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Soil Phosphorus Characteristics and Sources in Tree Islands of the Florida Everglades

Permanent Link: http://ufdc.ufl.edu/UFE0044860/00001

Material Information

Title: Soil Phosphorus Characteristics and Sources in Tree Islands of the Florida Everglades
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Irick, Daniel L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: everglades -- islands -- phophorus
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In many cases, soil phosphorus (P) in tree islands within the Everglades greatly exceeds concentrations of the surrounding P-limited marsh.  The magnitude of P contribution from specific sources to tree island soil is unknown.  This work was conducted to determine tree island soil P characteristics and assess potential nutrient contribution from animal wastes to tree islands.  Surface soils, bird guano, and animal bones were analyzed for chemical composition and mineral presence.  Soils and guano were analyzed for distribution of P pools by sequential chemical extraction and isotopic signature.  Soils were also physically fractionated and particle size fractions were analyzed for mass distribution, elemental composition, P pools, and soil minerals.  Total P concentrations of soils ranged from 0.05 – 8.81%.  Calcium bound P accounted for the majority (~85%) of total soil P in tree islands with elevated P concentrations.  Apatite was identified in soils with elevated P concentration suggesting an external source of P minerals.  Most (82 to 97%) of the P present in guano was also extractable by HCl. Guano had an average d15N value of 8.8‰.  Phosphorus concentrations and d15N were positively correlated in tree island soils (r = 0.95).  Deposition of guano-derived P and N can occur at rates much higher than other P and N sources at some tree island locations in the Everglades suggesting deposition of guano contributes to the accumulation of P at some locations.  Some tree island soils also contained bone fragments.  The P concentration of the sand sized soil was ~10% and more than twice the concentration of the silt+clay particle size fraction.  The presence of apatite and the P concentration in the sand sized soil indicated bioapatite may account for ~65% or more of the total P in the high P tree island soils. The source of bioapatite remains unresolved, but was likely derived from animal wastes due to wildlife utilization and historic anthropogenic occupation.  Animal waste derived P appears to potentially contribute large amounts of P to tree island soil.  The magnitude of these inputs will vary by island and stability of the material deposited will influence P accumulation in tree islands.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniel L Irick.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Li, Yuncong.
Local: Co-adviser: Inglett, Patrick.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044860:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044860/00001

Material Information

Title: Soil Phosphorus Characteristics and Sources in Tree Islands of the Florida Everglades
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Irick, Daniel L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: everglades -- islands -- phophorus
Soil and Water Science -- Dissertations, Academic -- UF
Genre: Soil and Water Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In many cases, soil phosphorus (P) in tree islands within the Everglades greatly exceeds concentrations of the surrounding P-limited marsh.  The magnitude of P contribution from specific sources to tree island soil is unknown.  This work was conducted to determine tree island soil P characteristics and assess potential nutrient contribution from animal wastes to tree islands.  Surface soils, bird guano, and animal bones were analyzed for chemical composition and mineral presence.  Soils and guano were analyzed for distribution of P pools by sequential chemical extraction and isotopic signature.  Soils were also physically fractionated and particle size fractions were analyzed for mass distribution, elemental composition, P pools, and soil minerals.  Total P concentrations of soils ranged from 0.05 – 8.81%.  Calcium bound P accounted for the majority (~85%) of total soil P in tree islands with elevated P concentrations.  Apatite was identified in soils with elevated P concentration suggesting an external source of P minerals.  Most (82 to 97%) of the P present in guano was also extractable by HCl. Guano had an average d15N value of 8.8‰.  Phosphorus concentrations and d15N were positively correlated in tree island soils (r = 0.95).  Deposition of guano-derived P and N can occur at rates much higher than other P and N sources at some tree island locations in the Everglades suggesting deposition of guano contributes to the accumulation of P at some locations.  Some tree island soils also contained bone fragments.  The P concentration of the sand sized soil was ~10% and more than twice the concentration of the silt+clay particle size fraction.  The presence of apatite and the P concentration in the sand sized soil indicated bioapatite may account for ~65% or more of the total P in the high P tree island soils. The source of bioapatite remains unresolved, but was likely derived from animal wastes due to wildlife utilization and historic anthropogenic occupation.  Animal waste derived P appears to potentially contribute large amounts of P to tree island soil.  The magnitude of these inputs will vary by island and stability of the material deposited will influence P accumulation in tree islands.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Daniel L Irick.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Li, Yuncong.
Local: Co-adviser: Inglett, Patrick.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044860:00001


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1 SOIL PHOSPHORUS CHARACTERISTICS AND SOURCES IN TREE ISLANDS OF THE FLORIDA EVERGLADES By DANIEL LYLE IRICK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREM ENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Daniel L yle I rick

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3 To my f amily and f riends

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4 ACKNOWLEDGMENTS I would like to thank Dr. Yuncong Li, who provided a balance o f patience, encouragement and challenge throughout my graduate studies. He recognized and fostered my potential as a scientist, making it possible for me to compile this dissertation. I thank Dr. Patrick W. Inglett for his continued guidance, willingness to openly discuss my work and the motivation he provided to advance this research This work would not have been possible without the help and direction I received from Dr. Binhe Gu, and I am thankful for many discussions we had about this research. I also thank other members of my supervisory committee; Drs. Kati Migliaccio, Michael Ross, and Alan Wright for their insight and direction. Drs. Willie Harris and Peter Frederick provided advice on many elements of this research and I am greatly appreciati ve of their help. I thank Ms. Guiqin Yu and Dr. Yigang Lou for their help with laborat ory navigation and analysis. I am grateful to my parents family and f riends for their support and encouragement. F ortunate ly, I have had a great companion with me dur ing this journey I thank Kelly Roberts for her strength and the foundation she provided for me to strive to successfully complete this dissertation.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 BACKGROUND AND OBJECTIVES ................................ ................................ ...... 12 Tree Islands in the Everglades ................................ ................................ ............... 13 Background: Island Characteristics and Classification ................................ ..... 13 Habitat Reduction ................................ ................................ ............................. 15 Soil Phosphorus ................................ ................................ ............................... 16 Research Objectives ................................ ................................ ............................... 21 2 SOIL PHOSPHORUS CHARACTERISTICS ................................ .......................... 28 Introduction ................................ ................................ ................................ ............. 28 Materials and Methods ................................ ................................ ............................ 30 Site Descriptions and Locations ................................ ................................ ....... 30 Elemental Analysis ................................ ................................ ........................... 31 Phosphorus Fractionation ................................ ................................ ................. 32 Mineralogical Analysis ................................ ................................ ...................... 33 Statistical Analysis ................................ ................................ ............................ 33 Results and Discussion ................................ ................................ ........................... 34 Soil Chemical Characterization ................................ ................................ ........ 34 Soil P Fractionation ................................ ................................ .......................... 37 Soil Mineral Identification ................................ ................................ .................. 40 Conclusion s ................................ ................................ ................................ ............ 43 3 WADING BIRD GUANO ENRICHMENT OF SOIL NUTRIENTS IN TREE ISLANDS ................................ ................................ ................................ ................ 54 Introduction ................................ ................................ ................................ ............. 5 4 Materials and Methods ................................ ................................ ............................ 57 Site Descriptions and Locations ................................ ................................ ....... 57 Soil and Guano Collection ................................ ................................ ................ 58 Elemental and isotopic Analysis ................................ ................................ ....... 58 Phosphorus Fractionation ................................ ................................ ................. 59 Guano Aging Exp eriment ................................ ................................ ................. 59 Elemental Mass Deposition Calculation ................................ ........................... 60 Atmospheric Deposition Data ................................ ................................ ........... 60 Statistical Analysis ................................ ................................ ............................ 61

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6 Results and Discussion ................................ ................................ ........................... 61 Guano Characterization ................................ ................................ ................... 61 Guano P Fractionation ................................ ................................ ..................... 63 15 N ................................ ........ 66 Tree Island Soil N and P Sources ................................ ................................ .... 67 Nutrient Deposition ................................ ................................ ........................... 69 Conclusions ................................ ................................ ................................ ............ 72 4 BIOAPATITE CONTRIBUTION TO SOIL PHOSPHORUS IN TREE ISLANDS ...... 82 Introduction ................................ ................................ ................................ ............. 82 Materials and Methods ................................ ................................ ............................ 84 Sample Collection and Site Description ................................ ........................... 84 Elemental Analysis and Inorganic Nutrients ................................ ..................... 85 Particle Si ze Separation ................................ ................................ ................... 86 Phosphorus Fractionation ................................ ................................ ................. 86 Mineralogical and Micro elemental Analysis ................................ ..................... 87 Statistical Analysis ................................ ................................ ............................ 87 Results and Discussion ................................ ................................ ........................... 88 Soil Chemical Characterization ................................ ................................ ........ 88 Particle Size Separation: Chemical Characteristics, P Forms and Minerals ..... 90 Bioapatite Contribution ................................ ................................ ..................... 93 Conclusions ................................ ................................ ................................ .......... 100 5 SYNTHESIS AND FURTURE RESEARCH ................................ .......................... 109 Chapter Summaries ................................ ................................ .............................. 112 Chapter 2 Soil P Characteristics ................................ ................................ .. 112 Chapter 3 Wading Bird Contribution to Soil Nutrients ................................ .. 113 Chapter 4 Bioapatite Contribution to the Soil P Pool ................................ ... 114 Suggestions for Future Research ................................ ................................ ......... 115 Nutrient Transport and Fate ................................ ................................ ........... 116 Cation accumulation ................................ ................................ ................ 116 Nitrogen loss from wading bird colonies ................................ .................. 116 Phosphorus Stability ................................ ................................ ....................... 116 Soil ................................ ................................ ................................ ........... 116 Animal wastes ................................ ................................ .......................... 117 APPENDIX : INVESTIGATION OF STABLE CARBON ISOTOPES IN BIOAPATITE AND SOIL ................................ ................................ ................................ ............. 119 LIST OF REFERENCES ................................ ................................ ............................. 125 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 138

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7 LIST OF TABLES Table page 1 1 Vegetation found within tree islands in the Everglades ................................ ...... 23 2 1 Sel ect soil properties from 26 tree islands with tropical hardwood hammock plant communities in the Florida Everglades. ................................ ..................... 45 2 2 Non order) for select tree island soil properties (n=26). ................................ ................................ ....... 46 2 3 Spearman correlation coefficients for water soluble phosphorus (WSP), 1.0 M KCl extractable P (KCl), 0.5 M NaOH extractable inorganic P (NaOH Pi) and organic P (NaOH Po), 0.5 M HCl extractable P (HCl), residual P ................ 47 3 1 Ch emical characteristics of wading bird guano (dry matter) from two colonies in the south central Everglades, Flor ida, USA. ................................ ................... 73 3 2 Stable nitrogen isotope ratio, total carbon, nitrogen and phosphorus weight based elemental ratios, and total phosphorus of fresh and aged bird guano, mammal scat, soil and pl ants (mean 1SD) from the Everglades, Florida ........ 74 3 3 Annual estimated mass deposition for six species of colonial wading birds that may nest or roost in tree islands in the Florida Everglades ........................ 75 4 1 Select soil properties from 22 high P (>10 g kg 1 ) tree islands with tropical hardwood hammock plant communities in the Florida Everglades. .................. 101 4 2 Select chemical properties of sand sized (2 mm 45 m), and silt+clay sized (<45 m) soil from tree islands tropical hardwood hammock plant communities in the Florida Everglades (n=3). Data represent mean 1 ........... 102 4 3 Molar ratio of calcium to phosphorus (Ca:P), and magnesium to P (Mg:P) for soil, animal bones, and different soil particle size classes (coarse fragments, sand, and silt+clay). Data represent mea n 1 s tandard deviation of the ........ 103

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8 LIST OF FIGURES Figure page 1 1 The geographic setting of the Florida Everglades, and general locations of the Water C onservation Areas (WCA 1, 2, and 3), and Everglades National Park (ENP). The yellow boundary is the estimated current extent of the ........... 24 1 2 A tree island in the southern Everglades. ................................ ........................... 25 1 3 The interior of a tree island in the southern Everglades where areas of different bedrock elevation are observable. ................................ ........................ 26 1 4 Three pr imary accumulation mechanisms described to influence soil phosphorus concentrations detected in tree island soils in the Florida Everglades and the general pattern of P redistribution within islands ................. 27 2 1 Site map depicting tree island locations with hardwood hammock plant communities where soil samples were collected in the Florida Everglades, USA. ................................ ................................ ................................ ................... 48 2 2 Depiction of soil phosph orus sequential chemical fractionation scheme. ........... 49 2 3 Distribution of phosphorus forms in tree island soil (0 10 cm). Bar values indicate mean and errors bars 1 SD. ................................ ............................... 50 2 4 Scatter plots depicting relationships between HCl extractable soil phosphorus concentration, and the concentrations of non carbon soil matter (NCM) and non carbonate calcium for tree island soil. Correlation coefficient ..................... 51 2 5 X ray diffraction patterns for particles <2 mm in size from three tree island soils (0 10 cm). The arrows indicate primary peaks for apatite and calcite. ...... 52 2 6 Stoichiometric ratios of total calcium (Ca) and non carbonate Ca to phosphorus (P) in comparison with non carbon matter and total phosphorus concentration in tree island soil (0 10 cm). Correlation coef ficient ..................... 53 3 1 Site map depicting tree island soil sample locations and general area of the bird colonies where samples were collected in the Florida Everglades, USA. .... 76 3 2 Picture of wading bird presence at an island in Water Conservation Area 3 in May 2011. ................................ ................................ ................................ ........... 77 3 3 Phosphorus form distribution in wading bird guano (a ll), and aged and fresh guano. Bar values indicate mean and errors bars 1 SD. ................................ 78

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9 3 4 15 N ) of aged and fresh guano while drying at 23 C. Data points represent mean value and errors bar indicate 1 standard deviatio n of triplicate sample analysis. .............. 79 3 5 Temporal variation in the total extractable NH 4 N aged and fresh gu ano while drying at 23 C. Data points represent mean value and errors bar indicate 1 standard deviation of triplicate sample analysis. Variation in aged and fresh .... 80 3 6 Temporal variation of the percentage of NH 4 N (inorganic N) of total N in aged and fresh guano while drying at 23 C. Data points represent mean value and errors bar indicate 1 standard deviation of triplicate sample ............ 81 4 1 Tree island soil sample locations in the Florida Everglades, USA. ................... 104 4 2 Soil phosphorus (P) forms of sand, and silt and clay particle size classes. The asterisks indicate sign ificant (P<0.05) differences between particle size classes. ................................ ................................ ................................ ............ 105 4 3 X ray diffraction (XRD) patterns for, (a) animal bones, (b) weather bone fragments, (c) sand sized soil, and (d) silt+clay s ized soil particles. ................. 106 4 4 Scanning Electron Microscopy (SEM) image and energy dispersive x ray spectroscopy (EDS) dot patterns for calcium and phosphorus for sand sized tree island soil. ................................ ................................ ................................ .. 107 4 5 Scatter plots of the relationships of the molar ratios of 1 M HCl extractable calcium to phosphorus (Ca:P) and magnesium to phosphorus (Mg:P) with 1 M HCl extractable phosphorus (Total I norganic P) and non carbon matter. ..... 108

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SOIL PHOSPHORUS CHARACTERISTICS AND SOURCES IN TREE ISLANDS OF THE FLORIDA EVERGLADES By Daniel Lyle Irick December 2012 Chair: Yuncong C. Li Cochair: Patrick W. Inglett Major: Soil and Water Science In many cases, s oil phosphorus ( P ) in tree islands within the Everglades greatly exceed s concentrations of the surrounding P limited marsh. T he magnitude of P contribution from specific sources to tree island soil is unknown This work was conducted to determine tree island soil P characteristics and ass ess potential nutrient contribution from animal wastes to tree island s Surface soil s bird guano, and animal bones were analyzed for chemical composition and mineral presence. Soils and guano were analyzed for distribution of P pools by sequential chemi cal extraction and isotopic signature Soils were also physically fractionated and p article size fractions were analyzed for mass distribution, elemental composition, P pools and soil minerals. Total P concentration s of soil s ranged from 0.0 5 8.81% Calcium bound P accounted for the majority (~85%) of total soil P in tree islands with elevated P concentrations Apatite was identified in soils with elevated P concentration suggesting an external source of P minerals Most (82 to 97%) of the P presen t in guano was also extractable by HCl. 15 Phosphorus 15 N were positively correlated in tree island soils (r = 0.95).

