the shallown ess of the sampling area, wave energy was certainly an issue. In fact sampli ng in both December 2006 and March 2007 was complicated by windy weather, whic h tends to dislodge the live Archaias from their phytal substrates and concentr ate them in bottom sediments. Thus, the incidence of breakage and repair that was found wa s likely was within normal limits. Dissolution and surface pits were both common features and were often found together. More than 40% of the archived sp ecimens exhibited one or both features and recent samples had percent abundance 6-40%. Specimens with dissolution were characterized by shallow pseudopore cups and removal of th e outer layer of rhombohedral plates. Some individuals exhi bited differential dissolution resulting in pseudopore cups that looked raised from the shell surface (Pl. 3.2 Fig. 2). The question should be asked how these foraminifers ar e dissolving in waters that are still supersaturated with respect to calcium carbona te. Yates and Halley (2003) measured coral-reef community metabol ism using a submersible habitat (Submersible Habitat for Analyzing Reef Quality—SHARQ) in Biscayne Bay, Florida Keys and South Molokai, Hawaii, and reported that dissolution ex ceeded calcification during darkness on many

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72 types of coral reef substrate types. Furthe r, they reported that the highest rates of dissolution occurred in sedime nts that had the highest pe rcentage of high-magnesian calcite. Another explanation for dissolution on the surface of these foraminifers is that the sea grass was covered with flocculent organic material, ther eby supporting micro organisms. Respiration by these micro orga nisms on the surface of the shells can cause localized drops in pH allo wing dissolution to occur. The surfaces of some individuals were riddled with microborings. Although microbioerosion is a natural process in cora l reef environments, its prevalence can be associated with nutrient pollution. Silva et al. (2005) and Chazottes et al. (2002) found higher bioerosion rates by microborers in r eefs subject to eutrophication compared to reefs in nutrient-poor areas. The Florida r eef tract has experien ced a shift from coralalgal dominated reef communities to algalsponge dominated hard-bottom communities over the past several decades (Dustan, 1999; Port er et al., 2002), a shif t that is reflected in changes in foraminiferal assemblages (C ockey et al., 1996; Hallock et al., 2003), and possibly in the foraminiferal shells themselves. Szmant and Forester (1996) and Szmant (2002) stated that eutrophication has in fact o ccurred in inshore waters. The much higher incidences of microbor ing in the specimens collected in 198283 (9-21%) as compared with the specim ens collected in 2006-07 (<3%) could be interpreted to indicate that eutrophication has diminished at the Pennekamp site over the past 25 years. This is quite possible, as management actions undertaken in the 1970s and early 1980s have improved water qua lity in some areas (Bottcher et al., 1995). However, another possibility for the difference in microboring is the difference in sampling approaches. Hallock et al. (1986) collected a rubble sample, a mixed algal-

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73 seagrass samples, and a predominantly seag rass sample each sampling date, while this study sampled primarily from seagrass. The rubble and mixed phytal samples may have been more commonly covered by flo cculent organic matter and dead seagrass, producing a local environment more conduciv e to microboring organisms and possibly to dissolution and surface pitting noted above. This possibility is consistent with the higher within-date variability of the 198283 samples and further supported by the somewhat higher percentages of microborings and microbial film seen in specimens from the December 2006 sample, as compared with other samples collected in 2006-07. Moreover, the incidence of dissolution and surface pits was also much higher in December 2006. As noted prev iously, most of the live A. angulatus had been dislodged from the phytal substrates and were in th e bottom sediments during that sampling. There were obvious size diffe rences in the occurrences of shell anomalies. Larger individuals have a greater surface ar ea on which environmental factors such as bioerosion can act. Further, the larger th e individual is, the l onger the individual has been in the environment and the more prom inent dissolution or bioerosional features can become. Irregular sutures and curling percentages were highest in the largest individuals because these two features mainly affected the outer rows of chambers. The largest specimens were found in Decem ber 2006, which may further explain the higher incidences of many anomalies as compared with other 2006-07 samples. 3.5 Conclusions Physical abnormalities observed included profoundly deformed, curled, asymmetrical, and uncoiled shells, irregular su ture lines, surface “blips,” and breakage

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74 and repair. Surface texture anomalies in cluded surface pits, dissolution, microborings, microbial biofilm, and epibont growth. Epibont growth included bryzoans, cyanobacteria and foraminifers. Eighty-six combinations of abnormalities and surface textures were observed and recorded. Shell anomalies were found in the Archaias angulatus population from Pennekamp State Park, Florida, in both the 1982-83 collections and the 2006-07 collec tions. Given that the site was originally chosen for study because A. angulatus were so abundant, the lack of significant change indicates that the variability of the geochemical habitat is still with in the range that A. angulatus can thrive. Some abnormalities, including curling, irre gular sutures, dissolution, pits and microborings increased in prevalence as test diameter increased. Dissolution and surface pitting were very prominent in the 1982 -83 samples, occurring in >40% of the specimens. Microborings were also more prev alent in the archived samples (9-22%). Conditions conducive to presence of microbor ings are similar to those that would produce dissolution and surface pitting. Dissoluti on, pits, irregular su tures, and curling tended to occur together in both archived and recent samples. The prevalence of anomalies observe d in samples collected in 1982-83 was highly variable within sample dates. Sa mples collected in 2006-07 were much more similar within dates but comparably variable overall.

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75 4. SHELL STRENGTH AND ULTRAS TRUCTURE IN DEFORMED ARCHAIAS ANGULATUS FROM THE FLORIDA KEYS (USA): IMPLICATIONS FOR SURVIVAL AND COASTAL SEDIMENTATION 4.1 Introduction The functional morphology of foramini feral shells is not well understood. However, it is likely that the shell functions similarly to other exoskeletons by providing protection against predators, a physical barrier to the external environment and support. It is reasonable to conclude that shell st rength and shape are important factors in the survival of Foraminifera (Wetmore and Plotnick, 1992). Furbish and Arnold (1997) investigat ed hydrodynamic strategies in the morphological evolution of spi nose planktonic foraminifers Orbulina universa and Globigerinoides sacculifer They reported that settling sp eed of planktonic shells varied with foraminifer shape and the presence of acicular spines produced two counteractive effects: spines increase the weight of a foraminifer, and therefore increased its settling speed, and the presence of spines also increase d the fluid drag on the foraminifer, thereby decreasing its settling speed. If growing spines is part of an evolutionary strategy to impede settling, then it is logical to presum e that the advantage of increasing drag by growing spines outweighs the disadvantage s of both increasing we ight (Furbish and Arnold, 1997) and added en ergetic expenditures. Correlations between sh ell strength, morphology, a nd habitat have been established for some larger benthic Foramini fera that host algal endosymbionts, including

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76 Archaias angulatus, Amphistegina gibbosa and Laevipeneroplis proteus (Wetmore and Plotnick, 1992), as well as for othe r smaller benthic species including Elphidiella hannai, Oolina borealis, Trochammina inflata, Buccella frigida, Elphidium tumidum and Elphidium frigidum (Wetmore, 1987). In 1987 Wetmore determined shell strength of foraminifers from the San Juan Islands, Washington, by measuring the force necessary to crush individual shells. She reported that shell strength increase d with size and with physical energy of the environment. Indi viduals from populations living in coarse unconsolidated sediment possessed stronger shells relative to their si ze than individuals living on algae or in finer-gra ined sediments. Further, morphological characteristics including overall shell shape and wall thickness, that determined the cross-sectional area over which the crushing force was distributed, affected shell strength more than shell composition or coiling morphology. Wetmore and Plotnick (1992) looked at correlations between shell morphology, crushing strength, and habitat of Archaias angulatus, Amphistegina gibbosa and Laevipeneroplis proteus from Bermuda. They stated th at shells of living individuals collected from high energy environments were remarkably harder to crush than similar sized shells from a low-energy seagrass bed. Wetmore (1988) suggested that the inner organic lining in benthic fora miniferal shells may provide mechanical strength against crushing. Wetmore and Pl otnick (1992) reported that not only were Archaias angulatus individuals from the energetic reef envi ronment more robust than similar-sized individuals from the more sheltered locality, but they also appeared to have a more robust inner organic lining.

