Nitrogen Dynamics during the Restoration of Calcareous Wetlands in the Florida Everglades

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Title:
Nitrogen Dynamics during the Restoration of Calcareous Wetlands in the Florida Everglades
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1 online resource (173 p.)
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english
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Liao, Xiaolin
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University of Florida
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Gainesville, Fla.
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Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Soil and Water Science
Committee Chair:
Inglett, Patrick
Committee Members:
Li, Yuncong
Sharma, Kanika
Cohen, Matthew

Subjects

Subjects / Keywords:
calcareous -- everglades -- fire -- hid -- limitation -- nitrogen -- nutrient -- pattern -- periphyton -- restoration -- seasonal -- wetlands
Soil and Water Science -- Dissertations, Academic -- UF
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Soil and Water Science thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Abstract:
In wetlands, the nitrogen (N) cycle is dynamically controlled by hydrology, fire and phosphorus (P), but despite its role as both a limiting nutrient and pollutant, N cycling has received little attention inmost of the Everglades system.  This dissertation investigated the spatial and temporal patterns of N cycling in the context of ecosystem restoration using the short-hydro period, marl prairies of the Hole-in-the-Donut (HID) region of Everglades National Park.  Specifically, two restored sites with high P and a native reference site with low P were followed seasonally and in response to a prescribed fire to assess the role of N in this landscape and in the recovery of these systems following restoration. N2 fixation by periphyton was significantly higher in the more N-limited restored sites, and most of this occurred during only 3 months in the early wet season. For the soil, NH4+ and NOX- dominated the wet and dry seasons, respectively, and accumulated NOX- levels were quickly consumed by denitrification at the onset of the wet season.  N mineralization and enzyme activity also revealed dramatic shifts in the abundance of bacteria and fungal communities during these hydrologic transitions. Fire resulted in immediate increases in soil N, and at the restored sites, increased rates of periphyton N2fixation suggesting either a direct input of N through charcoal or an indirect stimulation of mineralization/nitrification by P in ash. Overall, these findings illustrate the importance of hydrology and fire in maintaining N balance and fate in the Southern Everglades, and an increased need for consideration of N cycle processes during the management of restoration of P-impacted sites.
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In the series University of Florida Digital Collections.
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Statement of Responsibility:
by Xiaolin Liao.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Inglett, Patrick.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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1 NITROGEN DYNAMICS DURING THE RESTORATION OF CALCAREOUS WETLANDS IN THE FLORIDA EVERGLADES By XIAOLIN LIAO 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 Xiaolin Liao

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3 To my parents

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4 ACKNOWLEDGMENTS To my committee chair and advisor, Dr. Patrick W. Inglett, I would like to express my great thanks for his patience, trust, and encouragement during my pursuit of doctoral degree. To my committee members, Drs.Yu n cong Li, Kanika S. Inglett, and Matthew J. Cohen, I would like to give my sincere thanks for their suggestions on my research and review of the writing of my di ssertation. Drs. K. R a mesh Reddy and Andy Ogram, thank you for the in time suggestion when I was lost in my graduate study. I would also like to especially thank Miss Cassandra Medvedeff, Miss Anne Baker, Mr. Daniel Irick, Mr Ben Hogue for their accompany ing me in numerous tough field trips and their friendship in supporting many aspects of my research. I thank Miss Yu Wang, Mr. Gavin Wilson, and my many friends in the Wetland Biogeochemistry Laboratory for the laboratory assistance and generous forgivenes s o f my careless ness Anna Normand, lucky to have you as my officemate and driving teacher in my last year, and thanks for bringing fresh air, energy, and a lot of laughs to my office This research was also made possible in part through the funding by gr ant J5297 07 0276 from the US National Park Service and the Everglades National Park, Hole in the Donut Wetland Restoration Project. I thank L. Serra, C. Fisher and A. S. McKinley (US National Park Service) for the support in field sampling and Dr. Todd Os borne for help in the fire project. To all of these, I express my deepest thanks to my parents. Without their unconditional love, support, encouragement, and sacrifice, I c ould not be here and finish my doctoral study.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INT RODUCTION ................................ ................................ ................................ .... 15 Background ................................ ................................ ................................ ............. 15 Everglades Restoration and Nitrogen Cycle ................................ ........................... 18 Case Study and Objectives ................................ ................................ ..................... 20 Dissertation Format ................................ ................................ ................................ 21 2 INDICATORS OF NUTRIENT STATUS DURING THE RESTORATION OF CALCAREOUS WETLANDS IN THE FLORIDA EVERGLADES ............................ 23 Background ................................ ................................ ................................ ............. 23 Methods and Materials ................................ ................................ ............................ 26 Study Site ................................ ................................ ................................ ......... 26 Samplin g Methods ................................ ................................ ............................ 27 Plant Biomass and Nutrient Use/Resorption Efficiency ................................ .... 28 B iogeochemical Analysis ................................ ................................ .................. 29 Statistical Analysis ................................ ................................ ............................ 30 Results ................................ ................................ ................................ .................... 30 Basic Properties of the Soil, Periphyton, and the Plants ................................ .. 30 Plant Nutrient Use and Resorption Efficiency ................................ ................... 31 Extracellular Enzyme Activities ................................ ................................ ......... 32 Multivariate Analyses ................................ ................................ ....................... 32 Discussion ................................ ................................ ................................ .............. 32 TN:TP Ratio and Nutrient Limitation ................................ ................................ 32 Nutrient Use and Resorption Efficiency ................................ ............................ 33 Extracellular Enzyme Activities ................................ ................................ ......... 34 N 2 Fixation of Periphyton ................................ ................................ .................. 35 15 N) of Soil, Periphyton, and Plants ............................ 35 Conclusions ................................ ................................ ................................ ............ 37 3 SPATIAL PATTERNS OF PERIPHYTON NITROGENASE ACTIVITY IN THE CALCAREOUS WETLANDS ................................ ................................ .................. 49