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11 Deposition of guano derived P and N can occur at rates much higher than other P and N sources at some tree island loca tions in the Everglades suggesting deposition of guano contribut es to the accumulation of P at some locations. Some tree island soils also contained bone fragments. The P concentration of the sand sized soil was ~10% and more than twice the concentratio n of the silt+clay particle size frac tion. The presence of apatite and the P concentration in the sand sized soil indicated b ioapatite may account for ~ 65 % or more of the total P in the high P tree island soils. The source of bioapatite remains unresolved but was likely derived from animal wastes due to wildlife utilization and historic anthropogenic occupation An imal waste derived P appears to potentially contribute large amounts of P to tree island soil. The magnitude of these inputs will vary by isl and and stability of the material deposited will influence P accumulation in tree islands

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12 CHAPTER 1 BACKGROUND AND OBJECTIVES The Florida Everglades is an expansive (~10,000 km 2 ) subtropical ecosystem in southern Florida ( Figure 1 1; Davis 1994) Or ganic soil overlaid upon limestone bedrock supports wetland and terrestrial habitat (Loveless, 1959; Leighty and Henderson, 1958) Near the southern boundary of the system salinity gradients influence transitional patterns of marine emergent marsh and for ested habitat, intermixed freshwater emergent marsh, and shrub and forested habitat (Davis et. al., 2005). E mergent marsh is the primary wetland habitat type in the freshwater Everglades (Loveless, 1959). Vegetative composition of the m arsh is predomina ntly Cladium jamaicense (Sawgrass ) which is interspersed with areas of open water supporting floating aquatic species such as Nymphaea odorata (water lilly ; Loveless, 1959; Davis, 1994). Although early descriptions of the Everglades often emphasized an expansive sawgrass dominated marsh, areas of woody vegetation, including shrub and tree species, were also denoted in early land and ecological surveys (Davis, 1943; Loveless, 1959). Plant communities dominated by woody species dispersed within the emerg ent freshwater and marine marsh wetlands of the Everglades ecosystem are collectively described a s tree islands (Loveless, 1959). Most recently Wetzel (2002) advanced the general classification of patches of woody vegetation in strict comparison to the f reshwater emergent marsh of Everglades landscape describing these vegetative communities as tree islands and noting these habitats also occur in other ecosystems around the world

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13 D escri ption of soil characteristics and ecological function of tree island communities in the Everglades have become the focus of many research efforts in the past 10 15 years. R esearch from some of these studies indicates habitat diversity, specifically the presence of forested vegetation communities in the freshwater Everglade s, is indicative of differential patterns of chemical and physical soil characteristics and development (Orem et al 2002; Jayachandran et al 2004; Wetzel et al., 2005 ; Ross and Sah, 2011) Genesis and ecological role of these features in the Everglade s remains a point of discussion. Tree Islands in the Everglades Background: Island Characteristics and Classification T ree islands are visually recognizable as patches of tree s and shrubs within the emergent marsh wetland ( Figure 1 2 ). A n increase in top ography relative to adjacent marsh habitat is another characteristic of most tree islands. Soil surface elevations of tree islands have been reported to approach, and in some cases exceed, 2 m above adjacent m arsh soil ( Wetzel et al., 20 09 ; Ross and Sah, 2011 ). Numerous definitions and classifications for tree islands of the Everglades have evolved through continued research. Geomorphologic based classifications include fixed tree islands, pop up or battery tree islands and a less discussed island type, strand islands (Sklar and v an der Valk, 2002). Ecological or community level classifications are based on vegetative species composition and can be an indication of hydroperiod (Armentano et al., 2002 ; Ruiz and Ross, 2004 ). Tree islands that are elonga ted in a generally north south orientation and have a teardrop shape, which is visible in aerial photography, are currently classified as fixed islands (Sklar and van der Valk, 2002). Formation of these islands is proposed to have

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14 occurred by establishmen t of woody vegetation on an area of elevated bedrock or an out crop ( Figure 1 3 ; Loveless, 1959; W illard et al., 2002). Paleoecological investigations have reported tree island presence in the Everglades may have initiated as early as 3, 5 00 years ago (Wil lard et al. 2002 ; Willard et al., 2006 ). Fixed tree islands are present from Water Conservation Area 2 ( WCA2 ) south through Everglades National Park ( ENP ) The predominant island type present in Water Conservation Area 1 ( WCA 1 ) is identified as a pop u p or battery island ( St one et al., 2002; Willard et al., 2002). The terms pop up or battery are intended to describe patches of peat that detached from the organic soil substrate because of an increase in buoyancy and therefore provide an elevated medium suitable for establishment of woody vegetation. The increased buoyancy of the peat is associated with a localized accumulation of gases produced under anaerobic conditions that result in an area that is less dense then the surrounding peat (Wetzel, 2002). Once an area of peat is detached from the submerged peat and surfaces the newly emerged, shorter hydroperiod peat presents opportunity for colonization by woody vegetative species. Stone et al. (2002) also identify another island type in WCA 1 named stra nd islands. Strand islands are geometrically similar to fixed tree islands however they are hypothesized to have initiated on former sawgrass ridges instead of elevated bedrock (Stone et al. 2002). Ecological classifications such as bayhead, bayhead swam p, willow head and tropical hardwood hammock refer to island types and components of the tree island vegetation communities. Bayhead communities primarily refer to islands with Persea borbonia (redbay) or P. palustris ( swamp bay ) plant communities present ( Stone et al. 2002 ; Ruiz and Ross, 2004 ). A willow head refers to a patch of woody vegetation

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15 dominated by Salix caroliniana ( coastal plain willow; Armentano et al. 2002). V egetation data compiled for bayhead, bayhead swamp and tropical hardwood hammoc k tree island communities is outlined in Table 1 1 (Ruiz and Ross, 2004; Wang et al., 2011; Saha et al., 2010) Habitat Reduction Estimation of tree island percent cover in the Everglades ranges from <1.0 9.6% in sites mapped in ENP, 3.8% within Water Cons ervation Area 3 ( WCA 3 ) and 13.7% in WCA 1 (Ruiz and Ross, 2004; Wetzel et al., 2005; Brandt et al., 2000). Area s comprised of shrub and hardwood swamp in WCA 2A has been reported at 4.4% (Rivero et al. 2009). Throughout the region tree islands are est imated to cover approximately 3 14% of the land surface (Wetzel et al., 2005; Wetzel et al., 2009; Willard et al. 2006). Loss of tree island habitat from 1940 to 1995 in WCA 3 has been reported at 67% and attributed to alteration of the hydrologic regime (Hofmockel, 2008; Sklar and van der Valk, 2002; Patterson and Fink, 1999; Wetzel et al., 2005). Widespread loss of island habitat in areas of WCA 2 has prompted use of another ecological class of Everglades tree islands called s. The term has been used to refer to an island where vegetative species composition has been detrimentally altered and a community dominated by tree or shrub species is no longer present ( E we 2009). The majority of islands in WCA 2 are now considered ghost islands (E we, 2009). Sustained periods of elevated and decreased water table elevation have been postulated as mechanisms for reduction of quantity and area of tree island habitat through flooding and periodic episodes of fire (Hofmockel, 2008). Lo veless (1959) specifically reported tree island habitat may be adversely effected within the Water Conservation Areas (WCAs) and ENP due to decreases in water table

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16 elevations from regional water management strategies and subsequent alteration of the natur al fire regime. Loss of tree island area and abundance has resulted in reduction of unique habitat opportunity for plant and animal species within the ecosystem. Tree island habitat loss in the Everglades has become of increasing concern not only becaus e of effects to system wide biodiversity but also because of potential loss of ecosystem scale nutrient distribution mechanisms (Wetzel et al 2009). Ecological function and maintenance of potential nutrient distribution control mechanisms associated wit h tree islands in the Everglades has been a focus of research efforts as holistic ecosystem based restoration plans continue to become formulated and prioritized. Soil Phosphorus Soil nutrient studies in tree islands have primarily focused on ecological significance and function of these features within the Everglades regard ing phosphorus (P) accumulation Phosphorus is an essential element necessary for plants and is present in different forms, or pools, in soil. The total soil P pool is composed of or ganic and inorganic P forms which are continuously cycled through biogeochemical processes of mineralization and immobilization. Inorganic P, in the form of phosphate s (PO 4 3 ), is available for plant uptake. Unlike carbon (C) and nitrogen (N) P does not have a stable gaseous phase which contributes to loss or addition in a system. Phosphorus originates from weathered parent material, therefore insitu addition of P from weathered material, or translocated P via geologic, atmospheric or biological vectors are the primary mechanisms of P deposition. The Everglades is widely recognized as a historically oligotrophic wetland ecosystem limited by P (Noe et al. 2001). Miami Oolite and Fort Thompson formations,

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17 both limestones (CaCO 3 ), underlie the majority of the WCAs and ENP (Lodge, 2005). P resence of low P parent material and lack of P input s control regional P limitation (Noe et al., 2001) The system is ombrothrophic with low P content precipitation which is estimated to be 10 1 (Noe et al ., 2001). McCormick et al. (1999) reported soil total P (TP) concentrations range from 250 500 mg kg 1 in the P limited marsh. A meta analysis of available soil T P data from native marsh habitat throughout the Ever glades reported P average concentrations of 533 and 467 mg kg 1 for sawgrass and slough/wet prairie communities, respectively (Noe et al., 2001). Total P concentration of 500 mg kg 1 is generally considered the maximum for soil in the P limited marsh (Deb usk et al. 1994; McCormick et al., 1999). Drainage and flood control projects constructed from the late 19th century through the mid 20th century altered nutrient regimes in the Everglades (Davis, 1994; Light and Dineen, 1994). An area of marsh is describ ed as P enriched when soil total P concentrations exceed 500 mg kg 1 (McCormick et al., 1999; Debusk et al. 2001). Tree island ecosystems in the Everglades offer a particularly unique perspective of soil P distribution at the landscape and component scal e because of the oligotrophic status of the Greater Everglades Ecosystem related to P limitation and the relatively high P concentrations reported in some Everglades tree islands (Orem et al., 2002; Wetzel et al, 2005; Ross and Sah, 2011). Contrast of isl and and marsh soil total P concentration has influenced many recent studies focused on understanding identifying the mechanisms which control the accumulation of P in tree islands as compared to adjacent marsh areas (Wetzel et al. 2009). Nutrient accumul ation on tree islands is primarily attributed to three mechanisms: evapotranspiration mediated movement of

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1 8 groundwater and surface water to islands atmospheric deposition and deposition of animal waste ( Wetzel et al., 2005; Ross et al. 2006; Giv n ish et a l. 2008 ). Accumulation of P in tree island soil is theorized to occur principally in the head region where soil T P concentrations are genera lly greatest within the island ( Figure 1 4; Wetzel et al., 2005; Wetzel et al., 2009) Literature provides minima l information about the magnitude and extent of mechanisms controlling P distribution or the characteristics of P in tree island soil. In the past decade research indicates greater T P concentrations present in tree island soil as compared to adjacent ma rsh soil (Orem et al. 2002; Wetzel et al. 200 9 ). An initial study on this topic focused on two tree islands in Water Conservation Area 3B and reported T P soil concentrations as much as 12 times greater than concentrations in adjacent marsh soil (Orem et al. 2002). Wetzel et al. (2011) recently reported island:marsh soil TP ratio of greater than 200:1 at a location in WCA 3 Data indicat ing TP in tree island soil at concentrations greater than areas impacted by P from agricultural discharge is intrigui ng because of P limitation throughout the system. Wetzel et al. (2009) have proposed that tree islands may sequester greater than 65 % of the P for the central Everglades. Everglades wide tree island soil P data collected over the past 10 years reveals a wide range (~0.02 to >10%) of T P concentrations for tree island soil (Wetzel et al., 2011; Ross and Sah, 2011) The wide range in data reported indicates the pattern of very high soil P concentration in tree island soil as compared to the P limited marsh (island:marsh TP ratio >100 ; Wetzel et al., 2011 ) is not ubiquitous throughout the ecosystem and could be a function of different localized accumulation and storage mechanisms.

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19 Although an increasing number of studies have focused on the importance of tr ee islands in the Everglades with respect to system wide P dynamics, most of these studies have only focused on a few islands and primarily TP concentration Comparison of soil physical and chemical characteristics of different tree islands has been descr ibed by few individuals (Orem et al. 2002; Jayachandran et al. 2004; Hanan and Ross 2010; Wetzel et al. 2009; Ross and Sah, 2011 ). Soil from tree islands in Shark Slough and adjacent marl prairies have been reported to differ in characteristics such a s pH, bulk density, TP, TN, TC, and bicarbonate extractable P (Hanan and Ross 2010; Wang et al. 201 1, Ross and Sah, 2011 ). Additionally, Orem et al (2002) reported differences in dissolved P in porewater samples from the head region of two tree islands in WCA 3B. Wetzel et al (2009) showed a positive relationship between island head elevation and TP marsh:island ratio while noting the differences in bulk density between the marsh and island head alone, do not account for the wide difference in TP. Thi s finding was reiterated recently by Wetzel et al. (2011) based on review of bulk density and TP data for island head and marsh soil (0 10 cm) for 31 islands in WCA 3. Jayachandran et al. (2004) reported a general trend of decreasing soil and porewater TP concentration with increasing distance from tropical hardwood hammock vegetation communities in tree islands of Shark Slough and postulated P biogeochemistry could be influenced by reactions with Calcium (Ca). Similar patterns have been described of decr easing T P concentration with distance from the head region, or highest point of elevation, of tree islands ( Figure 1 4; Wetzel et al., 2009; Wetzel et al., 2011).

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20 The concentrations of Ca, chloride (Cl), magnesium ( Mg ) and sodium (Na) increased in groundwa ter beneath the head of a tree island in WCA 3 during the dry season (Wetzel et al., 2011). Notable shifts in groundwater geochemistry may indicate a seasonal flux of minerals including phosphate, from groundwater to tree islands as described for formati on and of soil minerals in islands in the Okavango Delta (MCarthy et al. 1993). However, with regard to P accumulation, l ittle attention has been given to the effect of anion accumulation in subsurface soils on the fate of dissolved P species. Presumabl y, an increase in anion concentration may influence P sorption through competition for exchange sites in soil and sediments. Additionally, the seasonal increase of Ca and Mg in groundwater reported by Wetzel et al. (2011) and precipitation of carbonates m ay influence P sequestration in tree island soil. Further study of nutrient leaching and transport in the tree island surface soil and vadose zone would help resolve questions regarding fate of soil P in tree islands. H eterogeneity or landscape patterni ng, within natural ecosystems provides opportunity for evaluation of ecological interactions of soil, water, nutrients, wildli fe use, and vegetation A visual indicator of landscape heterogeneity of an ecosystem is an interface of vegetative communities, s uch as the emergent marsh and tree island pattern present throughout the Everglades. Cohen et al. (2011) assert spatial pattern of t ree island s and marsh in th e Everglades are nonrandom, therefore implying tree island initiation and stability is a functio n of ecosystem scale mechanisms The timing of P accumulation with regard to island development remains unclear, and description of P characteristics in Everglades tree islands may help explain nutrient redistribution mechanisms in patterned landscapes.

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21 Research Objectives Accumulation of soil P in tree islands in the Florida Everglades appears to be influenced by mechanisms that are independent of, but interrelated to, components of contemporary P budgets for Everglades emergent marsh vegetation commu nities. The wide range (~0.02 to >10%) of P reported in tree island soil indicates variability in the magnitude of mechanisms controlling P accumulation (Ross and Sah, 2011 ; Wetzel et al ., 2011). Variation in ecological attributes among Everglades tree i slands, such as physical and chemical soil properties, size, vegetative species composition, wildlife use, fire frequency, anthropogenic alteration, and hydroperiod, cumulatively complicate the potential for generalized characterization of P forms and orig in. Elucidation of tree island P accumulation mechanisms will assist future land management decisions, specifically with regards to restoration and management activities that could influence mechanisms of soil P accumulation or release. On a larger scale the Everglades is a patterned landscape and mechanisms influencing the distribution of nutrients in this wetland may also influence nutrient cycles within and across other ecosystems The primary objective of this work wa s to determine if naturally depos ited animal wastes contribute nutrients to tree islands by chemical characterizing of tree island soil and animal waste products. This research was guided by pertinent unanswered questions regarding tree island soil P characteristics and mechanisms that c ould influence soil P accumulation. 1. What are the chemical characteristics of tree island soil and forms of soil P? 2. Does bird guano have similar chemical characteristics as tree island soil? 3. Does the contribution of bone fragments account for a large pr oportion of soil TP in tree island soil?

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22 Research focused on t hese questions was intended to clarify unexplained chemical characteristics of tree island soil and investigate a mechanism suggested to control differences in soil nutri en ts at different scale s within the Everglades (Wetzel et al., 2005; Coultas et al., 2008; Givinish et al., 2008; Ross and Sah, 2011). Three ancillary objectives were established to resolve these questions and support the primary objective of this research. The se objectives we re to: (1) determine elemental c omposition of tree island s oil and characterize the forms of soil P (2) chemically characterize wading bird guan o collected from the Everglades, and (3) describe the distribution of soil nutrients, P forms and minerals in different particle sizes to estimate the proportion of soil total P that may be from a biogenic P mineral.

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23 Table 1 1 Vegetation found within tree islands in the Everglades Vegetation Community Species Bayhead Anona glabra Chrysobalanus icaco I lex cassine Magnolia virginiana Myrica cerifera Persea borbonia Salix caroliniana Bayhead Swamp Anona glabra Chrysobalanus icaco Magnolia virginiana Persea palustris Salix caroliniana Tropical Hardwood Hammock Ardisia escalloniodes Burs era simaruba Celtis laevigata Chrysophyllum oliviforme Coccoloba diversifolia Eugenia axillaris Ficus aurea Lysiloma latisiliqua Myrsine floridana Nectandra coriacea Quercus virginiana Sideroxylon foetidissimum Sideroxylon salicifolium The spe cies list is s ummarized from Ruiz and Ross (2004), Wang et al.(2011) and Saha et al.(2010).

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24 Figure 1 1. The geographic setting of the Florida Everglades, and general locations of the Water Conservation Areas (WCA 1, 2, and 3), and Everglades National Park (ENP). The yellow boundary is the estimated current extent of the ecosystem, and was estimated from McVoy et al. (2011).

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25 Figure 1 2 A tree island in the s outhern Everglades.

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26 Figure 1 3 The interior of a tree island in the southern Evergl ades where areas of different bedrock elevation are observable.

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27 Figure 1 4. Three primary accumulation mechanisms described to influence soil phosphorus concentrations detected in tree island soils in the Florida Everglades and the general pattern of P redistribution within islands (Wetzel et al, 2011).

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28 CHAPTER 2 SOIL PHOSPHORUS CHAR ACTERISTICS Introduction Different forms of soil P are often designated as either inorganic or organic, and commonly further described as exchangeable, labile, and stabl e or recalcitrant ( Ruttenburg, 1992; Nair et al. 1995 ; Reddy et al., 1998). These descriptions imply differences among P forms with respect to reactivity in the environment. Freshwater aquatic ecosystems, such as much of the Everglades, are typically sen sitive to changes in P pools due to naturally low P availability. Detrimental ecological effects and undesirable vegetative community shifts have been attributed to increases in water column and soil P concentration (Koch and Reddy, 1992; Davis 1994; Deb usk et al., 2001). An element of Everglades restoration planning and management is focused on improving water quality and reducing ecosystem degradation Maintenance of a delicate balance of soil and water column P chemistry system wide is essential for t he success of long term ecosystem restoration Total P in tree island soil varies from concentrations not very different from adjacent marsh soil to levels more than 100 times greater (Wetzel et al., 2011). This variability has fostered interest in expla ining the ecological function of tree islands in the Everglades (Orem et al., 2002; Ross and Sah, 2011; Wetzel et al., 2011). Research has focused primarily on the development of theory to explain the pattern of general divergence of soil P concentration between the marsh and tree islands (Orem et al., 2002; Wetzel et al., 2005; Wetzel et al., 2009; Wetzel et al., 2011). Recent literature has also investigated heterogeneity of P concentrations among island soils ( Hanan and Ross, 2010; Ross and Sah, 2011 ). The wide difference between tree island and marsh

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29 soils and among tree islands, likely indicate s variability in the magnitude of abiotic and biotic mechanisms influencing P distribution across the landscape. Three primary hypotheses for the formation of soil P hotspots within the P limited Everglades marsh, or in similar oligotrophic wetlands in Belize (Macek et al. 2009), focus on the direct or indirect effects of vegetation (Weztel et al., 2005; Ross et al. 2006; Wetzel et al. 2011). High rates o f evapotranspiration by trees and shrubs, atmospheric deposition and accumulation of animal wastes are theorized to influence accrual of inorganic matter in tree island soil (Wetzel et al., 2005; Ross et al. 2006). Each of these mechanisms have been desc ribed as influential in transport of nutrients to island habitat s in other ecosystems around the world (McCarthy et al., 1993; Frederick and Powell, 1994; Wait et al., 2005; Krah et al., 2006; Ramberg and Wolski, 2008). Soil P chemistry in the Everglades m arsh has been investigated throughout the Water Conservation Areas (WCA 1, 2, and 3) with focus on the distribution of P forms (Koch and Reddy, 1992; Newman et al., 1997; Reddy et al., 1998; Bruland et al., 2006). Organic P dominates the soil P pool in the P limited Everglades marsh (Reddy et al., 1998; Bruland et al., 2006), while inorganic P forms are more prevalent in soils near sources of external P inputs such as surface waters in canals or water control structures (Newman et al., 1997; Reddy et al. 1998; Bruland et al., 2006). An increase in Ca bound P is frequently related to increase of inorganic P in marsh soil (Koch and Reddy, 1992; Reddy et al. 1998). Precipitation of Ca and P, and association with CaCO 3 is often described to explain charact eristics of inorganic P in Everglades soil (Koch and Reddy, 1992; Reddy et al., 1998).