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77 Hallock (1979) and Hallock et al. (1 986) investigated how the environment influenced the shell shape of Amphistegina spp. Hallock (1979) examined trends in shell shape with depth in Amphistegina lessonii and A. lobifera, reporting that shell sphericity decreased with increased habita t depth. In field samples of A. lessonii shell thickness-todiameter ratio decreased with increasing depth of habitat and with reduced wave exposure of habitat. However, A. lobifera responded primarily to habitat exposure. In laboratory cultures, A. lessonii and A. lobifera produced thicker shells when grown in high light regimes than under reduced light. Hallock et al. (1986) reported that light availability and water motion greatly in fluence thickness to diameter ratios of Amphistegina sp. grown in culture. Individuals s ubjected to water motion were as much as 50% thicker than individuals grown without water motion. Morphological abnormalities in foraminife ral shells due to natural variation and anthropogenic influences have been well docum ented. Industrial and domestic pollution (Alve, 1995; Yanko et al., 1999; Samir, 2000; Ge slin et al., 2002; Saraswat et al., 2004), heavy metals (Banerji, 1990; Yanko et al., 1994; Alve and Olsgard, 1999), low pH (Geslin et al., 2002; Le Cadre et al., 2003), and salinity (Stou ff et al., 1999a; Geslin et al., 2002) have been investigated in field and laboratory investigati ons. Although physical deformities are well documented, little is known if physical abnor malities affect shell strength. Archaias angulatus are porcelaneous foraminifers that produce high magnesium calcite shells covered with pseudopores (Ficht el and Moll, 1798). They host chlorophyte endosymbionts of the genus Chlamydomonus (Lee and Bock, 1976) and their shells are characterized by numerous chamberlets and pr onounced flaring in the outermost whorls

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78 of mature individuals (Loe blich and Tappan, 1988). Their robust shells are typically resistant to destruction and ar e thusly widespread and abunda nt in bioclastic carbonate sediments in many environments throughout the western tropical Atlantic Ocean (Martin, 1986). Recent studies have documented a suite of morphological and textural abnormalities in A. angulatus from the Florida Keys. Crev ison and Hallock (2007) (see Chapter 2) reported that surface texture a nomalies appeared to be unusually common among living Archaias angulatus individuals. Many specimens appeared to have a rough, etched finish to their shells. Under SEM, th ese individuals were a ffected by a variety of surface anomalies including pits, microborings microbial biofilm, and dissolution. Other individuals exhibited a range of physical abnormalities in cluding rows of mangledlooking chambers, ragged suture lines, and complete shell malformations. Chapter 3 discusses comparisons of specimens of A. angulatus collected from John Pennekamp Coral Reef State Park on Key Largo, Florid a, in 1982-83 with samples collected in 200607. Seven different types of morphological abnormalities and five different types of surface texture anomalies were identified. Many individuals had combinations of abnormalities and textures. The morphologica l abnormalities included irregular suture lines; profoundly deformed, curled, asymmetrical or uncoiled shells; surface ‘blips’; and breakage and repair. The surface texture anomalies included microbial biofilm, dissolution, surface pits, microborings, epifauna l growth. Some individuals were lost during the isolation process because they disi ntegrated when they were picked up using a small fine-haired paint brush. Other individuals lost a few outer rows of chambers during this process.

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79 The purpose of this investigation is to de termine if strength, wall fabric or Mg/Ca ratios in Archaias angulatus shells differ significantly in specimens that exhibit physical abnormalities as compared with shells that appear normal. 4.2 Materials and Methods Specimen Collection Bulk samples of living Archaias angulatus were collected from John Pennekamp Coral Reef State Park on Key Largo, Florid a, in May 2008 (Fig. 2.1). Samples collected in June, September and December 2006 and March 2007 were used to determine Mg/Ca ratios and they were gathered similarly to the bulk samples collect ed in 2008. The water was about 1.5 meters deep and sample s were collected while snorkeling. Archaias angulatus live attached to seagrass, ther efore three to f our handfuls of Thalassia testudinum blades were collected at 3 sites along the swimming area demarcation rope. The Thalassia was picked above the rhizome and pl aced into plastic bags underwater. Specimens were then scraped from the seagra ss blades and placed in to plastic one-liter wide-mouth containers filled with seawater for transport back to the laboratory. While in the laboratory, foraminifers were placed into Petri dishes and allowed to settle for 48 hours prior to picking. Indivi duals were examined under light microscopy for evidence of morphological anomalies. Approximat ely 55 normal and 55 abnormal individuals were tested. Maximum diameter of each spec imen was recorded as well as the type of abnormality present. Digital photographs were taken before specimens were crushed.

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80 Shell Strength Shell strength was determined by comp ression testing using methods previously reported by Wetmore (1987) and Wetmore and Pl otnick (1992). The force necessary to crush individual shells between two parallel flat surfaces was measured to determine the relative strength of foraminiferal shells. All measurements were collected on living individuals transferred directly from seawater to the crushing platform. The compressive force was applied to the sh ortest axis of the shell and specimens were immediately crushed. The measurements of crushing strength were made with a Lucas Schaevetz load cell (Fig. 4.1). The lo ad cell was mounted upside down opposite a moveable platform directly unde rneath the load cell. For eac h measurement, the platform was very slowly raised, pushing the shell agains t the probe. A strip chart was attached in series to the load cell and it recorded all vo ltages which were then converted to force in Newtons (N). To keep the loading rate as un iform as possible, the platform was raised at the same rate for all measurements of shell strength. The maximum force a shell bears before breaking was taken as its crushing strength. The output of the load cell was calibrated prior to crushing experiments by inverting the apparatus and placing known weights on the probe. Mg/Ca Ratios Magnesium/calcium ratios were dete rmined by ICP-OES (inductively coupled plasma optical emissions spectrometry). No rmal and abnormal she lls collected in 20062007 were weighed prior to the clea ning process. A minimum of 200 g of foraminiferal

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81 Figure 4.1. Set up of crushing apparatus; A inverted load cell and B movable platform material was required for each sample. Samp les were broken between 2 glass plates and then placed into 0.6 ml acid-leached centrif uge tubes. Samples were cleaned according the methods in Russell et al. ( 2004). The process was as follows: 1) 1 rinse in ultra pure H20 and placed in an ultrasonic cleaner with water for 30 seconds; A B

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82 2) 3 oxidation steps with hot buffered H2O2 (1:1 of 0.1 N NaOH and 30% H2O2) held at 70-80OC in a hot water bath for a half hour each step, the hot buffered H2O2 was pipetted off after each oxidation step; 3) 5 rinses with ultra pure H20; 4) 1 rinse with 0.001 N HNO3; 5) final rinse with ultra pure H20. Samples were then placed in a drying oven for one hour at 60OC and stored over night in closed centrifuge tubes. Right before ICP-EO S analysis, samples were placed into 10 ml plastic shell tubes and dissolved in 0.075 M HNO3 to a concentration of 1 ppm. Shell Ultrastructure Scanning electron microscopy was used to examine the morphology of broken normal and abnormal shells. A small number of shells were rinsed in buffered deionized water and allowed to dry for a week on a pale ontological slide. The maximum diameter of each shell was recorded. Five normal and five abnormal specimens were broken along the same axis using a razor blade. Abnormalities included one shell that exhibited breakage and repair, one asymmetri cal shell, and three shells th at had irregular sutures. Specimens were mounted onto aluminum SEM stubs using double-sided adhesive tabs and sputter coated with gold-palladium (approximately 10 nm thickness) using a Hummer 6.2 Sputtering System Specimens were then examined using secondary electron imaging on a Hitachi S-3500 N scanning electron microscope.

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83 Statistical Analysis An analysis of covariance (ANCOVA) was performed on the crushing data to test whether the slopes of the regression line s for normal and abnormal specimens were significantly different. A two factor ANOVA with replication was performed in Excel to test whether the Mg/Ca ratios in abnormal specimens differed significantly from the ratios in normal ones, or if the Mg/Ca ratios differed by sampling month. The significance level was p =0.05. 4.3 Results Under light microscopy, normal specimens had a smooth surface, concentric suture lines, uniform color and were free of any curling, asymmetry, or surface texture (Pl. 4.1). For these specimens, the relati onship between shell strength and maximum diameter (Fig. 4.2) was highly significant (y = 7.4x + 1.99), Pears on’s coefficient of determination, R2 = 0.82, N = 53) for specimens whose maximum diameter ranged from 0.3 to 2.35 mm. Table 4.1 summarizes the crushi ng strengths for normal specimens. Features defined as abnormalities included irregular suture lines, breakage and repair, missing shell wall, curled, asymmetr ical and profoundly deformed shells, and surface texture anomalies (Pl. 4.1, Table 4.2). Comparison of crushing strength to diameter (Figure 4.3) resulted in much more variable data (y = 4.6 + 1.13), R2 = 0.39, N = 55). Maximum diameter for these specimens ranged from 0.3 to 2.55 mm. To understand the high variability in cr ushing strengths of specimens exhibiting abnormalities, specimens with relatively minor features (i.e., irregular sutures, anomalous surface texture or both) were examined separately (Figure 4.4).