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30 Literature is limited regarding the chemistry stability and fate of tree island soil P. An i nvestigation of organic P forms with nuclear magnetic resonance (NMR) in tree island soil from a wading bird colony in WCA 3 revealed that orthophosphates were the primary form (El Rifai et al., 2008). Ross and Sah (2011) showed that increases in soil P concentration in tree island hardwood hammock communities might be related to the presence of inorganic P in association with non carbonate soil material. If P is not associated with soil carbonate tree island soil P concentration possibly indicates the presence of phosphate minerals, such as hydroxyapatite (apatite). Phosphat e minerals are rare in the Everglades (Harris, 2011), and their accumulation in tree island soil could represent a relatively s table reservoir of P, with implications for restoration. Determination of the elemental composition and P characteristics of tre e island surface soil will advance current hypotheses describing the ecological role of tree islands and enhance the spatial resolution of P characteristics in the Everglades. It is hypothesize d that apatite, a Ca phosphate mineral, is the pre dominant for m of P i n tree island soils characterized by elevated P. The objectives were to: (1) determine elemental characterization of soil in tree islands, (2) determine correlations among chemical constituents that may elucidate P characteristics, and (3) identif y the P minerals present in tree island soil. Materials and Methods Site Descriptions and Locations Composite soil samples, consisting of two surface soil cores each 0 10 cm deep, were collected from 26 tropical hardwood hammock plant communities within tr ee islands from 2005 2007 in the southern Everglades, Florida, USA (Fig ure 2 1). Hardwood hammocks are upland forests and differ from other forested areas in

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31 vegetative species composition and geomorphic setting in the Everglades landscape. Vegetation do cumented in the tree island hardwood hammocks includes tropical and subtropical tree and shrub species (Saha et al. 2010; Wang et al. 2010). On average, the soil surface is elevated ~0.9 m above the adjacent emergent marsh habitat and ~0.7 m above the av erage water table (Ross and Sah, 2011). Most hammock species are intolerant of prolonged periods of soil saturation and soils are typically well drained due to an elevated landscape position (Saha et al., 2010; Ross and Sah, 2011). Coultas et al. (2008) described nonhydric soils in two hammocks in the southern Everglades and categorized them as Mollisols. Hammock soils that are occasionally flooded, and located within the marl prairie regions of the southern Everglades are somewhat similar to the Histoso ls (Folists) found in dry hammock forests in the Florida Keys (Ross et al., 2003; Ross and Sah, 2011). Early categorizations of soils within Everglades tree islands describe them broadly as peats (Leighty and Henderson, 1958). Elemental Analysis Soil samp les were oven dried (70 C) and sieved (<2 mm) prior to analysis. S oil pH was determined on a slurry with water to soil ratio of 1:1. Total carbon ( T C) and nitrogen ( T N) were measured by dry combustion using an elemental combustion system (Costech Analyt ical Technologies, Inc. Valencia, CA ). Total P was determined by heating 200 mg of soil to 550 C for 4 hours then dissolving the residual material in 6 M HCl (Anderson, 1976) The solution was then analyzed for P concentration on a spectrophotometer (B eckman Instruments, Inc., Fullerton, C A ) using the ascorbic acid method of Murphy and Riley (1962) The concentrations of total aluminum (Al), calcium (Ca), iron (Fe), and magnesium (Mg) in the same solution were measured by inductively coupled plasma o ptical emission spectroscopy (PerkinElmer, Inc., Waltham, MA).

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32 Percent organic matter (OM) was determined by loss on ignition (LOI) during the combustion stage of the T P analysis. Total inorganic carbon (TIC) concentration was determined using a pressur e calcimeter, and calcium carbonate (CaCO 3 ) concentration was calculated from measured TIC a s described by Wang et al. (2011) The proportion of soil that was not accounted for by summation of OM and CaCO 3 comprised non carbon soil matter (NCM ; Ross and S ah 2011). The concentration of soil Ca associated with CaCO 3 was calculated with the assumption that Ca constitutes 40% of CaCO 3 by mass. Non carbonate Ca (NCCa) was determined by difference between the total soil Ca concentration and the concentration of Ca associated with CaCO 3 in soil. P hosphorus Fractionation Soil P was partitioned by sequential chemical extraction using a modified fractionation procedure (Figure 2 2) that differentiated six forms of P (Hieltjies and Liklema 1980; Ivanoff et al., 19 98; Reddy et al., 1998; Kou et al., 2009). The fractionation procedure consisted of four extracting solutions distilled deionized water (DDI), 1.0 M KCl, 0.1 M NaOH, and 0.5 M HCl. After the soil was treated successively with each of the extracting solu tions the remaining soil residue was analyzed for TP (residual P). Using a sample to solution ratio of 1:50, s oil and 25 mL of each extraction solution were combined in 50 m L centrifuge tube s and shaken with a reciprocating mechanical shaker. Sample solu tions were shaken for 1, 2, 17 and 24 hours for DDI, KCl, N aOH and HCl respectively. After equilibration, sample solutions were centrifuged at 2,100 x g for 15 minutes, and supernatants were filtered ( #42 Whatman ) and refrigerated at 4 C prior to P analy sis. Phosphorus measured in filtrates was assumed to be soluble inorganic P (Pi) and was determined colorimetrically as described previously Sodium hydroxide solutions were also measured for TP by persulfate

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33 digestion. Organic ally bound P in the NaOH solution (NaOH Po) was determined as the difference between dissolved P and T P in solution Phosphorus extracted by DDI is defined as water soluble P (WSP). Extractable P in the KCl solution is available P not solubilized by the DDI extraction. Cumula tively these fractions can be defined as available, exchangeable or labile P Phosphorus dissolved in the NaOH solutions (NaOH Pi) is considered Al or Fe bound P. The NaOH Po fraction is generally attributed to humic and fulvic acid P. Acid extractable P (HCl P) is presumed as Ca or Mg bound P. The composition of the residual P fraction is unknown, and commonly inferred to contain recalcitrant P associated with minerals and organic matter (Reddy et al., 1998). Mineralogical Analysis The first stage of mineralogical assessment was observation under a dissecting microscope. Subsequently mineral identification was conducted by x ray diffraction (XRD) using an automated XRD system (Rigaktu Corporation, The Woodlands, TX) equipped with stepping motor and gra phite crystal monochromator. Each sample was prepared for XRD analysis using a powder cavity mount (Harris and White, 2008). Samples were scanned from 0 Statistical Analysis All statistical analyses were conducted using SAS version 9.2 (SAS Institute, Inc.). Descriptive statistics were used to characterize the general soil properties and distribution of different forms of P. Correlation among soil properties was determined Order coefficients, with a mi nimum significance level of 0.05.

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34 Results and Discussion Soil Chemical Characterization E lemental composition of hardwood hammock soil varied widely as indicated by the range of values measured for each parameter (Table 2 1). Dissimilarity in measured T C and T P is not surprising due to the variety of concentrations reported for Everglades tree island soil (Gann et al., 2005; Hanan and Ross, 2010; Wetzel et al., 2011). Carbon, Ca, and P were the primary elements present, with mean concentrations of 20.0, 19.5 and 4.7%, respectively (Table 2 1). Total P concentration in tree island soil ranged from 760 to 88,1000 mg kg 1 (Table 2 1). Total Al, Ca, Fe and Mg concentrations for Everglades tree island soil were not found in the literature. Measured mean total Al and Fe soil concentrations were lower and total Ca and Mg soil concentrations were greater than marsh soil reported for WCA 3 (Craft and Richardson, 1997). Soil pH for tree island soils was slightly alkaline and the range measured was similar to soil pH reported for other tree islands in the Everglades ( Gann et al., 2005 ; Hanan and Ross, 2010). Average soil CaCO 3 concentration (~22.4%) was intermediate compared to tree island soil s described by Hanan and Ross (2010) and values estimated from inor ganic C reported by Ross and Sah (2011). Soil CaCO 3 measured in hammock soil was less than half the concentration reported for calcareous wetland soil in southeastern Everglades (Zhou and Li, 2000). Minimum T C measured in hammock soil was ~9.5% (Table 2 1). Wetzel et al. (2011) reported minimum soil T C concentration of 10.8 % and a mean of 38.4% from 31 tree islands in the central Everglades. Soil TC concentration reported in the predominantly peat soils of Shark Slough and southern WCA3 is approximatel y two

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35 times higher than the mean concentration measured in hardwood hammock soil (Table 2 1; Bruland et al., 2006; Hanan and Ross, 2010). Percent OM ranged from approximately 20 to 90% averaging ~40% (Table 2 1). Low TC values slightly alkaline pH and low OM concentration measured in the hammock soils suggests that CaCO 3 is a significant mineral component. The presence of limestone bedrock in south Florida and outcrops that underlie many tree islands supports this assumption. However, i f tree island s oils were pure CaCO 3 the T C concentration would be approximately 10% by weight based on the theoretical molecular mass of the mineral. Organic matter was present in measurable quantity in all samples therefore even at low TC concentrations a portion of the TC is attributable to organic C (Table 2 1). Total C concentration of the relatively shallow, CaCO 3 rich, marl prairie soil in the southern Everglades is ~15.0% (Hanan and Ross, 2010; Liao and Inglett, 2012). Ross and Sah (2011) found half of the soi l material in hammock soils of tree islands was comprised of non carbonate mineral matter. Non carbon matter of the hammoc k soil analyzed here averaged ~37%, rang ing from ~4.5 to 68% (Table 2 1). Difference in soil C:N ratio has been used to infer age, or degree of organic matter decomposition in relation to accumulation of inorganic material in forest soil (Obrist et al. 2011, Zhu et al., 2012). Lack of significant correlation (r=0.20, p=0.33) between OM and soil C:N (molar) ratio may suggest that low OM concentration is not caused by higher soil decomposition and mineralization, but rather by differential deposition or accumulation of mineral material The inference here would also support the theory that h igh soil non carbon mineral content in these subtropical dry forest

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36 ecosystems is suggestive of an exogenous source of inorganic matter that varies among islands. The high variability of OM, NCM, Ca and P are particularly interesting with respect to elucidation of tree island soil biogeochemistry. The range in soil OM, Ca and TP may reflect varying magnitudes of external contributions of inorganic elements derived from animal wastes to tree islands. Significant negative correlations were determined between soil organic matter, and P, Ca, NCCa, an d Fe (Table 2 2). Negative correlation (p<0.001) between OM and P indicates high soil total P is not likely due to the presence of organic bound P. Calcium, and particularly NCCa, is also not associated with o rganic matter (Table 2 2). Neither s oil CaC O 3 nor total Ca concentration w ere significantly correlated to TP (Table 2 2). Ross and Sah (2011) reported no significant correlation between TP and inorganic C, but a significant positive correlation between TP and NCM in hammock soils. They theorized that soil P may reflect the presence of Ca phosphate minerals such as apatite, or of P adsorbed to surfaces of non carbonate minerals (Ross and Sah, 2011). Zhou and Li (2000) showed P sorption with non carbonate clays in calcareous soils of south Florida was a significant characteristic of P chemistry. Significant positive correlation of soil NCM with P, NCCa and Mg indicate P chemistry in these hardwood hammock soils may be influenced by Ca and Mg minerals that are not carbonates (Table 2 2) Jayachan dran et al. (2004) reported a general trend of decreasing soil and porewater TP concentration with increasing distance from tropical hardwood hammock vegetation communities in tree islands of Shark Slough and postulated that P biogeochemistry could be infl uenced by reactions with Ca.

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37 Measurement of soil total elements and determination of elemental associations reported here provide new insight toward tree island soil chemistry and characteristics of P. Although these findings offer significant indication of P association, little can be inferred regarding P availability, distribution of P forms, relative stability, or the presence of specific P minerals. Soil P Fractionation Sequential chemical P fractionation revealed HCl P is the dominant phosphorus fo rm in these tree island soils, comprising on average ~85% of total P (Fig ure 2 3). Most of the P in the native marsh is organic bound P (Reddy et al. 1998, Bruland et al., 2006). Total P and HCl P were positively correlated (r=0.997, p<0.001) indicating that mechanisms influencing increases in Ca or Mg bound P are central to P accumulation and storage in tree islands and the Everglades (Table 2 3). Reddy et al. (1998) showed that high concentrations of total inorganic P were present in the most P rich Ev erglades peat soils. More than 65% of the total P pool in the Ap horizon of manure impacted Spodosol s was Ca or Mg bound P (Nair and Graetz, 1995). Water soluble and KCl extractable P, cumulatively exchangeable P, accounted for a very small proportion of the total P pool (Fig ure 2 3 ) and average P concentration in this pool equates to ~150 mg kg 1 of available P. This concentration of available P is approximately 1/3 the historic TP reported for Everglades marsh soil. The presence of relatively abundan t available P in hammock soil supports the theory that plant productivity in tree islands is enhanced by soil P availability, potentially leading to the expansion of the islands. The origin of the available P remains debatable and is likely the result of multiple sources (Wetzel et al. 2005; Ross et al., 2006).

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38 Both WSP and KCl extractable P were positively correlated (WSP, r=0.487, p<0.05; KCl extractable P, r=0.503, p<0.05) with TP (Table 2 3). An increase in available P with increasing TP suggests t ree island soil with high concentration of TP may release more P than soil with relatively lower P concentration. Water soluble P was also positively correlated (r=0.84; p<0.05) with KCl P and positively correlated (r=0.48, p<0.05) with HCl P (Table 2 3). The relationship between WSP and KCl extractable P suggests that WSP that is utilized by plants and microorganisms, or leached from the soil, may be replenished by the exchangeable P pool. The positive correlation between WSP and HCl P suggests that the dissolution of Ca or Mg P minerals may influence P availability in tree island soil. Concentration of water and NH 4 Cl extractable P was reported to show a similar trend as total P in soils impacted from dairy manure, where higher extractable P was detect ed in soil with increasing total P concentration (Graetz and Nair, 1995). W ater soluble P was the dominant type of available P in poultry litters and manure (Dail et al., 2007 ; Codling, 2006 ; He et al., 2010) accounting for as much as ~ 55% of the T P in poultry litter (Codling, 2006) Water extractable P in animal manures (cattle, swine, and poultry) has been used to indicate P runoff risk from soils amended with these waste products (Kleinman et al., 2005). Guano collected from wading birds in the Ever glades was found to contain~3,500 mg kg 1 of available P, which suggests large amounts of available P may be deposited at islands that birds visit frequently ( Chapter 3 ). Calcium or Mg bound P (HCl extractable P) accounted for most of T P pool in poultry l itter and manure (Dail et al., 2007; He et al., 2010), and ~88% of the TP in wading bird guano ( Chapter 3 ). Nair et al. (1995) reported that 80% of the Ca or Mg

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39 bound P in manure impacted soil may be subject to leaching based on repeated extraction with N H 4 Cl. Additionally, WSP was not significantly correlated with other forms of P, in particular NaOH Pi, indicating that Al or Fe bound P is not readily released (Table 2 3). Soil OM concentration was positively correlated (r=0.48, p<0.05) with NaOH Po, a nd negatively correlated (r= 0.86, p<0.001) with NaOH Pi (Table 2 3). Organic matter was also negatively correlated (r= 0.78, p<0.001) with HCl P (Table 2 3). The inverse relationship of NaOH Po and TP confirms the organic P is not the primary P form in tree island soils with high concentrations of P. Organic bound P extractable with NaOH comprised ~3.5% of the TP (Fig ure 2 3 ). No significant correlation was determined between residual P, and OM or total P (Table 2 3). This lack of correlation suggests the residual P is a relatively consistent and stable pool tree island soils. Cumulatively, HCl P and residual P comprise ~90% of the total P in the hammock soil, and based on the extraction sequence could be considered relatively stable forms of soil P (F ig ure 2 3 ). However further research is required to describe the potential rate of P release from these soils. The concentration of HCl P is positively correlated (r=0.98, p<0.001) with soil non carbon matter (Fig ure 2 4 ). These findings suggest that ele vated P in Everglades tree island surface soil is related to accumulation of Ca P or Mg P minerals, and these minerals are not likely carbonates. The total concentration of Ca and Mg in soil suggests Ca is more important in P dynamics because of the abund ance of Ca relative to Mg (~40 times greater). Additionally, the positive correlation (r=0.98, p<0.001)

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40 between NCCa and HCl P suggests that Ca P minerals are the dominant forms of P in tree island soil (Fig ure 2 4 ). Recruitment of dissolved minerals from the surrounding marsh to tree islands, through localized hydrologic pathways mediated by greater rates of evapotranspiration at island locations, is a potential mechanism for accumulation of mineral matter in tree island soil (McCarthy et al., 1993; W etzel et al., 2005; Ross et al., 2006). The presence of cemented carbonate soil layers in Everglades tree islands may be a product of this mechanism (Coultas et al. 2008). However our findings suggest that carbonate precipitation is not the dominant mec hanism of P retention. Recruitment of dissolved minerals from the surrounding marsh to tree islands, through localized hydrologic pathways mediated by greater rates of evapotranspiration at island locations is a potential mechanism for accumulation of min eral matter in tree island soil (McCarthy et al., 1993; Wetzel et al., 2005; Ross et al., 2006). Soil Mineral Identification Hydroxyapatite (apatite) ( Ca 5 (PO 4 ) 3 OH) and calcite ( CaCO 3 ) were present in abundance based on relative XRD peak intensities for eac h of the high P samples of tree island soil (Fig ure 2 5). No other phosphate minerals were observed in the soil using XRD. Quartz was identified in two of the samples and aragonite ( CaCO 3 ) was identified in one sample (data not shown). Observations unde r a dissecting microscope revealed that the presence of bone fragments in many samples with elevated P concentrations, while the presence of calcite in the samples was likely due to fragments of limestone or shell in soil. Aragonite was also probably rela ted to shell fragments, though geochemical analysis of surface and groundwater in a hammock in ENP have shown that tree island groundwater is frequently above critical saturation thresholds for

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41 these minerals, suggesting that they may precipitate (Sullivan et al., 2011). Quartz is not an uncommon component of nearby marl soil and has been documented to comprise 15 40% of the clay sized minerals of marl prairie soil in the region (Sodek et al., 1990). Investigation of different soil particle size classes m ay reveal more information regarding soil mineralogy. Comparison of molar ratios of total Ca and NCCa to P suggests that apatite may be the dominant mineral present as soil NCM and TP increase (Fig ure 2 6 ). Increase in NCM is negatively correlated ( r= 0 .97, p<0.001) with Ca:P and negatively correlated (r= 0.67, p<0.001) with non carbonate Ca:P ratios (Fig ure 2 6 ). The theoretical molar Ca:P ratio of hydroxyapatite is 1.67. As NMC and T P concentration in the hammock soils increases the ratio of Ca:P and NCCa:P converges upon and conforms to the Ca:P ratio of apatite (Fig ure 2 6 ). Average molar Mg:P ratio is < <1 further indicating that Mg phosphate interactions are not dictating P chemistry in these soils to the same extent as Ca phosphate minerals Cal cium phosphate minerals tend to dominat e in calcareous soil and Mg phosphates are generally not as abundant or common (Lindsay et al. 1989). Collectively soil elemental analysis, chemical fraction of P, physical identification of soil minerals, and stoic hiometric correlation indicate apatite presence in tree island soil is an important characteristic of Everglades P biogeochemistry. The mechanism for apatite accumulation or transport to tree island soil is likely biogenic based on the consistent presenc e of bone fragments in many soils with elevated P concentration. Apatite was not observed in the Biscayne, Pennsuco and Perrine Marl, and Chekika and Krome soil types (Sodek at al. 1990). Apatite has been detected in agricultural soil in south Florida kn own to be treated with P fertilizer (Harris,

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42 2011). Bates et al. (2010) reported the detection of amorphous apatite in soil from a wading bird colony in northern WCA 3 and attributed the apatite to guano deposition. Multiple apatite peaks were detected i n the hammock soil of the present study, indicating that the apatite present in these soils was relatively crystalline. Irick (2012) characterized P forms in wading bird guano from the Everglades. Crystalline P minerals were not detected in the bird guan o; however, most of the P in wading bird guano was Ca P ( Chapter 3 ). Hydroxyapatite has been proposed as a constituent of the Ca P pool in poultry litters, specifically when Ca:P ratios exceed 2 (Toor et al., 2005; Shober et al., 2006). Lindsay et al. (19 89) described Ca P mineral in terms of potential solubility in soil solution and apatite species are less soluble than monocalcium phosphates such as brushite and monetite. Sullivan (2011) recently found hydroxyapatite saturation indices of ground and sur face water chemistry of an Everglades hammock did not support in situ precipitation of apatite in tree island soil. Further they suggested that dissolution of inorganic P in surface soil likely contributes to P measured in groundwater (Sullivan, 2011). T he confirmation of apatite in multiple tree island soils and observation of bone fragments in these soils indicates that bioapatite contributes to the soil P pool. Coultas et al. (2008) reported gravel sized (>2 mm) fragments comprised ~22 36% of the soi l mass in the top 15 cm at two islands in the southern Everglades and that bone was observed in the gravel. They postulated guano and bone derived P could account for the elevated P observed in some tree island soil s (Coultas et al., 2008). Apatite pres ence may also be a result of historic anthropogenic use, as implied

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43 by the presence of buried bone and artifacts within the profile of tree island soil from the Everglades (Carr, 2002; Graf et al., 2008; Ross and Sah, 2011). The accumulation of bone in E verglades tree islands also may be an ongoing process related to wildlife use. Coultas et al. (2008) suggested in addition to anthropogenic focusing, wildlife utilization of islands could also be a vector for bone addition in tree islands. A qualitative assessment of wildlife use in Everglades tree islands noted the presence of avian, mammalian, reptilian, amphibian and invertebrate species ( Meshaka et al. 2002 ). Until quantitative estimates are developed for habits of animal use in tree islands, determi nation of potential rates of bioapatite deposition will probably remain unresolved. Additional geochemical analysis of soil porewater from a range of tree island soil P tiers could also provide valuable insight toward the phosphate mineral dynamics in thes e unique soils. Further investigation of phosphate mineral distribution and stability in tree island soils will help elucidate landscape scale mechanisms influencing differential patterns of nutrient accumulation in the Everglades. Protection of tree isl and habitat is likely an important management approach to maintain mechanisms that promote nutrient accumulation in tree island soil and minimize the potential for ecosystem eutrophication related to loss of island habitat. Conclusions Current theories s uggest tree islands are sentinels of nutrients within the oligotrophic Everglades wetland. Our empirical approach is the first to focus on P chemistry in tree island soil. Soil P characteristics in tree islands are dictated by different processes than the surrounding marsh, as evidenced by the high concentration of inorganic P in tree islands. The high proportion of HCl P observed in tree island soil coupled with a positive relationship between NCCa and TP concentration is evidence

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44 for an exogenous P sour ce. Adsorption of P to Ca in carbonate species is likely not a significant aspect of P chemistry in high P tree island soils. Positive correlation of NCCa with HCl P, and stoichiometry of total Ca:P, NCCa:P and Mg:P molar ratios is an indication that Ca phosphate minerals are the primary form of P in tree island soil. The presence of apatite and bone fragments in tree island soils confirms that deposition of biogenic apatite contributes to the soil P pool. The source, spatial extent, and timing of this contribution remain unknown. Further quantification of potential soil P constituents will advance development of tree island nutrients budgets and the dynamics of P distribution across the P limited landscape.

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45 Table 2 1. Select soil properties from 26 tre e islands with tropical hardwood hammock plant communities in the Florida Everglades. Parameter (g kg 1 ) Mean Median Minimum Maximum pH 7.96 8.08 6.36 8.42 Organic Matter 403 305 198 899 CaCO 3 224 206 56.3 646 Non carbon Matter 373 410 44.8 678 Tota l Carbon 200 166 95.5 407 Total Nitrogen 12.7 10.5 6.70 24.2 Total Phosphorus 47.2 52.2 0.76 88.1 Aluminum 2.56 2.11 1.20 7.15 Calcium 195 214 42.8 300 Iron 5.04 4.08 1.2 13.4 Magnesium 4.60 3.63 1.53 10.5 Data for pH are a subset from Ross and Sa h (2011).

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46 Table 2 2 Non order) for select tree island soil properties (n=26). Phosphorus Organic Matter Non carbon Matter Phosphorus 0.77*** 0.98*** CaCO 3 0.20 0.11 0.24 Aluminum 0.05 0.16 0.05 Calcium 0.39 0.70*** 0.31 Non carbonate Ca 0. 98 ** 0. 7 9 *** 0. 97 ** Iron 0.10 0.40* 0.21 Magnesium 0.44* 0.19 0.43* Correlation is s ignificant at the 0.05 probability level ** Correlation is s ignificant at the 0.01 probability level *** C orrelation is s ignificant at the 0.001 probability level

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47 Table 2 3. Spearman correlation coefficients for water soluble phosphorus (WSP), 1.0 M KCl extractable P (KCl), 0.5 M NaOH extractable inorganic P (NaOH Pi) and organic P (NaOH Po), 0.5 M HCl ext ractable P (HCl), residual P (Residual), with total P (TP) and organic matter (OM) for tree island soil (n=26). WSP TP OM WSP 0.487* 0.15 KCl 0.84*** 0.503* 0.13 NaOH Pi 0.337 0.896*** 0.86*** NaOH Po 0.003 0.475* 0.48* HCl 0.484* 0.997*** 0. 78*** Residual 0.234 0.136 0.11 Correlation is significant at the 0.05 probability level. ** Correlation is significant at the 0.01 probability level. *** Correlation is significant at the 0.001 probability level.

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48 Figur e 2 1. Site map depicting tree island locations with hardwood hammock plant communities where soil samples were collected in the Florida Everglades, USA.

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49 Figure 2 2. Depiction of soil phosphorus sequential chemical fractionation scheme.

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50 Figure 2 3 Distribution of phosphorus forms in tree island soil (0 10 cm). Bar values indicate mean and errors bars 1 SD.

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51 Figure 2 4 Scatter plots depicting relationships between HCl extractable soil phosphorus concentration, and the concentrations of non carbon soil matter (NCM) and non carbonate calcium for tree island soil. Correlation coefficient (Spearman), associated p value, and sample size denoted for each plot.

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52 Figure 2 5 X ray diffraction patterns for particles <2 mm in size from three tree island soils (0 10 cm). The arrows indicate primary peaks for apatite and calcite.

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53 Figure 2 6 Stoichiometric ratios of total calcium (Ca) and non carbonate Ca to phosphorus (P) in comparison with non carbon matter and total phosphorus conc entration in tree island soil (0 10 cm ). Correlation coefficient (Spearman), associated p value, and sam ple size denoted for each plot.

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54 CHAPTER 3 WADING BIRD GUANO EN RICHMENT OF SOIL NUT RIENTS IN TREE ISLAN DS Introduction Determination of mechanisms cont rolling nutrient transport and transformations in soil are essential for wetland restoration planning and management. Everglades r estoration activities are ongoing and mostly focus on water delivery and control of P in the oligotrophic marsh. Dramatic di fferences reported between tree islands and marsh soils may indicate that the mechanisms controlling P distribution between landforms may also influence distribution of P among islands. Accumulation of nutrients in tree island soils likely occurs at varia ble magnitudes among islands integrating the ecological complexity of interactions between local surface and groundwater hydrologic gradients, differential vegetation patterns and wildlife distribution. Natural deposition or accumulation of P in high quan tities in the contextual framework of the ecology of the Everglades suggests an emerging theory where high soil P and ecosystem health are no longer mutually exclusive (Wetzel et al., 2011). Identification of P source is often challenging because of the co mplexity of P biogeochemistry. Wildlife species, particularly colonial nesting birds, have been described as potentially significant biovectors for nutrient transport, especially in the oligotrophic Everglades, but also in other wetland habitats (Bildstei n et al., 1992; Frederick and Powell, 1994; and Post et al., 1998). In the Everglades, wading birds generally forage in the emergent marshes and roost in patches of trees and shrubs dispersed throughout the ecosystem. Spatial variation in foraging, and perching or nesting location s within the ecosystem suggest s a potential transport mechanism for nutrient redistribution

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55 (Frederick and Powell, 1994). Wading birds also seasonally nest in the Everglades in high density at many locations. Transport of mari ne derived N and P to terrestrial island environments by seabirds is a common mechanism for soil nutrient enrichment (Hutchison, 1950; Anderson and Polis, 1999; Wait et al., 2005). Similarly in the Everglades, transport of nutrients, particularly N and P, from marsh derived prey items through bird guano deposition has been hypothesized to influence the distribution of soil P throughout the ecosystem resulting in elevated soil P concentration in tree island soils (Orem et al., 2002; Wetzel et al., 2005). F or example, large nesting aggregations of birds may be capable of importing metric tonnes of P annually (Frederick and Powell, 1994). Nutrient transport alone does not comprise a mechanism for nutrient accumulation, unless the deposited nutrients have bee n transformed to a relatively stable form. Abiotic and biotic drivers of nutrient distribution and transformation can be investigated at various scales within an ecosystem. Deposition of high P content animal wastes such as guano, dropped food or carca sses in natural ecosystem settings can occur in discrete locations such as nesting sites of avifauna. Frederick and Powell (1994) suggested that where Everglades wading birds nest in high density, P deposition by avifuana may approach 3,000 times the atmo spheric P deposition rate, thereby contributing to nutrient redistribution. Deposition by avian species is one of the primary hypotheses offered to explain high concentrations of P in Everglades tree islands (Wetzel et al., 2005; Wetzel et al., 2011). Av ian species have been associated with nutrient focusing in wetlands and on islands located in marine environments (Post et al., 1998; Anderson and Polis, 1999; Wait et al., 2005; Macek et al., 2009). Anderson and

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56 Polis (1999) reported seabird guano deposi tion elevated soil P concentration up to six times higher than unaffected soil. Stable isotopes have been utilized to indicate deposition of wildlife waste products as a transport mechanism of marine derived nutrients into terrestrial environments, with in terrestrial environments and to explain increases of the soil stable N isotopic signature post animal waste deposition (Mitzutani et al., 1985; Mitzutani and Wada, 1988; Anderson and Polis, 1999; Frank et al., 2004; Macek et al., 2009). Investigation o f stable isotopes can illuminate interactions among plants, water, wildlife and nutrients, and has aided in interpreting the exchange of resources in ecology (Peterson and Fry 1987). Nitrogen has two stable isotopes 14 N and 15 N, with natural abundances o f 99.64 and 0.36%, respectively. Increase in the relative presence of 15 N to 14 N in a sample indicates a larger portion of total N in a sample consists of 15 N. Soil 15 N has been used as an indicator to demonstrate the influence of guano deposition by av ian species on island ecosystems (Wait et al., 2005). Although the investigation conducted by Wait et al. (2005) focused on N inputs from guano deposition, their data also indicated an ~18:1 difference in soil P observed in guano affected islands versus n on guano islands. 15 N signature of wading bird guano from the Everglades may provide insight regarding nutrient transport and fate within the ecosystem. It is hypothesized that c ontribution of guano to tree islands may be measure d through similarities between chemical properties of soil and guano. The objective s of this study were to: (1) chemically characterize wading bird guano collected

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57 from the Everglades, (2) investigate the stable N isotopic signature of wading bird guano a nd soil and (3) estimate mass deposition of nutrients from guano. Materials and Methods Site Descriptions and Locations Soil was collected from 46 tree islands (Figure 3 1) between 2005 and 2011, and guano samples from avian species were collected from tw o colonies (Figure 3 2) in the central and southern Everglades, Florida, USA in May 2011. All samples were collected from the head region of the tree island, which is the location most likely to have the highest concentration of soil P (Wetzel et al., 200 9). Surface soil in the head region of tree islands has been described as peat (Wetzel et al., 2009). Vegetation varied among tree island sample locations ranging from upland species intolerant of extended periods of soil saturation to swamp forest speci es capable of persisting during stages of soil saturation and standing water. Vegetative species typical of the upland forests include gumbo limbo (Bursera simaruba), white stopper (Eugenia axillaris), and false mastic (Sideroxylon foetidissimum). Swam p f orest species common in Everglades tree islands include red bay, swamp bay, coastal plain willow and coco plum (Chrysobalanus icaco). Wading bird species present at Colony 1 included white ibis (Eudocimus albus), tricolored heron (Egretta tricolor), snow y egret (Egretta thula), great egret (Ardea albus) and little blue heron (Egretta caerulea). In addition to the species observed at Colony 1, wood stork (Mycteria americana) were observed at Colony 2. The two locations differed in vegetative composition. Colony 1 was dominated by coastal plain willow with swamp bay and coco plum present. Colony 2 was primarily a monotypic stand of pond apple (Anona glabra).

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58 Soil and Guano Collection Soil samples (0 5 cm) from 17 tree islands and soil samples (0 10 cm ) from 29 tree islands were collected throughout the central and southern Everglades. The samples were oven dried and passed through a 2 mm mesh sieve prior to analysis. Soil samples collected from 0 5 and 0 10 cm depths were dried at 60 and 70 C, respe ctively due to collection and initial processing by different field survey personnel. Guano material was collected as a single composite sample from the colony on the day of each site visit. Three separate composite samples were collected from Colony 1 d uring one of the sampling events. The three composites included one composite of guano from the colony, one composite of aged guano, and one composite of freshly deposited guano. Fresh guano collected was moist to wet and aged guano was mostly dried. The aged material was very likely deposited within the same nesting season. Composite guano samples, except the aged and fresh, were homogenized, oven dried (60 C) and ground prior to analysis. Elemental and isotopic Analysis Excreta pH was determined in a 5:1 solution to dry matter ratio. Total carbon (C) and N were measured by dry combustion using an elemental combustion system (Costech Analytical Technologies, Inc., Valencia, CA). Total P was determined by heating 200 mg of soil or guano dry matter at 550C and dissolving the residual material in 6 M HCl. The P concentration in solution was then measured colorimetrically on a spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) as described by Murphy and Riley (1962). Percent organic matter wa s assessed by loss on ignition (LOI) during the combustion for P analysis. The concentrations of total aluminum (Al), calcium (Ca), iron (Fe), magnesium (Mg) and potassium (K) were analyzed by inductively coupled

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59 plasma optical emission spectroscopy (Perk inElmer, Inc., Waltham, MA). Extractable ammonium (NH 4 + ) and nitrate (NO 3 ) were determined by using EPA Methods 350.1 and 353.2 for NH 4 + and NO 3 respectively (EPA 1983 and 1993). Sample measurement inorganic N was conducted with an AQ2 auto analyzer ( SEAL Analytical, Inc., Mequon, WI). Analysis of total N stable isotopic ratios of guano and soil samples was performed by combustion using a Thermo Finnigan MAT DeltaPlus XL mass spectrometer (Bremen, Germany) at the University of Florida Soil and Water Science Department Stable Isotope Mass Spectrometry Laboratory (Inglett et al ., 2007). Sample isotopic ratio (R) of 15 N/ 14 2 where: 15 N Sample Sample /R Standard ) 1] 1000. The analytical precision for sample Phosphorus Fractionation Different forms of P were determined by a sequential chemic al extraction method which was modified based on techniques developed from soil and poultry litter P characterization (Hieltjies and Liklema, 1980; Ivanoff et al., 1998; Kou et al., 2009; He et al., 2010). The fractionation scheme employed sequentially se parated P pools using the fractionation procedure described in Chapter 2. Phosphorus recovery was within 10% of the total P concentration for each sample. Guano Aging Experiment Fresh and aged guano samples were placed into 50 ml glass beakers and ai r dried for 4 weeks. An initial sample of aged and fresh guano was collected to establish a baseline for changes in nutrients with time. After drying for 1, 2, 3, and 4 weeks

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60 samples of both types were collected and lyophilized prior to analysis Tripli cate samples were prepared for analysis for aged and fresh guano for each time period. Elemental Mass Deposition Calculation Total elemental deposition was calculated based on mass of guano dry matter per wading bird species. Specific guano dry matter m ass used in the deposition calculations for each wading bird species was based on dry weight guano mass per nest attempt described by Frederick and Powell (1994). D ata derived from analysis of guano dry matter were used to determine the mass of each eleme nt deposited per nesting attempt D ry matter nutrient concentration was multiplied by the mass of dry matter deposition per species where: kg nest 1 yr 1 = (nutrient (kg) / dry matter (kg))*(dry matter (kg ) / number of nest per yr) Annual area based loading rates were calculated by estimating nesting abundance at two continuously active wading bird colonies, one located in northeastern WCA 3 (~75 ha) and the other located in southwestern WCA 3 (~1.1 ha). Colony area was determined using aerial imager y. Annual nesting data for E. albus, E. tricolor, E. thula, A. alba and E. caerulea was compiled from technical reports for 1998 through 2010 (Gawlik, 1998, 1999, 2000, 2001, 2002; Crozier and Gawlik, 2003; Crozier and Cook, 2004; Cook and Call, 2005, 200 6; Cook and Herring, 2007; Cook and Kobza, 2008, 2009, 2010). Areal based deposition rate was calculated as : nutrient (g) m 2 yr 1 = (nutrient (g) nest 1 yr 1 ) (nest m 2 ) Atmospheric Deposition Data Phosphorus deposition reported by Davis (1994) was ut ilized for comparison of annual P loading rate. Annual averages for the time period of 1998 2010 from the