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84 The resulting regressi on line (y = 6.72x 0.501, R2 = 1, N = 23) was not significantly different from the regression for nor mal specimens (F = 0.063, F crit = 4, p = 0.05). However, the slope of the regression line for more anomalous specimens (y = 4.19 x + 0.92, R2 = 1, N = 22) was significantly different from that for normal specimens (F = 5.96, F crit = 4, p = 0.05). The results of the ANOVA on Mg/Ca ra tios are summarized in Table 4.3. The Mg/Ca ratios for both deformed and normal individuals were within normal parameters and ranged from about 12-15 mol % (Fig. 4.5). There was no significant difference in Mg/Ca ra tios between normal and abnormal shells ( p = 0.7). However, there were significant differences in Mg/Ca ratios among the sampling months ( p = 7.33 x 10-7). Although the subsample was small, abnormalities were found in the shell wall structure of abnormal A. angulatus (Pl. 4.2). Shell fabric of normal porcelaneous foraminifers is characterized by randomly arranged calcite need les overlain by calcite rhombohedral plates. The individuals that exhibited breakage and repair and irregular sutures had portions of their shells that l ooked as if the calcite needles were welded together (Pl. 4.2 Figs. 2 and 4). The indivi dual with breakage and repair also had an amorphous build-up overlying the calcite needles.

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85 Plate 4.1. Archaias angulatus ; 1 normal adult, 2 normal juvenile aperture, 3-4 profoundly deformed, 5 fragile broken outer chambers, 6 juvenile with irregular suture lines, 7 asymmetry, 8 uncoiled

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86Table 4.1. Results of crushing strength experiments for normal specimens listing maximum shell diameter (mm) and crushing strength (N), N = Newtons. Shell Max Diameter (mm) Crushing Strength (N) Shell Max Diameter (mm) Crushing Strength (N) 0.3 1.09 1.2 6.89 0.45 1.29 1.2 5.7 0.5 1.58 1.2 5.5 0.5 1.89 1.3 11.78 0.6 1.78 1.3 9.04 0.6 1.78 1.3 8.45 0.6 2.36 1.3 7.07 0.6 2.17 1.3 7.07 0.6 3.15 1.4 10.41 0.6 1.87 1.4 12.57 0.65 2.36 1.4 8.64 0.7 3.15 1.45 9.03 0.7 2.76 1.5 9.23 0.7 3.14 1.55 11.78 0.8 3.51 1.6 9.0 0.85 4.72 1.6 6.88 0.85 4.13 1.65 11 0.85 3.54 1.8 9.43 0.9 5.5 1.8 9.43 0.9 3.91 1.85 7.07 1 5.11 1.85 10.8 1 5.5 2 12.57 1 4.33 2 10.98 1.05 6.29 2 8.64 1.1 5.31 2.2 15.71 1.15 8.25 2.35 16.88 1.2 7.47

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87 Figure 4.2. Results of crushing experiments for normal shells with shell strength in Newtons plotted against maximum shell diameter (mm). Figure 4.3. Results of crushing experiments for ab normal shells with shell strength in Newtons plotted against maximum shell diameter (mm). Individual Test Size vs Test Strength (Normal)y = 7.4x 1.99 R2 = 0.820 2 4 6 8 10 12 14 16 18 00.511.522.5Maximum Test Diameter (mm)Test Strenth (Newtons) Individual Test Size vs Test Strength y = 4.6x + 1.13 R2 = 0.390 2 4 6 8 10 12 14 16 18 00.511.522.5Maximum Test Diameter (mm)Test Strength (Newtons) (Abnormal)

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88 Figure. 4.4. Results of crushing experiments of shell strength (N) plotted against maximum shell diameter (mm), N = Newtons; for normal individuals R2 = 0.84; for individuals with irregular sutures R2 = 0.69, and for deformed individuals R2 = 0.28 Individual Shell Size vs Crushing Strength0 5 10 15 20 00.511.522.53 Maximum shell diameter (mm)C rushing Strengt h (Newtons) Irreg sutures Deformed Normal Irreg sutures regr Deformed regr Normal regr

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89Table 4.2. Results of crushing experiments for abnormal individuals listing maximum shell diameter (mm), crushing strength (N), and abnormality, N= Newtons. Shell Max Diameter (mm) Crushing Strength (N) Abnormality 0.3 1.58 irregular sutures 0.7 2.32 irregular sutures 0.7 3.15 irregular sutures 0.7 1.97 irregular sutures 0.7 3.15 irregular sutures 0.75 4.72 irregular sutures 0.8 4.33 irregular sutures 0.9 5.1 irregular sutures 1 3.93 irregular sutures 1 5.11 irregular sutures 1.05 7.07 irregular sutures 1.1 10.60 irregular sutures 1.2 8.05 irregular sutures 1.4 7.86 irregular sutures 1.4 10.80 irregular sutures 1.5 10.21 irregular sutures 1.5 10.31 irregular sutures 1.65 14.92 irregular sutures 2.55 12.17 irregular sutures 1.1 9.43 irregular sutures, texture 1.4 6.88 irregular sutures, texture 1.4 9.43 irregular sutures, texture 1.4 11.39 irregular sutures, texture 0.6 3.15 irregular sutures, asymmetrical 1.85 13.35 irregular sutures, asymmetrical 1.4 9.62 irregular sutures, curled 1.7 11 irregular sutures, curled 1.85 8.25 irregular sutures, curled 2 11 irregular sutures, curled 2.2 11 irregular sutures, curled 2.4 5.9 irregular sutures, curled 0.7 3.15 asymmetrical 1.15 5.30 asymmetrical 1.2 11 asymmetrical 1.85 10.60 asymmetrical 1.7 7.66 breakage and repair 2 9.82 breakage and repair 1.7 16.49 breakage and repair, texture 0.4 1.19 irregular sutures, uncoiled 1.4 2.36 profound 1.5 5.9 profound 1.3 3.15 profound, texture 2.1 7.86 Shell wall missing, texture 1.7 5.11 uncoiled, curled 1.75 1.78 uncoiled, texture

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90Table 4.3. Results of the ANOVA for Mg/Ca ratios Source of Variation SS df MS F P-value F crit Normal v abnormal 115.0928 1 115.0928 3.490295 0.073986 4.259677 Sampling month 1776.97 2 888.4852 26.94413 7.33 x 10 -07 3.402826 Interaction 170.9875 2 85.49376 2.592677 0.095621 3.402826 Within 791.4022 24 32.97509 Total 2854.453 29 Fig. 4.5. Mg/Ca ratios for normal and abnormal shells Mg/Ca Ratios for Archaias angulatus110 120 130 140 150Jun-06Sep-06Dec-06Mar-07 Sampling DateMg/Ca Ratio (mmols/mol) Normal Abnormal

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91 Plate 42. Archaias angulatus ; 1 Normal shell wall, 2-3 individual with breakage and repair exhibited welded calcite needles and an amorphous build up, 4 individual with irregular sutures exhibited welded calcite needles 4.4 Discussion Shell Strength Crushing strengths of specimens with shell abnormalities were much more variable than those for normal individuals, in part reflecting the variety of abnormalities observed. Irregular sutures, even when anom alous surface texture was present, did not influence shell strength. A lthough the regressions indicated that most abnormal shells

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92 were generally weaker than normal shells, data points for 5 of the 22 most abnormal specimens fell above the regression li ne for normal shells (Fig. 4.4). Normal specimens of Archaias angulatus are planispiral and exhibit a disc-like geometry. In this study, normal specimens and those with only irregu lar sutures (with or without surface textural anomalies) were crushe d along the shortest axis of shell. Since Archaias is involute and planispiral, the strongest part of the test is presumably along the shortest axis of the shell which runs through the protoconch. This area thickens as the shell grows, so the high correlation between shell maximum diameter and shell strength for normal individuals and individuals with ir regular sutures is not surprising (irregular sutures were found mainly in the outer chambers). One explanation for the increased variability in test strength of abnormal shells is the fact that such shells are much more va riable in shape. These specimens were not necessarily crushed along the shortest axis because, in many instances, the shell did not exhibit the normal planispiral geometry. Th erefore changes in the structural properties due deformation rather than a change in the ma terial property of the calcite test is likely responsible for the highly va riable test strengths of deformed individuals. Comparisons of these results with th ose of Wetmore and Plotnick (1992) for Archaias angulatus from Bermuda revealed similar ranges of crushing strengths. Although Wetmore and Plotnick did not calcula te regressions, their crushing strength data from a lagoonal locality are quite similar in magnitude and variability to my data from abnormal specimens, Their data from a reef locality spanned a much smaller diameter range (~0.4 to 0.8 mm), the shell stre ngths appear to be more comparable to my data for normal specimens from Pennekamp