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61 National Trends Network atmospheric deposition database for south Florida station FL11 were used for comparison of loading rates for Ca, K, and Mg (N RSP 3, 2007). Statistical Analysis All statistical analyses were conducted using SAS version 9.2 (SAS Institute, Inc.). Descriptive statistics were used to characterize the colony composite guano samples. Distribution of P forms in aged and fresh guano samples were compared f or 15 N and N species distribution data were analyzed for statistical differences among aged and fresh guano using repeated measures analysis of variance (ANOVA). A Tuke y multiple comparison procedure was used for post hoc means separation. Correlation 15 Order coefficient. Results and Discussion Guano Characterization Chemical composition of bird guano varied as indicated by the range of data for each parameter (Table 3 1). Guano pH was neutral, which was in the range reported for seabird and oven dried samples of poultry manure and litter (Gillham, 1956; Codling, 2006; Dail et al., 2007). Organic matter conten t differed by approximately 35% between the minimum and maximum values (Table 3 1). Avian excrement consists of two parts : fecal matter and urine. Variable organic matter content may indicate different proportions of urine and fecal matter present in dif ferent samples of guano. Total Ca, N and P were high and cumulatively comprised 28% of the dry weight mass of the guano (Table 3 1). Frederick and Powell (1994) estimated Everglades wading bird guano contained 1.9% P, which is half of the mean value of 4.2% reported in this study but close to the measured minimum P concentration (Table 3 1). Scherer

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62 et al. (1995) estimated a bird guano P concentration of 1.87% based on literature of migratory waterfowl for determining P loading at an urban lake. A rang e of P concentrations 1.9 8.3% have been reported for colonial wading bird species (Stinner 1983; Bildstein et al. 1992). Veerbek and Boason (1984) reported alpine passerine species droppings were 3.7% P. Data for P reported in our study and by others a re similar to classical datasets compiled and reported by Hutchinson (1950) for a variety of bird guano. Mean Ca concentration (15.4%), and the range of N (7.8 10.6%) in the samples agree well with estimates of Ca and N used by Frederick and Powell (1994) Literature information regarding the concentration of other cations (Al, Fe, K and Mg) which may influence soil P dynamics and nutrient availability in wild bird guano (Table 3 1) is minimal Some data describing c ation concentrations has been reported for poultry litter; however, Everglades wading bird guano described here are much lower than recent data reported for poultry manure (Dail et al. 2007; He et al. 2010). Dietary differences among wild and domesticated species may explain these discrepan cies among elemental content. Cation characterization data presented here are utilized for calculation of nutrient deposition and loading rate. Bird guano, on average, was enriched in 15 15 N signature of guano differed am 15 N is understandable given the variety of bird species nesting at the colonies where samples were collected. E. albus primarily consume invertebrates and most other wading bird species are piscivorous. Although isotopic analysis of prey items was not included in this study, some inferences can be made regarding 15 N signature occurs in

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63 consumers with trophic level (Post 15 N values measured may provide an indication that species feeding at different trophic levels were likely present during guano collection. Guano P Fractionation There is no literature available for P forms in wild bird gua no from colonial wading bird species. Recent characterization studies related to poultry litter and manure and dairy manure have investigated the distribution of P in those respective materials (Nair et al., 2003; Codling, 2006; Dail et al., 2007; He et a l., 2010). In order to maintain some degree of comparability with our data, we used recent literature from poultry litter and manure research to explain and provide context to the observations made with regards to P form in wild bird guano. The primary for m of P in wading bird guano was HCl extractable P, which averaged 88.2% and ranged from 82.2 to 96.8% of the total P (Fig ure 3 3 ). Dail et al. (2007) also found HCl extractable P as the primary P form in oven dried poultry manure ; however the proportion was much lower and accounted for approximately 35 55% of the total P. He et al. (2010) reported HCl extractable P accounted for 40 45% of the total P in poultry litter and 60% of the P may be organic. Generally, HCl P is described as P bound with Ca or M g. It is likely the HCl extractable P in guano is mostly Ca P due to the wide difference in Ca and Mg concentrations measured in guano (Table 3 1). Stoichiometry provides an indication of which elemental associations are more likely dominant in the guano Mean molar ratios for Ca:P and Mg:P were >2 and <1, respectively. This rapid assessment based on elemental composition suggests that Ca P is the primary form, although some Mg P may be present. He et al. (2010) showed no significant relationship betw een extractable P and Mg in poultry litter across different

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64 extracting solutions which suggests Mg phosphate interactions may not be as significant in avian excrement as compared to Ca phosphate. Crystalline phosphate minerals were not identified in the bird guano using X ray diffraction (data not shown); however, Bates et al. (2010) observed amorphous, or more likely poorly crystalline, apatite present in tree island soil in northern WCA 3 and attributed the Ca P mineral to guano derived P. Using elemen tal analysis and X ray absorption near edge structure (XANES) spectroscopy Toor et al. (2005) reported that Ca:P ratios near 1 indicated dicalcium phosphate (CaHPO 4 2 O) as the dominant Ca P mineral in broiler and turkey litters while hydroxylapatite (C a 5 (PO 4 ) 3 OH) was dominant at Ca;P ratios >2. In another study of poultry litter, XANES spectroscopy revealed that non crystalline hydroxyapatite may comprise 50% of the total P present (Shober et al. 2006). Water soluble and KCl extractable P, i.e., labile or readily available P, cumulatively accounted for 9.65.8 % of the total P and are the second and third most abundant forms of P in bird guano, after the HCl extractable fraction (Fig. 3 2). Water soluble P comprised a higher proportion relative to KCl P in the composite guano samples. Approximately 3 5 00 mg of P per kg of guano dry matter is readily available for biological activity or mobilization in rain or surface water, based on mean guano total P concentration (Table 3 1) and proportion of water s oluble and KCl extractable P. These data provide new insight toward tree island formation hypotheses indicating birds not only focus nutrients but also bring an immediate supple of plant available P to nesting or perching habitat. Verbeek and Boason (198 4) suggested that P deposition by passerine species altered vegetation dynamics in an alpine ecosystem by creating

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65 and different vegetative characteristics relative to the low emergent surrounding vegetation. Tree species growth in northern ombrotrophic bogs responded positively at sites with bird guano nutrient inputs which reduced P and K limitation (Tomassen et al., 2005). Similar distribution of P among bioavailable P forms has been reported by others for poultry manure and litter where water soluble P was the dominant type of available P (Dail et al., 2007, Codling, 2006, He et al., 2010). Codling (2006) reported water soluble P accounted for 55% of the total P in p oultry litter. Very little (< 2%) guano P was alkaline extractable (Fig ure 3 3 ). Residual and alkaline extractable organic P on average was less than 3% of the total P. The 89.06.3 % of P deposited by wading birds in the Everglades is relatively stable and suggests that guano derived P would likely accumulate in tree island soils. The concentration of water soluble, KCl and alkaline extractable P was higher (p<0.001) in fresh guano as compared to aged guano (Fig ure 3 3 ). T his difference may be attributa ble to post depositional weathering of the guano. Leaching of soluble P and salt extractable P from fresh guano could lower the concentration of available constituents present in the material and thus increase the relative proportion of the remaining more recalcitrant forms of P. The dominant form in both aged and fresh guano was HC l extractable P, but the aged had a higher percentage of total P in this form compared to fresh guano (p<0.001). No significant difference in residual P was measured between a ged and fresh guano. The weathering of guano likely results in

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66 removal of soluble and available forms of P, while stable P remains relatively unchanged and therefore may accumulate in soil. 15 N Fresh and aged gua no samples were analyzed over time to observe post 15 N increased significantly (p<0.05) within 7 15 N signature of samples following Day 7 (Fig ure 3 4 ). A simi lar pattern was observed for total extractable NH 4 N (water and KCl extractable NH 4 N), which increased (p<0.05) by almost 1% after 7 days and did not change appreciably between Day 7 and 28 (Fig ure 3 5 ). Nitrate was not detected in aged or fresh guano ab ove the detection limit (0.02 mg L 1). The NH 4 N data is normalized to total N to show conversion of organic N to inorganic N (Fig ure 3 5 ). The distribution of inorganic and organic N changed significantly (p<0.05) during the first 7 days in the fresh gu ano (Figure 3 5 ). Urea hydrolysis is a plausible explanation 15 N nor extractable NH 4 N changed with time, suggesting the aged guano had been deposited for more than 7 da ys prior to collection (Fig ures 3 4 and 3 5 ). The assumption is logical given the P characteristics of the aged guano, which suggested the material exhibited a higher degree of weathering (i.e. loss of soluble P) relative to the fresh guano. An increase i 15 N and extractable NH 4 N provides some insight toward guano diagenesis and how post depositional changes in guano may influence soil N characteristics. Ammonia volatilization and concurrent conversion of organic N to the labile NH 4 + by microbial degradation can result in a preferential loss of light 14 N compared to 15 15 N in fresh guano during

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67 15 N signature due to volatilization of animal waste deriv ed ammonia has been described by others for avian and mammalian species (Mitzutami et al., 1985; Frank et al., 2004). Ammonia volatilization is a concern for nutrient management of poultry litter because of the high content of uric acid and protein in pou ltry manure (Nahm, 2003). Decomposition of uric acid and urea in the fresh guano would increase extractable NH 4 N concentration. Urea hydrolysis in soil and poultry litter, and subsequent ammonia volatilization has been described in the literature by oth ers (Overrien and Moe, 1967; Shankhayan and Shkula, 1975; Nahm, 2003). Measurement of NH 3 15 N NH 3 from soil following addition 15 N NH 3 signatur 15 N NH 3 signature increased, and both parameters equilibrated within 10 days after urine 15 N signature and inorganic N sugge st a similar pattern of N species alteration resulting in the 15 N signature of guano deposited in Everglades tree islands. The aged guano appears to have already reached equilibrium with respect to changes in the N isotopic signature. T ree Island Soil N and P Sources Guano deposition is one of the three main hypotheses explaining elevated P in tree island soil. Cohen et al. (2011) suggested animal waste, likely bird guano, as an 15 N in tree i slands compared to marsh soil 15 N and TP. A wider difference (2 15 N signature between tree island and marsh soil was found at two island

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68 locations in WCA 3 (Gu et al. unpublished data). Soil 15 N measured at tree islands in this study are positively correlated (n=46, r=0.95, p<0.001). A compilation of 15 N, TP, and C, N, and P ratios for soil and select N and P sources to tree island soil are presented in Table 3 3. Fukami et al (2006) reported soil total P concentrations were lower by roughly 50% at islands in a marine environment where presence of predators reduced bird use. Lack of guano deposition at islands where bird use has been reduced likely influences soil P concentra tion. 1 was 15 N signature and total P concentration as bird guano (Table 3 15 t 15 15 N value of low soil P islands (Table 3 2; Figure 3 4 15 N from high and low soil P 3 2). Wang et al. (2010) found a similar pattern in m ean 15 15 N = 15 N = 15 N signature between high and low soil P islands was suggested to indicate la ck of isotopic discrimination against 15 N during plant uptake due to N limitation (Wang et al., 2010). 15 N signature of wetland plants growing in areas of frequent wading bird use was soil P was also higher in areas of bird use (Maeck et al., 2009). Enrichment of wetland plant foliar and soil 15 N signatures have also been described in marsh habitat of the northern Everglades along a P concentration gradient (Inglett and Reddy, 2006; Inglett et al., 2007).

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69 The weight based ratio of N:P for guano from Everglades bird species and other wetland birds range from 2.6 to 8.7, which is similar to our guano characterization data (Bildstein et al., 1992; Frederick and Powell, 1994; Post et al ., 1998). Bird guano C:N ratios have been reported to range from 1.2 to 3.7, and relate well with C:N ratio of guano characterized in this study (Mitzutani et al., 1988; Bird et al., 2008). The C:N ratio of low P Everglades tree island soil is ~12 times higher than that of high P soil (Table 3 3). Island habitat affected by seabird guano derived nutrient inputs showed lower soil C:N ratio than islands where soil is unaffected by guano (Wait et al., 2005). A similar pattern of soil C:N ratio was observed between low and high P Everglades tree island soil (Table 3 3). Soil N:P ratios of high and low soil P from tree island samples were starkly different (Table 3 3). Fukami et al. (2006) reported near similar soil N:P ratios at islands with and without guano deposition. Soil N:P ratio at low soil P tree islands in the southeastern Everglades reported by Gann et al. (2005) was slightly higher (~49) than the ratio determined in low P concentration tree island soil in this study (Table 3 3). Both low and high P tree island soil N:P ratios are higher than the range of ratios for sawgrass and prairie soil of the Everglades marsh (Noe et al., 2001). Differences in soil N:P between marsh and tree island soil and among islands may be indicative of differing N:P ratio of sources and sequestration of N and P suggesting differential mechanisms of N and P deposition. Nutrient Deposition An estimation of annual mass deposition (g nest 1 ) of selected nutrients from guano at bird colonies in the Everglades is pres ented in Table 3 3. Annual loading rates calculated for two active wading bird colonies with different nesting densities

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70 provide a range of nutrient loads for a 12 year period (1998 2010). Available estimates of N and P loading at Everglades wading bird colonies range from 20 to 331 g N m 2 and 0.9 to 120 g P m 2 (Frederick and Powell, 1994). The estimated N and P loading rates rang ed from 3.3 to 37.2 g N m 2 and 1.7 to 19.6 g P m 2 from documentation of A. alba, E. albus, E. tricolor, E. thula and E. ca erula presence as described in the Materials and Methods. A limitation to these estimates is they are based on known nesting density and wading bird species composition at active colonies. The estimates do not take into account raptor species which also likely utilize Everglades tree islands. Many tree islands are either not continuously utilized for nesting or not utilized at all by wading birds for nesting habitat. However, tree islands provide locations for birds to perch or roost within the marsh. Annual monitoring of wading birds and hydrologic characteristics may be useful indicators of nutrient removal from marshes and redistribution if models can be developed to couple ecosystem scale hydrologic patterns with wildlife presence. Until quantitat ive estimates of other wildlife use habits in Everglades tree islands emerge, nutrient loading rates based on wading bird nesting density provide at minimum a range of potential nutrient inputs for comparison with other components in tree island nutrient b udgets. Troxler and Childers (2010) described the wet season N budget for a tree island in the southeastern Everglades with import rates for hydrologic, atmospheric and N 2 fixation of 26.8, 0.65 and 0.44 g N m 2 respectively. At the same location season al contribution of plant litter accounted for deposition of 4.93 g N m 2 (Troxler and Childers 2010). The N loading reported by Troxler and Childers (2010) may not be indicative of

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71 the N budget for all Everglades tree islands, but they provide a baseline for comparison of N sources which may influence the isotopic signature of soil N. The differences in potential annual N loading from birds at tree islands versus other potential N sources, and 15 N signature in tree islands likely reflects a significant contribution of guano derived N. wide atmospheric P deposition rate of 0.036 g m 2 was utilized to compare potential loading rate from guano P. These results indicated guano P loading at tree islands may have occurred at rates of 47 to 544 times higher than at tree islands only receiving atmospheric P. The highest deposition rate calculated for stable (i.e. HCl extractable and residual) P was18.4 g m 2 or 51 1 times atmospheric deposition. If atmospheric P is considered soluble and available for biological activity, a more appropriate comparison would be with the pool of guano P that is likely bioavailable. Guano derived available P loading rates ranged from 0.075 to 0.83 g m 2 which could be 2 to 23 times the atmospheric load. Guano deposition based on the distribution of P forms elucidates the potential for guano to serve as both an available P supplement that enhances soil fertility and also as a source of stable P that could lead to P accumulation. In addition to N and P loading, guano deposition rates were determined for Ca, K and Mg, which were also detected in relatively high concentrations in guano (Table 3 1). Annual deposition rates for guano deri ved Ca, K, and Mg ranged from 8.9 100.1, 0.2 2.3 and 0.1 1.0 g m 2 respectively. Average annual atmospheric deposition rates measured in south Florida from 1998 2010 for Ca, K, and Mg were 0.18, 0.09 and 0.10

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72 g m 2 respectively (NRSP 3, 2007). Depositi on of guano derived minerals may play an important role in tree island soil physical characteristics, fertility, and P chemistry. Conclusions Everglades tree islands appear to function similarly to islands in marine ecosystems where birds play an import ant role in nutrient transport from aquatic to terrestrial habitats; however, only a small percentage of tree islands are likely to receive significant guano deposition. Wildlife use may also result in accumulation of nutrients in tree island soil from bo nes, feathers, or other animal waste. This work provides new insight towards the guano contribution hypothesis by characterizing a P source in tree island soil and elucidating an isotopic indication of P source. Bird guano had high concentrations of P an d N, and guano N was enriched in 15 N. Approximately 90% of P deposited as guano was a relatively stable (HCl extractable or residual) form derived from biomass. Deposition of stable guano P and 15 N enriched N can occur at rates greatly exceeding other sou rces of P and N contributing to tree island soil. Nutrient transport and transformation from marsh areas to tree islands by wildlife is a mechanism that should be considered in restoration planning and management. Tree islands are focal points of nutrien t accumulation and loss of islands will reduce the capacity of islands to function as locales of nutrient storage within the ecosystem.

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73 Table 3 1. Chemical c haracteri stics of wading bird guano (dry matter) from two colonies in the south central Everglad es, Florida, USA. Parameter (unit) Mean Median Minimum Maximum pH 6.9 6.9 6.8 7.0 Organic Matter (%) 53.4 53.0 34.8 71.3 Total Carbon (%) 23.0 22.0 19.7 29.1 Total Nitrogen (%) 9.2 9.5 5.2 12.6 15 8.8 8.4 7.8 10.6 Total Phosphorus (%) 4.2 4.4 2.0 5.9 Aluminum (mg kg 1 ) 233.3 155.2 47.6 614.1 Calcium (%) 15.4 13.8 9.7 24.1 Iron (mg kg 1 ) 993.7 1,090.9 444.3 1,392.2 Potassium (mg kg 1 ) 5,700.3 5,729.5 1,216.1 9,752.2 Magnesium (m g kg 1 ) 2,535.9 2,432.6 866.0 4,245.2

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74 Table 3 2. Stable nitrogen isotope ratio, total carbon, nitrogen and phosphorus weight based elemental ratios, and total phosphorus of fresh and aged bird guano, mammal scat, soil and plants (mean 1SD) f rom the Everglades, Florida, USA. Sample Type (n) 15 N:P C:N C:N:P TP (%) Fresh Bird Guano (1) 8.20.1 4.00.9 2.50.2 9.8:4:1 2.00.1 Aged Bird Guano (1) 8.70.1 2.00.1 2.30.1 5:2:1 4.00.1 Mammal Scat (10) 2.01.0 8.55.5 16.63.5 157:9:1 0.50.2 High P Soil (33) 8.63.1 0.356.7 1.63 .2 5.4:0.4:1 4.13.2 Low P Soil (13) 2.21.4 31.07.5 20.53.8 631:31:1 0.07 0.02 High Soil P Plants § (10) 6.12.0 10.15.4 Low Soil P Plants § (8) 1.62.0 21.112.6 meanSD of triplicate analysis Irick et al. (unpublished data) § Data su mmarized from Wang et al. ( 201 1).