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93 Mg/Ca Ratios Toler and Hallock (2001) analyzed Mg and Ca in Amphistegina gibbosa shells collected from Conch Reef in the Florida Keys using Inductively Coupled Plasma Mass Spectrometry. They found normal Mg/Ca ratio s (2-5 mol %) in all specimens, including normal individuals collected in 1982 prior to the onset of the stress event, and both normal and broken specimens collected quart erly from afflicted populations in 1996 (Toler and Hallock, 2001). They al so reported normal Mg/Ca ratios for Archaias angulatus (10-14 mol %). The Mg/Ca ratios for my study were determ ined on a data set that was collected quarterly. As there were very few normal shells in the December 2006, there was not enough material to compare Mg/Ca ratios for sp ecimens collected in this month (Fig 4.4). Nonetheless, Mg/Ca ratios were within normal parameters (12-15 mol %) for both normal and deformed specimens for the other months, with expected seasonal lows. There were, however, significant temporal di fferences in Mg/Ca ratios, reflecting that Mg/Ca ratios are highly temperature dependent. The highe r values were found in June and September samples, the warmest sampling months. Shell Ultrastructure Morphological deformities in Foraminifera may coincide with abnormal shell wall structure. Geslin et al. (1998) investigated ultra structural deformation in Ammonia using SEM. The shell fabric of normal Ammonia is characterized by elongate calcite elements arranged normal to the wall. Howeve r, Geslin et al. (1998) reported that the

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94 walls of deformed Ammonia shells exhibited crystallite di sorganization and the presence of interlamellar spaces. Morphological abnormalities in Archaias angulatus appeared to affect the shell ultrastructure. The SEMs in Pl. 4.2 were all taken on freshly broken surfaces and yet in Figs. 2 and 4 there was evidence of dissolu tion, as areas of the exposed surface look welded together so that indivi dual crystals were barely visibl e. Individual crystals looked worn down, and as if the spaces between th e crystals had been infilled. The amorphous deposit in Pl. 4.2 (Fig. 3) was also found on a freshly broken surface. The amorphous deposit overlies the calc ite needles although the margins of the deposit clearly have calcite needles overlying it. This particular individual exhibited br eakage and repair and this could be repair gone awry. MacIntyre and Reid (1998) documented recrystallization in the shells of A. angulatus and found textural changes without mine ralogical alteration. They found that the original skeletal rods altered to dense minimicrite while the foraminifers were still alive. Not only did micritization increase w ith age, but basal layers and septal walls generally altered more rapidl y than lateral walls and pillars. They speculated that recrystallization could be due to changes in the p CO2 resulting from changes in patterns of respiration and photosynthesi s of the algal symbionts. Images 2 and 4 from Pl. 4.2 look similar to minimicritiza tion described by MacIntyre and Reid (1998). It is unknown if diagenesis of living foraminiferal shells compromises strength, although it is unlikely given the persistence of Archaias angulatus shells in sediments. Further, MacIntyre and Reid (1998) reported that studies of Archaias in sediments from the Bahamas and Florida indicated that even when Archaias appeared fresh, they were typically highly micritized.

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95 Possible Causes of Shell Abnormalities Cytological, mineralogical and environmen tal factors all influe nce the strength of a foraminiferal shell. Toler and Halloc k (1997) investigated shell breakage in Amphistegina gibbosa from the Florida reef tract. Anomalous shell breakage in A. gibbosa populations was first noticed in 1992 asso ciated with the onset of a new disease that was characterized by bleaching, reproductive dysfunction, and a suite of morphological abnormalities. Starting in 1993, occurrence of shell breakage and breakage and repair were recorded in samples collect ed monthly from Conch Reef. Their study also reported malformations, uneven extern al surfaces, abnormal shapes, bioerosion, and loss of outer chambers. Internal anomalies included poorly defined pore cups, excessive calcification, and minimal organic matri x. Talge and Hallock (1995) investigated cytological damage in A. gibbosa and found that abnormal indi viduals were characterized by loss of organelles crucial to synthesis of shell matrix macromolecules. Specifically, stressed individuals showed a loss of Golgi apparatus and endoplasmic reticulum, both of which are sites of glycoprotein and glyc osaminoglycan, two major organic matrix components. Reduced production of organic ma trix likely influences shell strength by controlling calcite crystal forma tion. It is unknown if deformed Archaias angulatus have a loss of Golgi apparatus and endoplasmic reticulum. The causes of the abnormalities reported in Chapters 2 and 3 are still unknown. Possibilities include influen ce of pollutants (e.g. heavy metals and pesticides) and low pH/carbonate saturation in the environmen t which the foraminifers were living. Although organisms have a multitude of adaptive mechanisms to protect them against foreign chemicals in their environment, xenobi otics can result in pa thological disruption

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96 of biological structures on molecular to eco system scales (Yanko et al., 1999). In fact, heavy metals and pesticides are an issue in south Florida environments, and it has been well established that they, ev en in low concentrations, can have profound effects on many organisms (as summarized in Chapter 3). Heavy metals can influence forami niferal shell chemistry, morphology and ultimately, strength (Alve, 1994; Alve and Olsgard, 1999; Yanko et al., 1999). Many studies have reported that deformed Forami nifera show elevated Mg/Ca ratios when compared to non-deformed specimens, particular ly in severely polluted areas. Several hypotheses may explain this. First, heavy meta ls may directly affect the calcite crystal structure or the foraminiferal cytoskeleton. Second, heavy metal toxicity could affect foraminiferal metabolism in ways that alter Mg/Ca ratios indirectly during calcification. Third, other pollution-related environmenta l effects somehow mediate Mg/Ca ratios (Yanko et al., 1999). Other metal ions, such as barium can also be included in the crystal structure of the shell (Lea and B oyle, 1989) and it is possible that Ca2+ binding sites can not distinguish among these ions (Yanko et al., 1999). Although heavy metals are an issue in south Florida environments, this de gree of pollution is far less severe than the regions discussed in Yanko et al. (1999), wh ich may help explain why Mg/Ca ratios appear unaffected in my samples. Miliolid shell morphology has previously been observed to be sensitive to environmental influences. Given the inherent solubility of their ma gnesian-calcite shell mineralogy, these foraminifers may be among th e most sensitive indicators of declining carbonate saturation in s eawater, which results locally from increasing benthic respiration rates and globally from rising concentration of atmospheric CO2.

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97 There is a great deal of eviden ce that even small changes in CO2 concentrations in surface waters can have significant negative impacts on marine calcifiers and oceanic biogeochemical cycles (Kleypas et al., 1999; Pecheux, 1999; Langdon et al., 2000, Langdon and Atkinson, 2005). Field and labor atory experiments on coccolithophorids showed malformed coccoliths, diminished calcification, incomplete coccospheres (Reibesell, 2004; Reibesell et al., 2000), and a decrease in the average coccolith and coccosphere size as p CO2 increased (Engel et al., 2005). Magnesian-calcite shells have an even higher solubility prod uct than aragonite (Weyl, 1967; Plummer and Mackenzie, 1974) and should be particularly sensitive to the declining saturation of CaCO3 in seawaters as a conseque nce of rising atmospheric CO2 (Kleypas et al., 1999). The s ynergistic effects of cytol ogical damage and decreased carbonate saturation could certain ly account for the variability seen in crushing strengths of deformed A. angulatus Compromised individuals not only have difficulty constructing their shell but maintaining it is more energetically expensive with decreased carbonate saturation Historically, Archaias angulatus has been considered a major contributor to foraminiferal assemblages and sediments in coral reef environments throughout the Caribbean and Atlantic (Marshall, 19 76; Martin, 1986; Cottey and Hallock, 1988), specifically Florida Bay (Bock, 1971), Flor ida Keys (Wright and Hay, 1971), and the Florida-Bahamas carbonate province (Ros e and Lidz, 1977; Lidz and Rose, 1989) because shells are thick-walled, robust, and are structurally reinforced by internal pillars (Martin, 1986). However, morphological a bnormalities and shell fragility will surely undermine their ability to pe rsist in nearshore environments. Thus, given the high