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75 Table 3 3. Annual estimated mass deposition for six species of colonial wading birds that may nest or roost in tree islands in the Florida Everglades. Species g nest 1 yr Ca K Mg N Available P Stable P M. a meric ana 3,649 135 60 2,186 83 926 A. alba 593 22 10 355 13 151 E. albus 495 18 8 296 11 126 E.tricolor, E. thula, and E. caerulea 272 10 5 163 6 69 Data calculated from mean nutrient concentration from Table 1 and dry matter deposition by species per nest from Frederick and Powell (1994).

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76 Figure 3 1. Site map depicting tree island soil sample locations and general area of the bird colonies where samples were collected in the Florida Everglades, USA.

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77 Figure 3 2. Picture of wading bird presence at an island in Water Conservation Area 3 in May 2011.

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78 Figure 3 3 Phosphorus form distribution in wading bird guano (all), and aged and fresh guano. Bar values indicate mean and errors bars 1 SD.

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79 Figure 3 4 Temporal variation in the stable nitrogen isotope values ( 15 N ) of aged and fresh guano while drying at 23 C. Data points represent mean value and errors bar indicate 1 standard deviation of triplicate sample analysis. The 15 N values observed for surface soil collected from the colon y where aged and fresh guano were collected. Variation in aged and fresh guano with time was analyzed by repeated measures analysis of variance. No significant difference was observed in aged guano. Significant difference (P<0.0001, F=54.2, df = 4) was observed in fresh guano.

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80 Figure 3 5 Temporal variation in the total extractable NH 4 N aged and fresh guano while drying at 23 C. Data points represent mean value and errors bar indicate 1 standard deviation of triplicat e sample analysis. Variation in aged and fresh guano with time was analyzed by repeated measures analysis of variance. No significant change in NH 4 N concentration was observed in aged guano. A significant (P < 0.05, F=16.9, df = 4) increase in extracta ble NH 4 N with time was observed in fresh guano.

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81 Figure 3 6 Temporal variation of the percentage of NH 4 N (inorganic N) of total N in aged and fresh guano while drying at 23 C. Data points represent mean value and err ors bar indicate 1 standard deviation of triplicate sample analysis. Variation in aged and fresh guano with time was analyzed by repeated measures analysis of variance. No significant change in the inorganic N content was observed in aged guano. A si gnificant (P < 0.05, F=5.0, df = 4) increase in the inorganic N content with time was observed in fresh guano.

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82 CHAPTER 4 BIOAPATITE CONTRIBUT ION TO SOIL PHOSPHOR US IN TREE ISLANDS Introduction The extent of contribution from specific P sources to tree isl ands soils in the Everglades remains unclear. The elevated patches of trees and shrubs dispersed throughout the Everglades provide unique habitat opportunities within the predominantly emergent marsh wetland. Tree islands have been utilized by humans for refuge (e.g. hunting camps) and crop cultivation ( Craighead; 1971; Carr, 2002; Griffin, 2002; Coultas et al., 2008; Graf et al., 2008). Historic anthropogenic use of Everglades tree islands was documented as early as the mid da land surveys (Carr 2002, Griffin 2002). Concentration of soil P can be an indication of historic anthropogenic activity or occupation, attributable to deposition of waste and refuse, presence of livestock, or application of fertilizer (Holliday and Gartner, 2007). An investigation of techniques of soil P analysis in archaeological studies described a midden site in Washington State that was composed of bones, shells and other waste, and that exhibit ed soil P concentrations an order of magnitude high er than other sites where similar materials were not observed (Holliday and Gartner 2007). Bone is composed of ~70% biogenic hydroxyapatite (bioapatite), a calcium (Ca) phosphate compound that is 18.5 % P by mass based on the theoretical molecular formul a of the mineral (Ca 5 (PO 4 ) 3 OH). Lima et al. (2002) detected bioapatite fragments >100 m in anthropogenic soil horizons (0 30 cm) of anthrosols in Amazonia in which soil P ranged from 0.3 to 1.3 %. Presence of bone material in Everglades tree island soil could also be influenced by wildlife utilization (Coultas et al., 2008). Tree islands are used by animals and

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83 qualitative assessments of wildlife in Everglades tree islands identified avian, mammalian, reptilian, and amphibian presen t (Meshaka et al. 200 2; Ruiz, 2004). Tree islands have also been described as essential habitat for some small mammal populations in the Everglades (Gaines et al., 2002). Notably, colonial nesting birds utilize some tree islands as nesting sites and may be present in high de nsities (Frederick and Powell, 1994). Skeletal remains have been document ed in soils from seabird colonies in Antarctica and were found in soil layers from former nesting sites (Heine and Speir, 1989). The potential scale or extent of bone deposition by wildlife or anthropogenic utilization of tree islands has not been quantified in literature R esearch described in Chapter 1 has revealed most P in tree island hardwood hammock communities is inorganic P, likely Ca bound P, and not associated Ca fixed wi t h inorganic C A patite and bone fragments were also observed in high P tree island soil. Deposition and accumulation of bioapatite in tree island soil could represent a large proportion of the soil P pool, with implications for ecosystem restoration and management. Determination of P contribution from biogenic apatite to the soil P pool in tree islands will enhance descri ptions of mechanisms for nutrient accumulation and the ecological role of tree islands in t he Everglades landscape Modification of sample processing procedures, such as soil sieving, may be necessary to retain bone fragments from small animals and fish, and a 1.5 to 1 mm sieve may be appropriate to enhance recovery of small bone material (Ross and Duffy, 2000). Physical separation of tree island soil, and physical and chemical analyses of different particle size classes, may help quantify bioapatite contribution to tree island soil. The presence of small bones or bone fragments in tree island soil may be

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84 distinguishable based on the chemical characteristics of the soil particle size class. It is hypothesize d that the presence of bioapatite in tree island soil can account for a high proportion of the total P in tree island soils with elevated P concentration. The objective of this st udy was to: (1) determine elemental composition of inorganic soil nutrients, (2) chemically characterize the forms of P and identify minerals present in different particle sizes, and (3) estimate the proportion of soil total P that may be from a biogenic P mineral Materials and Methods Sample Collection and Site Description Composite soil samples, consisting of two soil cores (0 10 cm) each, were collected from 22 tropical hardwood hammock plant communities within tree islands from 2005 2007 in the south ern Everglades, Florida, USA (Figure 1). Bone samples were also collected from skeletal remains of unknown species of bird, fish and snake in Water Conservation Area 3A, in Everglades from 2008 2012. The snake and bird samples were collected from tree is lands. The tropical hardwood hammock communities of tree islands are dry upland forests comprised of tropical and subtropical tree and shrub species (Ross and Sah, 2011). Hammock communities vary in areal extent and species composition, with northern Eve rglades hammocks consisting of a smaller proportion of tropical species (Ross and Sah, 2011). Hammock soils are generally well drained, and described broadly as peats (Lieghty, 1958; Wetzel et al., 2009; Ross and Sah, 2011). Soil mineral content is varia ble and Coutlas et al. (2008) classified two nonhydric hammock soils as Mollisols.

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85 Elemental Analysis and Inorganic Nutrients Soil and bone samples were oven dried (70 C) and soils were sieved (<2 mm) prior to analysis. Coarse fragments (>2 mm) which a ppeared to be bone were removed and retained for nutrient analysis. Soil pH was determined in a 1:1 ratio slurry of water and soil (Ross and Sah, 2011). Animal bones and weathered bone fragments were powdered with an automated steel ball mill Total P w as determined by combusting 200 mg of sample (i.e. soil, coarse soil fragments or bone) at 550 C for 4 hours, followed by dissolution of the ash in 6 M HCl (Anderson, 1976) The ascorbic acid method used was to analyze P concentration in all solutions us ing a spectrophotometer (Beckman Instruments, Inc., Fullerton, California) as described by Murphy and Riley (1962). Total bone Ca and magnesium (Mg) were measured in the same solutions by inductively coupled plasma optical emission spectrometry (ICP OES PerkinElmer, Inc., Waltham, MA) Total inorganic P, Al, Ca, Fe, and Mg in soil samples were determined by extraction with 1 M HCl, and a 1:50 sample to solution ratio (Reddy et. al, 1998). Soil and 25 ml of extracting solution were shaken in a 50 ml cent rifuge tube for 3 hours, centrifuged (2,100 x g) for 15 minutes, and filtered (#42 Whatman). Phosphate in the solutions was determined as described above, and cations were measured by ICP OES. Organic matter (OM) was calculated from mass loss during sam ple combustion for the analysis of total P. Total inorganic carbon (TIC) was determined using a pressure calcimeter, and calcium carbonate (CaCO 3 ) equivalent was estimated from TIC as described by Wang et al. (2011). Soil mass not accounted for by OM or CaCO 3 is defined as non carbon soil matter (NCM).

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86 Particle Size Separation A subset of soils with total P concentration >6%, by mass, were separated into two particle size classes using a wet sieving procedure (Harris, 2008). Approximately 20 50 g of so il (<2 mm) was elutriated with deionized (DI) water over a 45 m (325 mesh) sieve. Material retained on the sieve is defined as sand sized particles (2mm 45 m), and soil that passed through the sieve are defined as silt+clay (<45 m). The mass of the ov en dried sand fraction deducted from the total mass was attributed to silt+clay. Weight percent of each fraction was calculated based on the relative proportion each particle size class contributed to the total soil mass. All chemical analyses of each pa rticle size fraction were conducted in triplicate. Phosphorus Fractionation Soil P was partitioned by sequential chemical extraction using a modified fractionation procedure that differentiated five forms of P (Ivanoff et al., 1998; Reddy et al., 1998). The fractionation procedure consisted of three extracting solutions : 1.0 M KCl, 0.1 M NaOH, and 0.5 M HCl. Soil residue remaining upon completion of the sequential extraction procedure was analyzed for TP, and is defined as residual P. Soil and 25 ml of each extraction solution (sample to solution ratio of 1:50, wieght:volume) were combined in a 50 ml centrifuge tube, shaken with a reciprocating mechanical shaker and allowed to equilibrate. Sample solutions were shaken for 2, 17 and 24 hours for DDI, KCl NaOH and HCl respectively (Reddy et al., 1998). After each equilibration period, sample solutions were centrifuged at 2,100 x g for 15 minutes, supernatants filtered (#42 Whatman), and refrigerated at 4 C prior to P analysis. Phosphorus measured in fi ltrates is assumed as soluble inorganic P (Pi) and was determined colorimetrically as described above. Sodium hydroxide solutions were also measured

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87 for TP by persulfate digestion. Organic bound P in the NaOH solution was determined by difference from d issolved P and total P in solution. Available, exchangeable, or labile P is defined as P extracted with the KCl solution (Kuo et al., 2009). Organic P in the NaOH solution (NaOH Po) is P derived from humic and fulvic acid (Reddy et al., 1998). Inorgani c P extracted with NaOH (NaOH Pi) is defined as P bound with Al or Fe (Reddy et al., 1998). Acid (0.5 M HCl) extractable P (HCl P) is assumed to account for dissolution of Ca and Mg P minerals (Reddy et al., 1998 ) The residual P faction contains P miner als that were not dissolved in the alkaline or acid solutions and recalcitrant organic P (Reddy et al., 1998). Mineralogical and Micro elemental Analysis M ineral identification was conducted by x ray diffraction (XRD) using an automated XRD system (Riga ktu Corporation, The Woodlands, TX ). Each sample was powdered and prepared for XRD analysis using a powder cavity mount (Harris and White, 2008). The samples were x rayed from 0 ectron microscopy (SEM) and energy dispersive x ray spectroscopy (EDS) were utilized to for qualitative investigation of materials present in the sand sized soil and association of Ca and P. Statistical Analysis All statistical analyses were conducted u sing SAS version 9.2 (SAS Institute, Inc.). Descriptive statistics were used to characterize general soil properties and distribution of different forms of P. Correlation among soil properties was determined Order coefficients. Dif ference between chemical properties, test. An analysis of variance (ANOVA) was conducted to determine significant differences

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88 among Ca:P, and Mg:P ratios of bone, coarse s oil fragments, and sand and silt+c la y 0.05). When necessary data were log transformed to meet normality assumptions prior to means comparison analysis. Results and Discussion Soil Chemical Characterization The predominant component of the soils was NCM, which comprised on average ~43% of the soil by mass (Table 4 1). Organic matter accounted for ~33% of the soil mass, with the remaining portion (24%) attributable to carbonates (Table 4 1 ). Soil pH was alka line. Total P concentration was 55.5 g kg 1 (Table 4 1). Total inorganic P was positively correlated (r = 0.99, p <0.01) with total P, and accounted for 93% of the P present in these soils. The high proportion of inorganic P measured by a single acid extr action in these high P tree islands soils is corroborated by previous P fractionation studies where most (~85%) of the tree island soil P was suggested to be inorganic, Ca bound, P ( Chapter 2 ). Ross and Sah (2011) reported a negative correlation with tota l P and organic carbon in tree island soil providing further support to the relationship between increasing inorganic P and high P tree island soils. Little information describing characteristics and concentrations of cations in tree island soil s is ava ilable Data from Chapter 2 indicate total Ca and Mg concentrations in tree island soil were elevated as compared to concentrations reported for marsh soil in the central Everglades (Craft and Richardson, 1993; Bruland et al. 2006). Acid extractable Al, Ca, Fe and Mg were reported by Reddy et al. (1998) for marsh soil in the central Everglades (i.e. WCA 3A) provide context for the data presented here for inorganic cations in tree island soils (Table 4 1). The values for acid extractable Al and

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89 Fe, were ~ 1 2 g kg 1 lower in the high P hammock soils than reported for marsh soil, and Ca and Mg ~2 4 times greater in the hammock soil as compared to data for the marsh soil (Reddy et al., 1998). Chemistry data describing groundwater beneath the head region of tw o islands showed similar trends to the soil data, indicating Ca and Mg concentrations were elevated in comparison with marsh surface waters (Wetzel et al., 2011; Sullivan et al., 2012). Total inorganic P showed positive correlation (r = 0.97, p <0.001) wi th NCM, and negative correlation (r = 0.81, p <0.01) with TIC. Previous studies reported no significant correlation between TIC or soil CaCO 3 suggesting interactions between P and Ca associated soil CaCO 3 were not influenc ing P release or retention in t ree island surface soil (Ross and Sah, 2011; Chapter 2 ). The positive correlation with NCM, and negative correlation with TIC in these high P tree island soils indicates that inorganic P dynamics are likely controlled by Ca or Mg phosphates. Ross and Sah (2011) proposed that the positive correlation between total P and NCM in hardwood hammock soil may indicate the presence of Ca phosphate minerals, such as apatite. Additionally, apatite presence has been documented in tree island hardwood hammock soils w ith total P concentrations exceeding ~60 g kg 1 and also reported in soil from a tree island in the north central Everglades (Bates et al., 2010; Chapter 2 ). Bates et al. (2010) described detection of amorphous apatite, and attributed the presence of poo rly crystalline apatite to deposition of P rich guano. Apatite identified in hardwo od hammock bulk soils (<2 mm) in Chapter 2 suggested the mineral was present in a relatively crystalline form based on the presence of multiple diagnostic apatite peaks, ob served by XRD. Bone fragments were also observed in the hardwood

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90 hammock soils and likely contributed to the apatite observed in tree island soil ( Chapter 2 ). The reports of both poorly crystalline and crystalline apatite in tree island surface soils ma y indicate multiple sources of this phosphate mineral. From a broad p er spective deposition of P rich animal wastes is a likely mechanism for presence and accumulation of apatite in these soils. M ost of the P in Everglades wading bird guano was likely Ca bound P, although no crystalline phosphate minerals were observed by XRD (Chapter 3) Non crystalline apatite has been described as a component of poultry manure and litters (Toor et al., 2005; Shober et al. 2006). Coultas et al. (2008) postulated guano deposition and bone could account for high P in tree island soils. Particle Size Separation: Chemical Characteristics, P Forms and Minerals Sand sized soil particles on average comprised 48% of the soil mass in high P tree island samples. Coultas et al. ( 2008) reported sand sized particles accounted for 62 and 87% of the mass of surface (0 15 cm) soil at two islands in the southern everglades. Although the mass of soil material was distributed relatively evenly between the sand and silt+clay particle siz e classes, the concentration of OM, total P, and acid extractable Ca of the particle size classes were dissimilar (Table 4 2). Organic matter concentration was significantly (t=3.4, P=0.027) greater in the silt+clay fraction, accounting for ~68% of total soil OM (Table 4 2). High proportions of organic matter in the silt+clay soil particle size classes has been well described in literature, and most OM in the fine soil fractions is attributed to humic and fulvic acids (Anderson et al., 1981; Catroux and Sc hnitzer, 1987). The total P concentration of the sand fraction was significantly (t=5.3, P=0.006) greater than, and approximately twice as high as, the silt+clay sized soil material (Table

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91 4 2). The high P concentration of the sand sized soil contrasts other studies, where most soil total P has been reported in association with clay sized particles (Day et al., 1987; Pierzynski et al., 1990; Lienweber et al., 1997) Total P present in the sand fraction accounts for 67% of the P present in these high P s oils by mass. A similar trend was observed in the concentration of acid extractable Ca, which was significantly (t=7.2, P=0.001) higher in the sand fraction (Table 4 2). While acid extractable Ca was higher in the sand fraction, TIC concentrations were n ot significantly different (Table 4 2). If TIC concentration is assumed as a measure of CaCO 3 content then higher concentration of acid extractable Ca present in the sand sized soil may suggest an additional source of inorganic Ca. On average NCM was ~4 0% higher in the sand fraction as compared to the silt+clay fraction (Table 4 2). The general pattern of high P and NCM concentration in the sand fraction suggests non carbon phosphate mineral presence in the sand sized soil. High total P, NCM and acid e xtractable Ca concentration, coupled with similar TIC concentration between particle size classes suggests P in the sand fraction is associated with Ca phosphate minerals. The relatively low acid extractable Mg concentration, as compared to Ca, present in the sand fraction further substantiates this theory. Soil P fractionation data for the sand and silt+clay particle size classes revealed most of the P in both size classes was extractable by 0.5 M HCl, and therefore likely associated with Ca (Figure 4 2 ). The proportion of P extracted with HCl during the fractionation sequence was significantly (t=7.8, P=0.001) greater in the sand fraction