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98 percentage of miliolids in sediments from the Florida Keys, decreased shell strength could have profound effects on sedimentati on. Crevison et al. (2006) reported the foraminiferal assemblage in a series of short push cores from the back reef of the Florida reef tract. In the upper keys, miliolids acc ounted for about 60% of the total assemblage and their numbers ranged from 3500-7000 indivi duals per gram of sediment. In the lower keys, they accounted for 40-60% of the total assemblage with 5500 individuals per gram of sediment. Although it is unknown if smaller miliolids are experiencing problems with shell strength, deformities have been reported in other taxa from the Florida Keys including Miliolinella, Quinqueloculina and Triloculina (Crevison and Hallock, 2001). 4.5. Conclusions Shell strength was more vari able among abnormal specimens of Archaias angulatus as compared to normal individuals. The presence of irregular sutures and surface textures did not influence shell streng th compared to normal individuals. Mg/Ca ratios were within normal parameters for all individuals although a seasonal trend was evident. Some abnormal individuals exhibi ted shell ultrastructure anomalies including dissolution, infilling, an d amorphous deposits.

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99 5. CONCLUSIONS Deformed shells and unusual shell-surface features were observed in juvenile and adult Archaias angulatus and other miliolids with algal endosymbionts collected live along the Florida reef tract. Calcification anomalies includ ed mineralogical projections and lacy crusts. Features typically consid ered taphonomic included microborings, pitted surfaces, bacterial infestation, and dissoluti on; evidence of shell repair was also documented. Prevalence of such features may indicate that these foraminifers experienced environmental stress. In 2006, a comprehensive study was undertaken to see if the occurrence and types of morphological abnormalities have changed in A. angulatus from the Florida Keys over the past 2.5 decades. Archived samples of A. angulatus collected live in 1982-83 from John Pennekamp Coral Reef State Park on Key Largo, Florida, were available for comparison to recent samples. Eighty-six combinations of abnormalities and surface textures were observed. Physical abnorm alities included profoundly deformed, curled, asymmetrical, and uncoiled shells, irregular su ture lines, surface “blips,” and breakage and repair. Surface texture anomalies in cluded surface pits, dissolution, microborings, microbial biofilm, and epibiont growth. Epibiont growth included bryzoans, cyanobacteria and foraminifers. Shell anomalies were common in the Archaias angulatus population from Pennekamp State Park, Florida, in both the 1982-83 collections and the 2006-07

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100 collections. The continued abundance of these foraminifers and the absence of significant change in occurrence of shell anom alies indicate that the variability of the geochemical habitat is still within the range that A. angulatus can thrive. Some abnormalities, including curling, irre gular sutures, dissolution, pits and microborings exhibited trends as test diamet er increased. Dissolution and surface pitting were very prominent, occurring in >40% of the specimens in the archived samples. Only the December 2006 samples exhibited equivalent percentages of dissolution and surface pitting. Dissolution, pits, irre gular sutures, and curling tende d to occur together in both archived and recent samples. The prevalence of anomalies observed in samples collected in 1982-83 was highly variable within sample dates. Samples collected in 2006-07 were much more similar within dates but comparably variable overall. Shell strength was more vari able among abnormal specimens of Archaias angulatus as compared to normal individuals. The presence of irregular sutures and surface textures did not influence shell strength compared to normal individuals. Shell strength increased linearly with maximum diameter of the shell; for normal individuals R2 = 0.84; for individuals with irregular sutures R2 = 0.69, and for deformed individuals R2 = 0.28. Mg/Ca ratios were within normal pa rameters for all individuals, with an evident seasonal trend. Some deformed i ndividuals exhibited shell ultrastructural anomalies including dissolution, infi lling, and amorphous deposits. Assessing environmental change can be a difficult task because baseline information is often lacking. My investiga tions have provided information that can be used by resource managers to assess the impacts of climate change and ocean acidification on miliolid Foraminifera. Given the inherent solubility of high-magnesian

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101 calcite, miliolid Foraminifera are potentially sensitive indicators of declining carbonate saturation associated with climate cha nge or increased organic matter due to eutrophication Further, the documentation of surface textural anomalies on living foraminifers can have profound implications for the fossil record in terms of taphonomy and interpretation of paleoenvironmental conditions. Researchers examining the taphonomy of foraminiferal shells should be aware that such modification can occur to the tests while the protists are still alive. Future Work An entire suite of morphological anomalies were documented in Archaias angulatus from the Florida Keys. Several questions arose during this investigation which set the stage for future work. First, are morphological anomalies as prevalent in other soritid foraminifers or other smaller miliolids from Pennekamp Coral Reef State Park? Second, what are the percentage s and types of anomalies in A. angulatus further off Key Largo toward Molasses Reef? Third, can any of these abnormalities be reproduced in the laboratory? Fourth, are the environmenta l conditions responsible for abnormalities nearshore reaching further out toward the reef? Based on thes e four questions, I recommend the following: Characterize heavy metals in the sediment s of Pennekamp Park both in terms of species and concentrations, Monitor populations of A. angulatus in Pennekamp Park on decadal time scales to assess impact of ocean acidif ication on these Foraminifera,

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102 Re-examine archived specimens from Molasses Reef for abnormalities and compare these to newly collected sa mples from the same transect/area, Conduct laboratory experiments on affects of lowered pH, pesticides, and changes in salinity on A. angulatus or Amphistegina gibbosa (because this species is easily cultured in the laboratory) to s ee what, if any, anomalies occur.

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103 6. REFERENCES Alve, E., 1995, Benthic foraminiferal responses to estuarine pollution: a review. Journal of Foraminiferal Research, v. 25, p. 190-203. _____ and Olsgard, F., 1999, Benthic foraminife ral colonization in experiments with Cu-contaminated sediments. Journal of Foraminiferal Research, v. 29, p. 186195. Angell, R. W., 1980, Shell morphogenesis (cha mber formation) in the foraminifer Spiroloculina hyaline Schultz. Journal of Foraminiferal Research, v. 10, p. 89-101. Banerji, R. K., 1990, Heavy metals and benthic foraminifera l distribution along Bombay coast, India. Benthos, p. 151-157. Bock, W. D., 1971, A handbook of the benthoni c foraminifera of Florida Bay and adjacent waters, in Jones, J. I. and Bock, W. D. (eds.), A Symposium of Recent South Florida Foraminifera, Miami Geological Society, Memoir 1, p. 1-72. Braiser, M. D., 1975, Morphology and habitat of living benthonic foraminiferids from the Caribbean carbonate environm ents. Revista Espanola de Micropaleontologia, v. 7, no. 3, p. 567-578. Callahan, M. K., 2005, Distribution of Clionid Sponges in the Florida Keys Marine Sanctuary, (FKMS) 2001-2003, Unpublished Masters Thesis, University of South Florida, 88 p. Chazottes, V., Alsumard, L. C. T., Clausade M. P., and Cuet, P., 2002, The effects of eutrophication-related alterations to coral reef communities on agents and rates of bioerosion: Coral Reefs, v. 21, no. 4, p. 375-390. Clarke K. R., Warwick, RM (2001) Change in marine communities: an approach to statistical analysis and in terpretation. PRIMER-E, Plymouth Cockey, E. M., Hallock, P. M. and Lidz, B H., 1996, Decadal-scale changes in benthic foraminiferal assemblages off Key Largo, FL. Coral Reefs, v. 15, p. 237-248.

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104 Cottey, T. L. and Hallock, P., 1988, Shell surface degradation in Archaias angulatus Journal of Foraminiferal Research, v. 18, p. 187-202. Crevison, H. L. and Hallock, P., 2001, Fora minifera as Bioindicators: Key Subtropical Western Atlan tic and Caribbean Taxa, cd ROM, University of South Florida, St. Petersburg, Florida Crevison, H. and Hallock, P., 2007, Anomalous features observed on the shells of live Archaiasine foraminifers from the Fl orida Keys. Journal of Foraminiferal Research v. 37, no. 3, p. 223-233 Crevison, H. L. and Hallock, P., and McRae, G., 2006, Sediment cores from the Florida Keys (USA): is resolution suffi cient for environmental applications? Journal of Environmental Mi cropaleontology, Microbiology, and Meiobenthology, v. 3, p. 61-82. Debenay, J. P., Guillou, J. J., Geslin, E., and Lesourd, M., 2000, Crystallization or calcite in foraminiferal shells. Mi cropaleontology, 46 Supplement v. 1, p. 8794. Debenay, J. P., Geslin, E., Eichler, B. B., Duleba, W., Sylvestre, F., and Eichler, P., 2001, Foraminiferal assemblages in a hypersaline lagoon, Araruama (R.J.) Brazil: Journal of Foraminiferal Research, v. 31, no. 2, p. 133-151. Dodge, R. E. and Vaisnys, J. R., 1977. Co ral populations and growth patters: responses to sedimentation and turbidity associated with dredging. Journal of Marine Research, v. 35, p. 715-730. Dustan, P., 1999. Coral reefs under stress: so urces of mortality in the Florida Keys. Natural Resources Forum, v. 23, p. 147-155. Dustan, P., and Halas, J. C., 1987, Changes in the reef-coral comm unity of Carysfort Reef, Key Largo, Florida-1974-1982: Coral Reefs, v. 6, no. 2, p. 91-106. Engel, A., Zondervan, I., Aerts, K., Beaufo rt, L., Benthien, A., Chou, L., Delille, B., Gattuso, J. P., Harlay, J., Heeman, C., Ho ffman, L., Jacquet, S., Nejstgaard, J., Pizay, M. D., Newell, E. R., Schneider, U., Terbrueggen, A. and Riebesell U., 2005, Shelling the direct effect of CO2 concentration on a bloom of the coccolithophorid Emiliania huxleyi in mesocosm experiments: Limnology and Oceanography, v. 50, no. 2, p. 493-507. Erez, J., 2003, The source of ions for biom ineralization in foraminifera and their implications for paleoceanographic proxies. In Biominieralization, P. M. Dove, J. J. De Yoreo and S. Weiner, ed s. Mineralogical Society of America v. 54, Washington D. C.

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105 Fichtel, L. and Moll, J. P. C., 1798, Sh ellacea microscopica aliaqueminuta ex generibus Argonauta et Nautilus ad natu ram picta et descripta: Pichler, Vienna, 123 p. Francini, R.B., Moura, R.L., Fabiano, L ., Thompson, C., Reis, R.M., Kaufman, L., Kikuchi, R.K, and Leao, Z., 2008, Diseases leading to accelerated decline of reef corals in the largest South Atlan tic reef complex (Abrolhos Bank, eastern Brazil). Marine Pollution Bulletin, v. 56, no. 5, p. 1008-1014. Furbish D.J., and Arnold, A. J., 1997, Hydrodynamic strategies in the morphological evolution of spinose planktonic foramini fera. Geological Society of America Bulletin, v. 109, no. 8, p 1055-1072. Gattuso, J. P., Frankignoulle, I., Bourge, S., Romaine, S., and Buddemeier, R. W., 1998, Effect of calcium carbonate sa turation of seawater on coral calcification. Global Planetary Change, v. 18, p. 37-46. Geslin, E., Debenay, J. P., Delubia, W., and Bonetti, C., 2002, Morphological abnormalities of foraminiferal shells in Brazilian environments: a comparison between polluted and non-polluted ar eas. Marine Micropaleontology, v. 45, p. 151-168. _____, Debenay, J. P., Lesourd, M., 1998, Abnormal wall textures and shell deformation in Ammonia (hyaline foraminfer). Journal of Foraminiferal Research, v. 28, p. 148-156. Gladfelter, W. B., 1982, White-band disease in Acropora palmata : implications for the structure and growth of shallow reef s. Bulletin of Marine Science, v. 32, p. 639-643. Glynn, P. W., 1984, Widespread coral mort ality and the 1982-1983 El Nio warming event. Environmental Conservation, v. 11, p. 133-146. _____, 1996, Coral reef bleaching: facts, hypothesis, and implications. Global Change Biology, v. 2, p. 495-509. Griffin, D. W., Gibson III, C. J., Lipp, E. K., Riley, K., Paul III, J. H., and Rose, J. B., 1999, Detection of viral pathogens by reverse transcriptase of PCR and of microbial indicators standard methods in the canals of the Florida Keys. Applied Environmental Microbiology, v. 65, p. 4118-4125. Grigg, R. W., and Dollar, S. J., 1990, Natu ral and anthropogenic disturbance on coral reefs, in Coral Reefs (ed. Dubinsky, Z.), pp. 439-452. Elsevier, Amsterdam.

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106 Hallock, P., 1979. Trends in shell shape with depth in large, symbiont-bearing Foraminifera. Journal of Foraminiferal Research, v. 9, no. 1, p. 61-69. _____, 1981, Production of carbonate sedime nts by selected large benthic foraminifera on two Pacific coral reefs. Journal of Sedimentary Petrology, v. 51, p. 467-474. _____, P., 1997, Reefs and reef limestones in earth history in Life and Death of Coral Reefs, Chapman Hall, p. 13-42. _____, 2000, Larger foraminifera as indi cators of coral-reef vitality, in Marine Micropaleonotolgy, 15: 121-150. _____, 2001, Coral reefs, carbonate sediment s, nutrients, and global change, in The History and Sedimentology of Ancient Reef Systems, Kluwer Academic Press, New York, p. 387-427. _____, Cottey, T. L., Forward, L. B., and Halas, J., 1986, Population biology and sediment production of Arch aias angulatus (Foramin iferida) in Largo Sound, Florida. Journal of Foraminiferal Research, v. 16, p.1-8. _____, Forward, L. B., and Hansen, H. J., 1986. Influence of environment on the shell shape of Amphistegina Journal of Foraminiferal Research, v. 16, no. 3, p. 224-231. _____, Lidz, B. H., Cockey-Burkhard, E. M., and Donnelly, K. B., 2003, Foraminifera as bioindicators in cora l reef assessment and monitoring: The FORAM Index: Environmental Mon itoring and Assessment, v. 81, no. 1-3, p.221-238. _____, Muller-Karger, F. E., and Halas, J. C., 1993. Coral reef decline. National Geographic Research, and Exploration, v 9, p. 358-378. _____, Talge, H. K., Cockey, E. M., and Mull er, R. G., 1995, A new disease in reefdwelling foraminifera: implications fo r coastal sedimentation. Journal of Foraminiferal Research, 25: 280-286. Hardie, L. A., 1996, Secular variation in seaw ater chemistry: An explanation for the coupled secular variation in the mineralo gies of marine limestones and potash evaporites over the past 600 my. Geology, v. 24, p. 279-283. Harney, J. N., Hallock, P., Fletcher III C. H., and Richmond, B., 1999, Standing crop and sediment production of reef -dwelling foraminifera on O’ahu, Hawai’i. Pacific Science, v. 53, p. 61-73.

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107 Hay, M. E., and Kubanek, J., 2002, Community and ecosystem level consequences of chemical cues in the plankton: Journal of Chemical Ecology, v. 28, no. 10, p. 2001-2016. Hein, M., and Sand-Jansen, K., 1997, CO2 increases oceanic primary production: Nature, v. 388, no. 6642, p. 526-527. Hoegh-Guldberg, O., 1999, Climate Change, co ral bleaching, and the future of the world’s coral reefs. Marine a nd Freshwater Resources, v. 50, p. 839-866. Hoegh-Guldberg, O., 2004, Coral reefs in a cen tury of rapid environmental change. Symbiosis, v 37, p. 1-31 Kearns, K. D., and Hunter, M. D., 2000, Gr een algal extracellula r products regulate antialgal toxin production in a cyanob acterium: Environmental Microbiology v. 2, no. 3, p. 291-297. Kleypas, J. A., Buddemeir, R. W., Ar cher, D., Gattuso, J. P., Langdon, C., and Opdyke, B. N., 1999, Geochemical conse quences of increased atmospheric carbon dioxide on coral reefs: Science, v. 284, no. 5411, p. 118-120. _____, Feely, R. A., Fabry, V. J., Langdon, C., Sabine, C. L., and Robbins, L. L., 2006, Impacts of Ocean Acidification on Coral Reefs and Other Marine Calcifiers: A Guide for Future Resear ch: Report of a workshop held 18–20 April 2005, St. Petersburg, FL, sponsored by NSF, NOAA, and the U.S. Geological Survey, 88 p. Langdon, C., Takahashi, T., Sweeny, C., Chip man, D., Goddard, J., Marubini, F., Aceves, H., Barnett, H., and Atkins on, M. J., 2000, Effect of calcium carbonate saturation state on the calcification rate of an experimental coral reef: Global Biogeochemical Cycles, v. 14, no. 2, p. 639-654. _____, Broecker, W. S., Hammond, D. E., Gle nn, E., Fitzsimmons, K., Nelson, S. G., Peng, T. H., Hajdas, I., and Bonani G., 2003, Effect of elevated CO2 on the community metabolism of an experiment al coral reef: Global Biogeochemical Cycles, v. 17, no. 1, p. 1101-1114. _____, and Atkinson, M. J., 2005, Effect of elevated pCO2 on photosynthesis and calcification of corals and inter action with seasonal change and temperature/irradiance and nutrient enrichment: Journal of Geophysical Research, v. 110, no. c9, p. 1-16. Langer, M. R., 1993, Epiphytic Foraminifera. Marine Micropaleontology, v. 20, p. 235-265.