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92 (Figure 4 2). Phosphorus extracted by HCl accounted for 97% of the total P in the sand sized soil and 91% of total P in the silt+clay fraction. No significant differences were determined between particle size classes for other forms of inorganic P, such as KCl extractable P or NaOH Pi (Figure 4 2). The quantity of P extracted by KCl may have been affected by the el utriation procedure. Presumably some water soluble P may have been removed from the soil during the particle size separation. Water soluble and KCl extractable P comprise d a very small proportion (i.e. < 3 %) of the total P pool in Everglades tree island h ardwood hammock soils ( Chapter 2 ). Removal of a portion of this pool would likely not have a large effect on the overall distribution of P. Mass balance calculations, based on mean weight percent of the sand and silt+clay size classes and concentration o f each P fraction, confirmed P recovery was greater than 90% of the total P measured in these soils. Residual P was the second largest pool of soil P in both particle size classes, comprising approximately 3 and 8% of the total P in the sand and silt+cla y fractions, respectively (Figure 4 2). The SC fraction had a significantly (t=8.2, P=0.001) higher proportion of residual P in the soil P pool indicating silt+clay sized particles have a larger role than sand sized particles in P stability in tree island soils (Figure 4 2). Organic bound P extractable by NaOH was also a more significant (t=9.0, P=0.005) portion of the silt+clay fraction (Figure 4 2). An increase in the proportion of NaOH Po and residual P in the silt+clay sized soil may be explained by the relatively higher concentration of OM. Organic P is the dominant form in soils of native areas of the P limited Everglades marsh (Reddy et al., 1998; Bruland et al., 2006). Investigation of mechanisms and rates for transformation of inorganic soil P into more stable organic P

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93 forms may help elucidate the theory put forward by Wetzel et al. (2009) describing tree island soils as a landscape sink for P in the Everglades. Apatite was observed in both the sand and silt+clay particle size fractions (Figure 4 3). Other crystalline phosphate minerals were not observed in these soils. Harris (2011) reported phosphate minerals are rare in Everglades soils. Calcite (CaCO 3 ) and quartz (SiO 2 ) were also observed in sand and silt+clay sized soil (Figure 4 3). Ar agonite (CaCO 3 ) was identified in a previous study of tree island hardwood hammock bulk soil (<2 mm), but not detected in these soil samples ( Chapter 2 ). Calcite presence in each particle size class may be attributed to small pieces of limestone or shell in soil. Recent geochemical modeling of tree island surface and groundwater suggests calcite precipitation may occur in these soils and be a process influencing pedogen sis (Sullivan et al., 2011 ). Quartz has also been documented in the region within the clay sized soil particle class (Sodek et al., 1990). Detection of apatite in the SD fraction supplements the soil chemistry data and suggests most of the P in this particle size f r action is associated with non carbon Ca phosphate minerals. Further, obser vation of bone fragments in the coarse soil (>2 mm) suggests the apatite, and subsequently the P, in the sand fraction of these high P tree island soils is derived from bioapatite. Presence of bone in the sand fraction was confirmed using light microscopy SEM and EDX (Figure 4 4). Although the SEM image and EDX data are observational data of bone and presence of both P and Ca in the sand sized soil, they provide context to the chemistry data. Bioapatite Contribution The concentration of total P (10.2%) measured in the sand fraction of these soils was similar to P concentrations (~10 18%) reported for chemical assays of bone from

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94 various vertebrate species (Blitz and Pellgino, 1969, Pate et al., 1989; Farlow and Argast, 2006) Average total P concentrat ion of animal bones collected from skeletal remains in the central Everglades was 11.6 1.3% ( 1 standard deviation) Weathered bone fragments (>2 mm), collected from these and other high P tree island soils (n=5) had an average total P concentration of 13.3 0.4% ( 1 standard deviation). Gravel sized (>2 mm) fragments were reported to comprise ~20 35% of the surface (0 15 cm) soil horizon by mass at two islands in the southern Everglades, and contained some bone (Coultas et al., 2008). Depositi on and weathering of bone could explain patterns of high P, Ca, and NCM concentration in the sand fraction of these soils. The theoretical Ca to P (Ca:P) molar ratio of apatite is 1.67, a weight based equivalent of 2.15 is also reported in the literature (White and Hannus, 1983). The degree of substitution of ions for Ca, PO 4 or OH, would affect the elemental ratios of apatite in soils or bones. The molar Ca:P ratio for bone is reported to range from 1.54 to 1.89 (Blitz and Pellgino, 1969; White and Hann u, 1983; Farlow and Argast, 2006). The Ca:P ratios for animal bones, coarse fragments, and sand sized soil were significantly (F=18.72, P<0.001) less than SC sized soil (Table 4 0.05) differences were determined for Ca:P ratios among animal bones, coarse fragments and sand sized soil, which had mean values that ranged from 1.56 to 1.87 (Table 4 3). The Mg to P (Mg:P) molar ratios for the same materials r evealed a similar pattern where the animal bones and coarse soil fragments were significantly (F=8.59, P=0.003) lower than the silt+clay determined among animal bones, coarse fragments and sand sized soil (Table 4 3).

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95 The low Mg:P molar ratio of the sand and silt+clay sized soil indicates Mg bound P is not a domina n t form of soil P in these tree island soils. Average Mg concentrations of bones from a variety of vertebrate species have been reported in literature to range from 0.36 to 0.83% and have Mg:P molar ratios of 0.04 to 0.05 (Blitz and Pellgrino, 1969; Farlow and Argast, 2006; Blincoe et al.. 1962, Pate et al., 1989). The mean Mg:P molar ratios of Everglades animal bones, coarse fragments and sand sized soil agree with the data available in literature (Table 4 3). The high P concentration and apatite detected in sand sized soil coupled with similarity of Ca:P and Mg:P molar ratios among the animal bones, coarse fragments, and sand sized soil suggests most of the P in the sand fraction is derived from bioapatite. If all of the P extractable by 0.5 M HCl during the fractionation sequence in the sand sized soil is assumed as bioapatite ~ 65 % of the T P could be attributed to bone in these high P t ree island soils. This is based on the weight fraction (48%) of the sand sized soil of the total soil mass and the proportion of the soil total P accounted for by P extracted with 0.5 M HCl during the extraction procedure from the sand sized soil The to tal P concentration of the silt+clay fraction was 4 .6 % by mass (Table 4 2). Average total P concentration measured in guano collected from the Everglades was 4. 2 % and had an average Ca:P molar ratio of 2.84 (Chapter 3) which was also similar to the avera ge Ca:P molar ratio (2.96) of the silt+clay (Table 4 3). Similarity of these chemical properties between guano and silt+clay soil particles may indicate influence of different nutrient sources to different particle size classes of soil. T otal P concentra tion in ornithogenic soils from penguin rookeries in Antartica ranged from 2.6 to 5.4 % (Speir and Cowling,1984) M ost (>90%) of the soil total P was described as inorganic P

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96 immobile and suggested to accumulate (Speir and Cowling, 1984 ). Guano derived n utrients, in particular P, have been reported to accumulate in soil and continue to influence productivity in wetlands even after colony abandonment (Oliver and Schoenberg, 1989). Previous studies have shown higher concentrations of TP in clay sized parti cles, relative to silt or sand sized particles, in soils that have had fertilizer, sewage sludge, or manure ap plications. Apatite presence, P fractionation data, and low Mg:P molar ratio suggests most of the P in the silt+clay fraction is also Ca bound P It is likely that a portion of this apatite is derived from bioapatite suggesting more than 65% of the soil TP could be attributed to bone. Apatite has also been identified in similar ornithogenic soils from seabird colonies in Antarctica (Tatur and Ke ck, 1990). It is unknown what proportion of this fraction might be attributable to guano or excrement from other animals. The potential for in situ precipitation of secondary apatite is not known for these soils, however recent research focused on ground and surface water geochemistry at a high soil P (~4% P by mass) hardwood hammock tree island suggested apatite genesis in surface soil is unlikely (Sullivan et al., 2011 ). The results described by Sullivan et al. ( 2011 ) suggest apatite presence in tree i sland soil is due to deposition of exogenous P rich material. The XRD data for animal bones, coarse fragments, and sand and silt+clay sized soil provides some basis to theorize bone deposition may be a P redistribution mechanism in the Everglades and may be an ongoing process. Diffraction peaks for the known bone samples revealed relatively poor crystallinity with low resolution of the primary peaks for hydroxyapatite (Figure 4 3). An increase in the resolution of

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97 diagnostic apatite peaks was observed a long the following gradient: animal bone
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98 was suggested to indicate weathered bioapatite (White and Hannus, 1983). Historic anthropogenic use of tree islands is well documented and evidenced by presence of buried bone and artifacts within the profile of tree island soil from the Everglades (Carr, 2002; Griffin, 2002; Coultas et al., 2008; Graf et al., 2008). These r eport s of bone within the soil profile of tree islands are direct confirmation of bone burial with soil accretion. Physical events, such as treefall could serve as a mechanism to resurface buried bone and thus potentially further weather and release mineral P. Regardless of source, historic anthropogenic or ecological focusing by wildlife, the XRD patterns suggest an increased weathering gradient persistent from animal bone to apatite in the sand and silt+clay sized tree island soil (Figure 4 3). Further micromor p h ological investigations of apatite in the silt+clay sized soil fraction may revea l more conclus ive determination of bioapatite contribution to silt+clay sized soil fraction. Tatur and Keck (1990) described apatite in guano as poorly crystalline in a specific comparison with fossilized penguin bone. The relative abundance of crystalli ne apatite observed in the silt+clay fraction (Figure 4 3) may indicate a large proportion of this apatite could be derived from weathered bone. In addition to further investigation of the silt+clay fraction s ite specific geochemical equilibrium models wo uld be useful to determine mineral stability and potential for secondary precipitation. Isotopic analysis may provide some indication of apatite source, however little information can be extrapolated regarding the extent of apatite contribution in soil. In order to assess how bioapatite may influence soil P concentrations across a range of high P tree island soils, Ca:P and Mg:P ratios of inorganic Ca, Mg and P were compared with concentrations of total inorganic P and NCM. T otal inorganic P and

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99 NCM were negatively correlated (r = 0.96, p<0.001, inorganic P; r = 0.98, p<0.001, NCM) with Ca:P molar ratio (Figure 4 6 5 ). As total inorganic P concentration in the high P hammock soils increases the ratio of inorganic Ca:P decreases and begins to approach th e molar ratio of apatite (Fig. 4 6 5 ). The theorictal apatite ratio is within the range of Ca:P ratios determined for Eveglades animal bones and coarse fragments (Table 4 3). Data from Chapter 1 showed decreasing total Ca:P ratios with increasing NCM and P and theorized this relationship indicated an increase in apatite in tree island soil. The inorganic Mg:P ratio follow a similar pattern of negative correlation with inorganic P (r = 0.78, p< 0.001) and NCM (r = 0.73, p< 0.001) suggesting Ca, Mg and P m ay be intricately linked in these soils and from a similar source (Figure 4 6 5 ). With increasing inorganic P and NCM the Mg:P is near and within the range of Mg:P molar ratio of animal bones and coarse fragments (Table 4 3, Figure 4 6 5 ). These data suppo rt the results from the chemical P fractionation of the sand sized soil and suggest that most of the P in high tree island soil is likely biologically derived apatite. Investigation of total inorganic cations and P ratios may be a good technique for futur e investigations of Everglades tree island soil to quickly elucidate potential P source. The magnitude and spatial extent of modern and historic sources of bioapatite to tree island soils remains an open question. Further studies which quantify potential depositional rates verses historic material will greatly elucidate questions related to mechanism for high P in Everglades tree islands. Regardless of source, modern or historic, post depositional weathering of bioapatite and potential P release or retent ion has implications for Everglades ecology because of the P limited nature of the ecosystem.

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100 Conclusions This study is the first to quantify a source of P to tree islands as a proportion of the soil P pool. The importance of integrating multiple sourc es of soil P in development of future tree island P budgets is also implied by these data. Numerous mechanisms likely contribute P accumulation in tree islands, each playing greater or lesser roles in different islands across the Everglades landscape. Th e magnitude of a particular P source may vary across different islands. The high proportion of inorganic Ca and P observed in the sand sized fraction in tree island soil coupled with apatite presence and observation of bone is empirical evidence of bioapa tite as a source of P in high P tree island soil. Sequential chemical fractionation of P in the sand fraction of high P tree island soils indicates ~65% or more the soil P pool may be attributable to bioapatite. The relationship of inorganic Ca, Mg and P stoichiometry with increasing inorganic P and NCM concentration is an indication that bioapatite contribution influences increasing P concentration in these tree island soils. Further investigation of sources of bioapatite to tree islands, and bioapatite dissolution rates will advance ecological theory regarding P sequestration in tree island soils, and concerning mechanisms for nutrient distribution across the Everglades patterned landscape.

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101 Table 4 1. Select soil properties from 22 high P (>10 g kg 1 ) tree islands with tropical hardwood hammock plant communi ties in the Florida Everglades. Data presented in a s ubset from Ross and Sah (2011) Parameter (unit) Mean 1 SD pH 8.15 0.2 Inorganic Carbon (%) 2.97 1.7 Non carbon Matter (%) 42.8 18 Organic Matter (%) 32.5 9.4 Phosphorus (g kg 1 ) 55.5 25 1 M HCl Extractable Aluminum (g kg 1 ) 0.83 0 .5 Calcium (g kg 1 ) 172 19 Iron (g kg 1 ) 1.93 1 4 Magnesium (g kg 1 ) 3.85 1.9 Phosphorus (g kg 1 ) 51.5 23

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102 Table 4 2. Select chemical properties of sand sized (2 mm 45 m), and silt + clay sized (<45 m) soil from tree islands tropical hardwood hammock plant communities in the Florida Everglades (n=3). Data represent mean 1 standard deviation. Different letter within eac h row indicate significant Soil Particle Size Class Parameter (%) 2 mm 45 m <45 m Total Phosphorus 10.2 2.0 a 4.64 0.7 b Organic Matter 19.0 8.8 a 36.5 1.3 b Non carbo n Matter 62.5 15 a 44.9 6.7 a Inorganic Carbon 2.43 0.5 a 2.38 0.6 a 1 M HCl Extractable Calcium 21.9 1.6 a 14.4 0.9 b Magnesium 0.33 0.2 a 0.45 0.3 a

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103 Table 4 3. Molar ratio of calcium to phosphorus (Ca:P), and magnesium to P ( Mg:P) for soil, animal bones, and different soil particle size classes (coarse fragments, sand, and silt + clay). Data represent mean 1 standard d eviation of the mean. for log transformed data using the Bonferroni multiple means comparison method. Sample Type (n) Ca:P Mg:P Animal Bones (5) 1.56 0.15 a 0.04 0.01 a Weathered Bone Fragments ( 5 ) 1.79 0.06 a 0.03 0.01 a Sand (3) 1.87 0.24 a 0.05 0.03 ab Silt + Clay(3) 2.96 0.62 b 0.14 0.10 b Ratios are based on total elemental analysis. Ratios are based on 1 M HCl extractable Ca, Mg, and 0.5 M extr actable from the P fractionation sequence

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104 Figure 4 1. Tree island soil sample locations in the Florida Everglades, USA.

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105 Figure 4 2. Soil phosphorus (P) forms of sand, and silt and clay particle size classes. The asterisks indicate significant (P<0.05) differences between particle size classes.

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106 Figure 4 3 X ray diffraction (XRD) patterns for, (a) animal bones, (b) weather bone fragments, (c) sand sized soil, and (d) silt + clay sized soil part icles.

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107 Figure 4 4. Scanning Electron Microscopy (SEM) image and energy dispersive x ray spectroscopy (EDS) dot patterns for calcium and phosphorus for sand sized tree island soil.

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108 Figure 4 5 Sc atter plots of the relationships of the molar ratios of 1 M HCl extractable calcium to phosphorus (Ca:P) and magnesium to phosphorus (Mg:P) with 1 M HCl extractable phosphorus (Total Inorganic P) and non carbon matter. Solids lines represent the Ca:P mola r ratio for hydroxyapatite (Ca:P = 1.67) and Mg:P measured for select Everglades animal bones (Mg:P = 0.04). Non p value denoted for each plot.