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108 Lapointe, B. E., Barile, P.J., and Matz ie, W. R., 2004, Anthropogenic nutrient enrichment of seagrass and coral reef communities in the Lower Florida Keys: Discrimination of local versus regi onal nitrogen sour ces: Journal of Experimental Marine Biology and Ecology, v. 308, no. 1, p. 23-58. Lea, D.W. and Boyle, E.A., 1989, Barium c ontent of benthic foraminifera controlled by bottom water composition. Nature, v. 338, p. 751-753. Le Cadre, V., Debenay, J. P., and Les ourd, M., 2003, Low pH effects on Ammonia beccarii shell deformations: implications for using shell deformation as a pollution indicator. Journal of Fo raminiferal Research, v. 33, p. 1-9. Lee, J. J. and Bock, W. D., 1976, The importa nce of feeding in two species of soritid foraminifera with algal symbionts. Bu lletin of Marine Science, c. 26, p. 530537. Lidz, B. and Rose, P. R., 1977, Diagnostic foraminifera assemblages of Florida Bay and adjacent shallow waters. Bullet in of Marine Science, v. 44, p. 399-418. Lipps, J., 1973, Shell structure in forami nifera. Annual Review of Microbiology, v. 27: p. 471-488. Loeblich, A. R. and Tappan, H., 1988. Foramini feral Genera and their Classification. New York, Van Nostrand Reinhold, 970 pp. MacIntyre, I. G., and Rei d, R. P., 1998. Recrystalliz ation in living porcelaneous foraminifera (Archaias angulatus): textural changes without mineralogic alteration. Journal of Sedimentary Research, v. 68, p. 11-19. Marshall, P. R., 1976, Some relationships between living and total foraminifera faunas on Pedro Bank, Jamaica, in Schafer, C. T., and Pelletier, B. R. ( eds. ), First Symposium on Benthic Foraminifera of Continental Margins. Part A, Ecology and Biology: Maritime Sedime nts Special Publication 1, p. 61-70. Martin, R. E., 1986, Habitat and distribution of the foraminifer Archaias angulatus (Fichtel and Moll) (Miliolina, Soritid ae), northern Florida Keys. Journal of Foraminiferal Research, v. 16, p. 201-206. Marszelak, D. S., 1969, Aspects of chamber formation by Archaias angulatus a foraminifer (Recent). Ph.D. Thesis, University of Illinois, Urbana. McConnaughey, T. A., 1994, Calcification, photo synthesis, and global carbon cycles. Bulletin de l’Institut Oceanographi que, Monaco, Special 13, p. 137-161.

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110 Porter, J.W., Kosmynin, V., Patterson, K., Porter,K. G., Jaap, W. C., Wheaton, J., Hackett,K. E., Lybolt, M., et al., 2002, Detection of coral reef change by the Florida Keys coral reef monitoring proj ect, in The Everglades, Florida Bay, and coral reefs of the Florida Keys: An Ecosystem Sourcebook, Porter, J. and Porter, K., Eds., CRC Press, Boca Raton. pp. 749-769. Precht, W. F., and Miller, S. L., 2006, Ecol ogical shifts along the Florida reef tract: the past as a key to the future, in Aronson, R. B. (ed.), Geological Approaches to Coral Reef Ecology, Springe r Verlag, New York, p. 237-312. Riebesell, U., 2004, Effects of CO2 enrichment on marine phytoplankton: Journal of Oceanography, v. 60, no. 4, p. 719-729. _____, Zondervan, I., Rost, B., Tortell, P. D., Zeebe, R. E., and Morel, F. M. M., 2000, Reduced calcification of marine phyt oplankton in response to increased atmospheric CO2: Nature, v. 407, no. 6802, p. 364-367. Rose, P. R. and Lidz, B., 1977, Diagnostic foraminiferal assemblage s of shallow water modern environments: South Florida and Bahamas: Sedimenta VI, University of Miami, Miami, Florida, 55 p. Russell, A.D., Honisch, B., Spero, H. J., Lea, D. W., 2004, Effects of seawater carbonate ion concentration and temper ature on shell U, Mg, and Sr in cultured planktonic Foraminifera. Geochmica et Cosmochimica Acta, v. 68, no. 21, p. 4347-4361. Samir, A. M., 2000, The response of benthic foraminifera and ostracods to various pollution sources: as study from two lagoons, Egypt. Journal of Foraminiferal Research, v. 30, p. 83-98. _____., and El-Din, A. B., 2001, Benthic foraminiferal assemblages and morphological abnormalities as polluti on proxies in two Egyptian bays. Marine Micropaleontology, v. 41, p. 193-227. Santavy, D. L., Summers, J. K., Engle, V. D., and Harwell, L. C., 2005, The condition of coral reefs in South Fl orida (2000) using coral disease and bleaching as indicators. Environmen tal Monitoring and Assessment, 100: 129-152. Saraswat, R., Kurtarkar, S. R., Mazumder A., and Nigam, R., 2004, Foraminfers as indicators of marine pollution: a cu lture experiment with Rosalina leei. Marine Pollution Bulletin, v. 48, p. 91-96.

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111 Sen Gupta, B. K., 1999, Systema tics of modern foraminifera. In Modern Foraminifera, B. K. Sen Gupta (ed.). Kluwer Academic Press, Great Britain, p. 7-36. Sharifi, A. R., Croudace, I. W., and Aus tin, R. L., 1991, Benthic foraminiferids as pollution indicators in South Hampton water, Southern England, United Kingdom: Journal of Micropaleontology, v. 10, p. 109-113. Silva, M. C., McClanahan, T. R., and Kiene, W. E., 2005, The role of inorganic nutrients and herbivory in contro lling microbioero sion of carbonate substratum: Coral Reefs, v. 24, no. 2, p. 214-221. Stouff, V., Debenay, J. P., Lesourd, M., 1999a, Origin of double and multiple shells in benthic foraminifera: observations in laboratory cultures. Marine Micropaleontology, v. 36, p. 189-204. _____, Geslin, E., Debenay, J. P., Lesour d, M., 1999b, Origin of morphological abnormalities in Ammonia (foraminifera): studies in laboratory and natural environments. Journal of Foraminiferal Research, v. 29, p. 152-170. SWFWMD website, 2008, http:// www.swfwmd.state.fl.us/data/ Szmant, A. M., 2002, Nutrient enrichment on co ral reefs: is it a major cause of coral decline?: Estuaries, v. 25, no. 4b, p. 743-766. Szmant, A. M., and Forrester, A., 1996, Water column and sediment nitrogen and phosphorus distribution patterns in the Fl orida Keys, USA: Coral Reefs, v. 15, no. 1, p. 21-41. Talge, H.K., Hallock, P., 1995. Cytological ex amination of symbiont loss in a benthic foraminifera, Amphistegina gibbosa. Marine Micropa leontology, v. 26, p. 107–113. Talge, H. K. and Hallock, P., 2003, Ultrastr uctural responses in field-bleached and experimentally stressed Amphistegina gibbosa (Class Foraminifera). Journal of Eukaryotic Microbiology, v. 50, p. 324-333. Talge, H.K., Williams, D.E., Hallock, P., Harney, J.N., 1997. Symbiont loss in reef foraminifera: consequences for affected populations. Proc. 8th Int. Coral Reef Symp., Panama City 1, 589–594. Ter Kuile, B., 1991, Mechanisms for calci fication and carbon cycling in algal symbiont-bearing, in Lee, J. J. and Anderson, O. R. (eds.), Biology of Foraminifera, Academic Press, New York, p. 73-89.