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109 CHAPTER 5 SYNTHESIS AND FURTUR E RESEARCH Descr iption of nutrient distribution mechanisms throughout the Everglades will continue to evolve as research efforts focus on specific ecological interactions of abiotic and biotic drivers at different scales This study is the first to describe tree island s oil P chemistry and chemical characteristics of potential sources of nutrients to tree islands and to elucidate soil chara cteristics that indicat e potential sources of nutrients Chapters 2, 3 and 4 present approach es that address questions about nutrient characteristics and sources of nutrient contribution to soils in a patterned landscape Increase in TP concentration in tree island s urface soils is a result of the accumulation of Ca bound P. Deposition of bird guano and bioapatite at tree islands are s ources of P that can explain P accumulation in tree island soil. These t wo potential sources of P were investigated, both of which could also contribute Ca bound P to tree island soil. Bird guano has been suggested as a contributor of P to tree island so ils and concentrations of Ca bo u nd P that range from 1.6 to 5.6% by mass for guano analyzed in this study Guano also appears to be a potential source of available P ( 3,500 mg kg 1 ) and deposition could lead to the plant growth and island expansion Acc umulation of guano derived P is tree island soil may also occur because most of the P in guano required acid extraction. Post depositional weathering of guano would likely increase the proportion of P in guano and thus increase the potential scale of cont ribution to tree island soil. The guano was ~50% OM by mass and decomposition of this OM could potentially lead to preferential accumulation of recalcitrant guano P forms in soil, such as Ca bound P. Preferential accumulation of guano in tree soil would likely occur in the fine (i.e silt+clay) particle size class. The specific form of guano

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110 derived P which could accumulate is unknown at this time. However, based on the P concentration, dominant P pool (i.e. Ca bound P), and Ca;P molar ratio measured in bird guano in this study it is possible amorphous apatite was present and could influence P accumulation in soils where birds nest or visit frequently. The characterization of wading bird guano suggests guano deposition alone likely cannot account for TP concentrations observed in some tree island surface soils This assertion is based on the maximum T P concentration measured in bird guano ( 5.9% ) and maximum TP concentration measured ( 8.8% ) in soil in this study. This suggestion may be overly simplistic ; however it provides a tangible justification to consider other sources of animal derived P to tree island surface soils that may also be related to animal or anthropogenic use of the islands The deposition and presence of bioapatite can reconcile P c oncentration in tree island soil that may not be accounted for by bird guano deposition. Analysis of skeletal remains and weather bone fragments from tree islands soils indicated samples were ~11 13% P by mass and were therefore great enough to support th e high concentration s of P measured in some tree island soils. Deposition of bioapatite would not likely provide a significant source of available P as compared to guano; however, bioapatite is likely to accumulate and could represent a slowly available r eservoir of inorganic P in tree island soils. Separation of guano derived nutrients and deposition of bioapatite becomes challenging because of the potential for concomitant deposition of guano and apatite derived from bone. Presumably soils at islands affected by guano deposition would also have other nutrient inputs from avifuana, such as bones from carcasses or dropped

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111 prey items, which would be sources of bioa p atite. The proportion of the magnitude of each of these specific sources of P deposition t o tree islands remains unknown. The presence of bioapatite may also be an indicator of historic anthropogenic utilization of tree islands. Relative to historic use it can be assumed that current anthropogenic inputs from subsistence living at island sit es are negligible. Observation of skeletal remains on the soil surface at islands indicates a recent deposition associated with wildlife activity, and also very likely deposition of animal excrement or bird guano. The magnitude of historic inputs of nutr ients to tree islands relative to current sources is a topic open for investigation and debate. I t is reasona ble to theorize that current nutrient accumulation may not reflect past conditions in the ecosystem if most of the P that has accumulated in tree island soil within the Everglades was deposited via historic mechanisms, such as larger wading bird populations or anthropogenic foraging. T he significance and timing (i.e. current or historic) of mechanisms that influence the distribution of animal deriv ed nutrient depo s ition and accumulation in tree island soil remain unresolved and are open areas of future research The results of this study provide new insight toward resolution of questions regarding specific nutrient sources to tree islands in the E verglades and P forms present in tree island soils. These results can help guide restoration planning for tree islands in the Everglades by providing information about chemical characteristics of potential nutrient sources, and forms of P that may be sequ estered in tree island soil F uture research projects that aim to further describe transport and transformation of nutrients within and between ecosystems may also benefit from the results of this work

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112 to help explain accumulation of soil P and mechanisms that may lead to nutrient redistribution. Chapter Summaries Chapter 2 Soil P Characteristics The results discussed in Chapter 2, advanced the current knowledge of tree island soil ch emistry and P characteristics This work presents new data describin g total cation (Al, Ca, Fe and Mg) concentrations, P fractionation, and mineral identification of tree island soil. The results of the soil and P chemistry indicate most of the soil P in tree islands is in an inorganic form. This high concentration of in organic P provides the basis to suggest mechanism s dictating P biogeochemistry in tree islands is different than the surrounding marsh Most of the inorganic P is present in a form that is very slowly available (i.e. extractable by HCl during the fraction ation sequence) and is suggestive of an external source. These data confirm current hypotheses regarding accumulation of soil P from external sources. Although the P fractionation data do not directly describe which types of P minerals are dominant in t he Ca /Mg P pool, the concentrations of NCM and cations provide a good indication of P mineral association. The stoichiometry of total Mg:P indicates Mg phosphate minerals are not dominant because the ratio is << 1. Further, the positive relationship of NC M, NCCa with P in the Ca /Mg P pool indicate two chemical characteristics of these soils: (1) P association with carbonate species is likely not a significant aspect of P chemistry in high P tree island surface soil and (2) Ca phosphates are the primary f rom of P in these tree island soils The observation of a patite in these soils with XRD confirmed the characteristics inferred by the chemical

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113 analyses. Other Ca phosphate minerals were not detected in these soils suggesting apatite is the dominant form of P in high P tree island soil. Chapter 3 Wading Bird Contribution to Soil Nutrients Avian species are a source of nutrients f o r tree islands in the Everglades In particular, wading birds transport nutrient s from the marsh to resting or nesting loca tions in t ree islands Guano deposition can be a significant source of nutrients to tree islands utilized by wading birds, although few locations likely receive continued deposition of guano. Other potential nutrient inputs from wading bird use are bones from carcasses dr opped prey items, feathers, and eggs. R esults discussed in this chapter provided new insight regarding chemical characteristics of a P source to tree islands and an isotopic indication of the soil P source. The guano from Everglades w ading birds was high in Ca (~15%), N (~9%), and P (~4%). G 15 Tree islands soils with high total P 15 15 N showed a significant (P<0.001) positive (r=0.95) relat ionship. Most of the P in the guano required acid extraction. Cumulatively, stable P accounted for ~90% of the total P in the bird guano suggesting potential for guano derived P to accumulate in tree island soil at locations bird frequently utilize. E stimation of potential guano nutrient loading indicates guano deposition of P 15 N value can occur at rates much great er than other P and N sources to tree islands in the Everglades. Loss of tree island habitat will reduce the potential ar ea within the Everglades where nutrients removed from the marsh by wading birds may be transported to tree islands. Reduction of the nutrient transport mechanism s will reduce the potential for nutrient storage and accumulation. Restoration and

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114 management planning should incorporate measures to assess n utrient transport to maintain the function of tree island nutrient storage in the Everglades Chapter 4 Bioapatite Contribution to the Soil P Pool The determination of apatite as the dominant form of soi l P in tree island soil s with high (>60 g kg 1 ) P concentration and observation of bone fragments in soil from the work presented in Chapter 1 indicated bioapatite contributes to the soil P pool. T he extent of bioapatite contribution to high P island soil was addressed in this chapter and the results are the first to quantify a potential source of tree island soil P as a proportion of the total P pool. Multiple sources influence P accumulation in tree islands in the Everglades including deposition of ani mal excrement and skeletal remains, and the post depositional stability of these materials The contribution of bioapatite may play a larger role in the tree island soils described in this chapter as compared to other islands within the ecosystem. As men tioned in Chapter 3, regarding deposition of guano derived nutrients, t he scale of specific P source is likely variable across islands throughout the Everglades This variability among islands would certainly apply to bioapatite deposition, which may be i nfluence d by animals and, or, historic anthropogenic utilization Data from this chapter describe some specific indices which may help future research identify if bioapatite should be considered as a potential source of apatite and P to the soil P pool. Physical separation and subsequent chemical analysis appears to yield promising data indicative of the presence of bone in tree island soil. Specifically high concentrations of inorganic Ca and P and apatite detection with XRD in the sand size tree isla nd soil coupled with observation of coarse bone fragments is a strong indication of bioapatite as a source of P in high P tree island soil. Similarity of the

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115 stoichiometric ratios of Ca:P and Mg:P of bone and bone fragments found in soil with sand sized soil is an indication that bioapatite is likely the primary source of P in these high P tree island soils. Sequential chemical fraction of P in the sand fraction of high P tree island soils indicates ~ 65 % or more of the soil P pool may be attributable to bioapatite. The source and fate of bioapatite remain debatable ; however based on the XRD data from bone, weathered bone fragments and sand and silt+clay sized soil, bone deposition may be an ongoing process influencing P accumulation in tree islands. F urther investigation of recent wildlife derived bone and historic anthropogenic deposition inputs will help clarify the current potential for ongoing P accumulation in tree island soil. Suggestions for Fut ure Research The results of this work demonstrate the necessity for further study into nutrient transport and fate and P stability in tree islands in the Everglades. Further assessment of nutrient stability and fate in tree islands will advance theories about nutrient accumulation and redistribution in a patterned landscape. As restoration efforts move forward throughout the Everglades estimation for potential P mobility from tree islands will enhance the ability of land managers to predict how P stored in tree islands will behave under changing hydrolo gic conditions Additional ly further research into the stability of specific P sources will give an indication of which materials may accumulate in relatively larger quantities Further assessment of accumulation of additional plant nutrients (i.e. othe r than N and P) would further the knowledge of tree islands function in the Everglades. Also research regarding the fate of accumulated nutrients would help elucidate landscape scale pathways of nutrients within the ecosystem.

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116 Nutrient Transport and Fate Cation a ccumulation Thus far little attention has been given to the concentrations of plant nutrients besides N and P in tree island soil. Elemental data for bird guano described in Chapter 3 suggest, in addition to P, large amounts of plant nutrients (i .e. Mg and K) may be transported from the marshes to tree islands. Future research may seek to investigate elemental composition other macro and micronutrient s of tree island soil to evaluate how addition of cations may influence plant growth and soil ge ochemistry. Nitrogen loss from wading bird colonies A future study that could emerge from the Chapter 3 results is an investigation of N loss from wading bird colonies. Ammonia volatilization from guano and soil at tree islands could be quantified to es timate ecosystem wide N loss from the Everglades due to wading bird presence. Also the fate of ammonia could be investigated to determine how N is gained or lost from soil at wading bird colonies. Does guano derive d ammonia redistribute after deposition? Is foliar absorption of ammonia a significant pathway of N redistribution to plants during the nesting seasons? Is ammonia lost from tree islands and redistributed to nearby marsh areas? Research into these questions will advance the description of N p athways in the Everglades ecosystem. Phosphorus Stability Soil The soil P fractionation data from Chapter 2 establishes different forms of P based on chemical extraction. The high concentration o f inorganic P, in particular Ca bound P, in tree island soi l warrants analysis of P stability. Geochemical equilibrium model s can predict which P species may control P release in soil based on porewater

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117 geochemistry and mineral solubility. Application of an equilibrium model acro ss a range of tree island soil P concentrations would be helpful to determine if different P minerals control P retention. An experimental approach based determination of P species stability from an equilibrium model of soil porewater chemistry coupled with analysis of clay mineralogy co uld further describe P characteristics in tree islands. Although much of the P requires acid extraction, this pool may be prone to leaching. The positive relationship of total P with WSP and available P, as described in Chapter 2, is an indication that hi gh P tree island soils may be localized sources of P. An investigation of potent ial leaching from surface soil would help explain the stability of soil P in tree islands. A study that utilizes a modified extraction sequence by repeating water and weak sa lt solution extractions until P can no longer be detected, then proceding with alkaline and acid extractions would allow for determination of changes in the Ca P pool. If repeat extractions reduced the Ca P then estimates for potential leaching could be d eveloped. Concentration of cations (Al, Ca, Fe and Mg) could also be determined on the extracting solutions to see if P associated cations are also potential loss due to leaching. Investigation of the s tability of Ca bound P in tree island soil is also w ill advance ecological theory regarding P sequestration in tree islands soil and nutrient distribution in the Everglades. Animal wastes The guano P fractionation data presented in Chapter 3 reveal most of the guano P is inorganic and likely bound with Ca. Additionally, the presence of bone in tree island soil also indicates another inorganic P source, and primarily a Ca phosphate mineral. Further investigation of the stability of P in these materials wou ld advance the knowledge of how they contribute to p ost depositional P release or retention. Other

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118 potential P source material, such as feces from mammalian species, could be included to further describe how animal wastes may react in tree island soil. Animal wastes could be subjected to a modified P fract ionation procedure as described above for soil to determine how much of P could be extracted with water or a neutral salt solution. These data would be helpful for determining if P derived from certain animal wastes is more or less likely to accumulate. As described above for soil, cations such as Al, Ca, Fe, K, Mg, Mn and Zn could also be analyzed in the extracting solutions to determine the potential contribution of important plant nutrients from these materials.

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119 APPENDIX INVESTIGATION OF STA BLE CARB ON ISOTOPES IN BIOAP ATITE AND SOIL 13 C apatite values are commonly utilized for dietary reconstruction studies of fossilized bone and may be a useful to trace bioapatite presence in soil. Carbon has two stable isotopes 12 C and 13 C, with natural abundances of 98.89 and 1.11%, re spectively. Increase in the relative presence of 13 C to 12 C in a sample indicates a larger portion of total C in a sample consists of 13 C. Bioapatite can contain very small amounts of CO 3 2 that may be substituted for PO 4 3 or OH in the crystal structu re (Legeros et al., 1969; Koch et al., 1997). A concern for isotopic investigation of bioapatite is geochemical changes in the original elemental composition that would obscure interpreting C isotope data for reconstruction of historical ecological condit ions (Schoeninger and DeNiro, 1982). Soil is a dynamic environment and interaction of bioapatite with soil solution is suggested as a mechanism for post depositional alteration 13 C apatite values due to carbonate adsorption to surfaces and substit ution for anions in the crystal structure (Schoeniger and DeNiro, 1982; Kueger, 1991; Lee Throp and van de Merwe, 1991). Kueger (1991) suggested exchange of solution carbonate 13 C apatite values are useful if pedogenic carbonates are removed. These concerns certainly apply to bioapatite present in these alkaline tree island soils that appeared highly weathered, which is implied from reduction in particle size and resolution of the diffrac tion peaks ( Chapter 4 ). The purpose of this investigation was to utilize a stable isotopic technique to trace biogenic apatite in soil Total Carbon, 13 C Analysis

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120 Animal bones, weathered bone fragments, and soil samples were analyzed for TC and stable isotope ratio of TC Soil samples were also prepared for OM stable C isotopic analysis, by removal of IC using an ac id fumigation method (Hedges and Stern, 1984; Komada et al. 2008). Briefly, 1.0 to 4.0 mg soil was placed into silver capsules and exposed to concentrated HCl inside a glass desiccator for approximately 6 hours (Komada et al., 2008). Sample isotopic ratio (R) of 13 C/ 12 Sample = [(R Sample /R Standard ) 1] 1000. Analysis of TC 13 C TC ) and 13 C OM ) was performed by combustion using a Thermo Finnigan MAT DeltaPlus XL mass spectr ometer (Bremen Germany) at the University of Florida (UF) Soil and Water Science Department Stable Isotope Mass Spectrometry (SIMS) Laboratory. Sample measurements were substantiated using standard wheat flour as described by Inglett et al. (2006). The analytical precision for 13 C TC and 13 C OM 13 C apatite ) analysis was based on modification of common techniques applied in archeological bioapatite 13 C isotope investigations (Koch et al., 1997; Eagle et al., 2011). The p reparation method is also similar to common methods for soil carbonate and OM removal utilized to concentrate soil matter prior to mineralogical (e.g. XRD) or chemical analysis (Soukup et al., 2008; Shang and Zelazny, 2008). Pedogenic carbonates were diss olved in 1 M sodium acetate solution buffered to pH 4.6 with glacial acetic acid for 14 hours (Koch et al., 1997; Shang and Zelazny, 2008; Eagle et al., 2011). After carbonate dissolution OM was oxidized in a 2% sodium hypochlorite (NaOCl) solution adjust ed to pH 9.5 (Koch et al, 1997; Soukup et al., 2008). The NaOCl treatment was conducted over 72

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121 hours and consisted of solution renewal following 4 and 24 hours of the initial treatment. The samples were rinsed three times with distilled DI water and oven dried (70 C) overnight prior to isotopic analysis. Carbonate removal efficacy was evaluated by post 13 C apatite value of treated bone and bone/CaCO 3 mixture (9:1) was also compared to confirm carbonate dissolution (Koch et al. 1997). Bone samples from uniden tified species of fish and snake were only treated for OM removal with the NaOCl solution as described above. 13 C apatite was measured by grinding, and homogenizing, dried samples and reacting samples in 100% orthophosphoric acid at 70C usi ng a Finnigan MAT Kiel III (Bremen, Germany) carbonate preparation device. The evolved CO 2 gas was measured online with a Finnigan MAT 252 mass spectrometer at the UF Geological Sciences Department SIMS Laboratory. Analytical precision for sample analysi 13 C apatite Sample measurements were verified using NBS 19 standard. Results 13 C apatite values for animal bones were highly variable ranging from 8.3 to 13 C apatite for carnivores, herbivor es, and omnivores ranging from roughly, 13 to 13 C apatite values in the soil (<2 mm), weathered bone fragments, and sand and silt+clay size soil were all within the range of 13 C apatite values (Figure A 1 13 C ap atite values from soil and animal bones may not necessarily provide particularly useful information regarding specific species contribution. However, collection of animal bones from some Everglades tree islands, observation of bone fragments in soil, and m 13 C apatite

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122 values provide a foundation to theorize bioapatite contribution is a continuous ecological event, rather than only derived from historic anthropogenic use. 13 C OM values for bulk soil (<2 mm), and sand and silt+clay size soi l were 26.6, 26.6, and (Table A 1) Cerling and Quade (1993) showed an enrichment of 13.5 across many soil types and climate. These estimates are based on respired CO 2 of OM con trolling the carbonate isotope signature in the soil environment. The isotope signature of CO 2 in surface soil is also influenced by atmospheric CO 2 which has a 13 13 C value ranging from roughly 10 to 13 C OM value. Inter estingly, the similarity among weathered bone fragments, bulk soil (<2 mm) and sand and silt+clay 13 C apatite values may suggest that apatite in the silt+clay size soil is derived from bioapatite (Figure A 1). If soil carbonates substitute for PO 4 3 or OH during bone 13 C apatite values of coarse fragments, bu lk soil (<2 mm), and sand and silt+clay sized soil may suggest this process occurs before bone weathers to <2 mm in size.

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123 Table A 1. Total carbon (TC), and stable C isotope values for TC ( 13 C TC ) and organic matter ( 13 C OM ) for soil, animal bones, weathered bone fragments, and sand and silt+ clay size soil ). Data represent mean 1 standard deviation of the mean. Sample Type (n) T C (%) 13 C TC 13 C OM Animal Bones (5) 14.4 2.7 24.5 1.9 Weathered Bone Fragments ( 9 ) 4.8 0.9 23.0 1.6 Soil (22) 15.8 4.5 25.2 0.5 26.6 0.8 Sand (3) 9.7 3.8 20.8 1.2 26.6 0.7 Silt + Clay (3) 18.7 1.3 25.7 1.5 26.4 0.4

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124 Figure A 1. 13 13 C apatite ) for animal bones, weathered bone fragments (>2 mm) soil (<2 mm), sand, and silt+ clay size soil. Bar heights indicate mean values and error bars represent 1 standard deviatio n.

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138 BIOGRAPHICAL SKETCH Daniel Lyle Irick was born in Selme r, Tennessee to Centhia and Dennis Irick. He lived with his parents in their home in Corinth, Mississippi until the following year when they moved to Iowa City, Iowa. His brother, only sibling, and lifelong friend was born that year and the family soon m oved to Wichita, Kansas. Before he graduated high school, he and his family had moved five more times living in Texas, Montana and Missouri. As a youth he was active in sports and other activities outdoors which elevated his interest in his surroundings and nature. After high school he began a long path to earning a b Missouri and ultimately to Seattle, Washington where he earned a Bachelor of Science from the University of Washington Both ell He began h is post baccalaureate career as an environmental consultant in Honolulu, HI and quickly decided to pursue a graduate degree. A search for graduate degree programs revealed a m Water Science that best suited his academic interest. He began his graduate studies in 2007 and continued his professional career, which eventually took him back to the Se attle area. He earned a Master of Science from the University of Florida in 2009. While pursuing the m d egree he became more interested in research and was fortunate to have an opportunity to extend his education after completion of the m career change for Daniel in January 2010 when he moved from Washington State to Gainesville, FL to become a full time graduate student and begin work for a doctoral deg ree. Persistence to achieve a personal dream and a little luck has resulted in completion of this dissertation.