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113 Williams, D. E. and Hallock, P., Talge, H. K., Harney, J. H., and McRae, G., 1997, Responses of Amphistegina gibbosa populations in the Florida Keys (USA) to a multi-year stress event (1991-1996). J ournal of Foraminiferal Research, v. 27, p. 264-269. _____ and Hallock, P., 2004, Bleaching in Amphistegina gibbosa d’Orbigny (Class Foraminifera): observations from la boratory experiments using visible and ultraviolet light. Marine Biology, v. 145, p. 641-649. Williams, E. H. and Bunkley-Williams, L., 1990, The world-wide coral reef bleaching cycle and related sources of coral mortality. Atoll Research Bulletin, v. 335, p. 1-71. Wolfe, G. V., 2000, The chemical defense eco logy of marine unicellular plankton: constraints, mechanisms, and impact s: Biological Bulletin, v. 198, no. 2, p. 225-244. Wright, R. C. and Hay, W. W., 1971, The abundance and distribution of foraminifers in a back-reef environment Molasses Reef Florida, in Jones, J. I. and Bock, W. D. (eds.), A Symposium of Recen t South Florida Foraminifera, Miami Geological Society, Memoir v. 1, p. 121-174. Yang, Y., and Gao, K., 2003, Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta): Journal of Applied Phycology, v. 15, no. 5, p. 1-11. Yanko, V., Kronfeld, J. and Flexer, A., 1994, Response of benthic foraminifera to various pollution sources: implicati ons for pollution monitoring. Journal of Foraminiferal Research, v. 24, p. 73-97. _____, Ahmed, M., and Kaminski, M., 1998, Mo rphological deformities of benthic foraminiferal shells in re sponse to pollution by heavy me tals: implications for pollution monitoring. Journal of Fora miniferal Research, v. 28, no. 3, p. 177200. _____, Arnold, A. J., and Parker, W. C., 1999, Effects of marine pollution on benthic foraminifera. In Modern Foraminifera. Kluwer Academic Publishers, p. 217238. Yates, K. K., and Halley, R. B., 2003, M easuring coral reef community metabolism using new benthic chamber technol ogy. Coral Reefs, v. 22, p. 247-255.

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115 APPENDICES

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116 Appendix A: Detailed Desc ription of Morphological and Textural Anomalies Found on Archaias Angulatus from The Florida Keys Microborings Some microborings were straight while others were curved and they often formed dense networks with a dendritic appearance. They were about 1 m wide and up to 50 m in length. They had fairly smooth edges and avoided contact with pseudopores. Some individuals were completely covered with mi croborings while others exhibited sporadic smaller patches. Surface Pitting Surface pits were variable in appearance. Some pits were as small as 25 m and circular in shape with ragged edges. Others looked like sink holes on the surface of the shells, and when they coalesced into large pockmarks (100 m), a crumbly appearance was evident. Pitting was often found in co mbination with dissolution giving a smooth polished look to the pits Microbial Biofilm The bacteria were capsule-shaped and approxi mately 1 m in length. In some areas, bacteria looked melted to the shell surface. Dissolution Dissolution looked highly vari able. Some specimens exhibited extremely shallow pseudopores, so shallow that the bottoms of the pseudopores were visible. In other individuals, the outer layer of rhombohedral plates was removed exposing the underlying layer of randomly arrange calcite needles. Di fferential dissolution was also present. The outer layer of rhombohedral plates and some of the calcite needles were dissolved away allowing the pseudopores to look raised. Cons equently, the surface of the shells looked as though they were covered in donuts. Epibionts Bryzoan and foraminiferal growth on the su rface of the shells was sparse. Bryzoans appeared encrusting and l obate and were about 150 m maximum length. The foraminifers were small rotaliid species about 100 m diameter. Cyanobacteria were long filaments and formed a dense network on the surface of the shell, so dense the pseudopores were often obscured. Irregular Sutures Irregular sutures were anything that deviated from the typical straight concentric geometry. They included suture lines that merged together or bi furcated, were wiggly, dense, or stopped and started suddenly. If pr esent on one side of the shell, they usually were not present along the same suture line on the other side. Irregular sutures mainly appeared on the outer rows of ch amberlets of larger individuals.

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117 Curling Curled shells exhibited a wide range in mor photypes. Some individuals were very curled and bowl-shaped. Other individuals ha d undulating margins whereas other looked creased and warped to one side. The outer rows of chamberlets of la rger individuals were more severely curled than juveniles. Asymmetry Asymmetrical individuals had a trochospiral instead of the normal planispiral geometry. These individuals were flat on one side and convex on the other. Profoundly deformed Profoundly deformed individuals were highly variable in appearance. They had no planispiral characteristics what so ever a nd possessed obscured apertures. They were often spheroid in shape. Uncoiled These individuals were characterized by a norma l juvenile portion of the shell. However as rows of chamberlets were added, the i nvolute characteristics were lost and the individuals looked long, slender, and uncoiled. Surface Blips Surface blips were structural, not precipitated structures that prot ruded from the surface of the shell. They varied in length from 250-500 m in length and were often in combination with irregular sutures. Mineralogic Projections Mineralogic projections prot ruded from the pseudopores. Th ese protrusions varied in length (about 5-25 m) and girth, and some app eared slightly curved while others were straight. Crystal faces were visibl e on nearly all of the projections. Lacy crust The lacy crust was a build up on the surface of the test. The crust appeared thicker in some areas, completely obscuring the pse udopores, whereas in other areas, pseudopores could be discerned through the lacy outer layer. It look ed structural rather than something that had prec ipitated on the surface. Breakage and repair Breakage and repair looked as if the surface of the shell was a patchwork, so the interface between the original shell and th e area of repair was very ra gged. In some individuals, large pits within a single row of chamberlets were repaired by regrowth from a different row of chamberlets. Consequently, areas of these individuals look smeared.

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About the Author Heidi Crevison Souder received and BA in art from Ohio Domincan College in 1990 and MS in marine science from the Univ ersity of South Florida in 2001. She has been involved in conservation, education and outreach and considers herself a champion for the environment. After entering the Ph.D. program at the University of South Florida in 2004, she has continued educate the public about climate change and ocean science issues. She was an NSF GK-12 Fellow and taught earth, life and physical science to middle school students and has authored several science and ed ucation papers. She is a member of the Geological Society of America and the National Science Teachers Association.


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ABSTRACT: Historically, Archaias angulatus has been a major contributor to foraminiferal assemblages and sediments in coral-reef environments throughout the Caribbean and tropical Atlantic. A variety of anomalous features were observed in the tests of A. angulatus individuals collected live from the Florida reef tract in 2004 and 2005. Six types of anomalies were documented using scanning electron microscopy: microborings, microbial biofilm, pitted surfaces, dissolution, calcification abnormalities, and growth abnormalities. Calcification abnormalities included mineralogical projections, lacy crusts, and repair marks. These abnormalities were found among both juvenile and adult A. angulatus, and similar features were also found among Cyclorbiculina compressa and Laevipeneroplis proteus specimens collected live in the same samples. In 2006, a comprehensive study was undertaken to see if the occurrence and types of morphological abnormalities have changed in A. angulatus from the Florida Keys over the past 2.5 decades.Archived samples of A. angulatus collected in 1982-83 from John Pennekamp Coral Reef State Park were compared to recent samples. Seven different types of morphological abnormalities and 5 different surface texture anomalies were documented. Eighty-six combinations of abnormalities and surface textures were observed. Physical abnormalities included profoundly deformed, curled, asymmetrical, and uncoiled tests, irregular suture lines, surface "blips," and breakage and repair. Surface texture anomalies included surface pits, dissolution, microborings, microbial biofilm, and epibiont growth. Epibiont growth included bryzoans, cyanobacteria and foraminifers. The archived samples were not obviously more pristine than the recent samples indicating stress was well underway in the early 1980s. Test strength was compromised in deformed specimens.Crushing strength of abnormal individuals was much more variable compared to individuals with irregular sutures and normal specimens. Deformed individuals also exhibited abnormal test wall structure including dissolution and infilling. Mg/Ca ratios for normal and deformed specimens were within normal parameters (12-15 mol/mol). Implications of these observations are at least twofold. First, in studies of fossil assemblages, damage to tests and changes in test-surface textures should not be assumed to have occurred postmortem, and may provide evidence of environmental stressors acting upon living populations. In addition, we speculate that test dissolution in larger miliolid foraminifers when alive can indicate declining carbonate saturation in seawater, which can result locally from salinity changes or increasing benthic respiration rates, as well as globally from rising concentration of atmospheric CO.
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