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6 Background ................................ ................................ ................................ ............. 49 Materials and M ethods ................................ ................................ ............................ 51 Study Site ................................ ................................ ................................ ......... 51 Sampling Methods ................................ ................................ ............................ 52 Nitrogenase Analysis ................................ ................................ ........................ 53 Chemical and Isotopic Analysis ................................ ................................ ........ 54 Water Chemistry ................................ ................................ ............................... 54 Statistical Analysis ................................ ................................ ............................ 55 Results and Discussion ................................ ................................ ........................... 55 Water Chemistry ................................ ................................ ............................... 55 Nutrient Composition ................................ ................................ ........................ 55 Nitrogenase Activity ................................ ................................ .......................... 56 15 N ................................ ................................ .................. 59 15 N .................... 60 Conclusions ................................ ................................ ................................ ............ 62 4 SEASONAL PATTERN OF PERIPHYTON NITROGENASE IN THE SHORT HYDROPERIOD, CALCAREOUS WETLANDS ................................ ...................... 70 Background ................................ ................................ ................................ ............. 70 Materials and Methods ................................ ................................ ............................ 73 Study Site ................................ ................................ ................................ ......... 73 Field Sampling ................................ ................................ ................................ .. 74 Nitrogenase Analysis ................................ ................................ ........................ 74 Chemical and Isotopic Analysis ................................ ................................ ........ 75 Statistical Analysis ................................ ................................ ............................ 76 Results ................................ ................................ ................................ .................... 76 Periphyton Properties ................................ ................................ ....................... 76 Nitrogen Fixation ................................ ................................ .............................. 77 Seasonal Patterns ................................ ................................ ............................ 78 Relationship between N 2 15 N ............................ 79 Discussion ................................ ................................ ................................ .............. 80 Spatial and Sea sonal Patterns of Periphyton N 2 Fixation ................................ 80 Relationship of N 2 Fixation with Nutrient Status ................................ ............... 82 Relationship of N 2 Fixation with 15 N ................................ ............................... 84 Conclusions ................................ ................................ ................................ ............ 86 5 SEASONAL DYNAMICS OF SOIL NITROGEN PROCES SING DURING THE RESTORATION IN SHORT HYDROPERIOD, CALCAREOUS WETLANDS ......... 98 Background ................................ ................................ ................................ ............. 98 Methods and Material ................................ ................................ ........................... 100 Study Site ................................ ................................ ................................ ....... 100 Field Sampling ................................ ................................ ................................ 101 Soil Physical and Chemical Analysis ................................ .............................. 101 Denitrification Enzyme Activities ................................ ................................ ..... 102 Extracellular Enzyme Activities ................................ ................................ ....... 103

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7 Statistical Analysis ................................ ................................ .......................... 103 Resul ts ................................ ................................ ................................ .................. 104 Spatial Patterns of Soil Properties ................................ ................................ .. 104 Seasonal Patterns of Soil Properties ................................ .............................. 105 Discussion ................................ ................................ ................................ ............ 10 6 Seasonal Pattern of Available Nitrogen ................................ .......................... 106 Seasonal Patterns of Microbial Biomass Carbon and Nitrogen ...................... 108 Seasonal Pattern of Extracellular Enzyme Activities ................................ ...... 109 Seasonal Patterns of Denitrification ................................ ............................... 110 Conclusions ................................ ................................ ................................ .......... 112 6 FIRE EFFECTS ON THE NITROGEN CYCLE IN CALCAREOUS WETLANDS .. 121 Background ................................ ................................ ................................ ........... 121 Materials and Methods ................................ ................................ .......................... 123 Study Site and Sampling Methods ................................ ................................ 123 Biogeochemical Analysis ................................ ................................ ................ 125 Statistical Analysis ................................ ................................ .......................... 125 Results ................................ ................................ ................................ .................. 126 Fire Effects on Soil Nitrogen Availability ................................ ......................... 126 Fire Effect s on Microbial Activities ................................ ................................ .. 126 Fire Effects on Denitrification ................................ ................................ .......... 127 Fire Effects on Periphyton N 2 Fixation ................................ ............................ 127 Discussion ................................ ................................ ................................ ............ 127 Responses of Nitrogen Availability to the F ire ................................ ................ 127 Fire Effects on Denitrification ................................ ................................ .......... 130 Effects of Fire on Microbial Activities ................................ .............................. 130 Fire Effects on Periphyton N 2 Fixation ................................ ............................ 132 Conclusions ................................ ................................ ................................ .......... 133 7 SUMMARY AND SYNTHESIS ................................ ................................ .............. 144 Summary ................................ ................................ ................................ .............. 144 Synthesis ................................ ................................ ................................ .............. 147 LIST OF REFERENCES ................................ ................................ ............................. 151 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 172

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8 LIST OF TABLES Table page 2 1 Summary of two way ANOVA tests for effects of season (seas.), site, and their interaction on basic properties of soil and periphy ton ................................ 39 2 2 Basic properties of soil and periphyton at Res00, Res03, and Reference sites (mean SE, n =90) ................................ ................................ ............................. 40 2 3 Soil extractable nutrients at Res00, Res03, and Reference sites in the dry and wet season (mean SE, n =15) ................................ ................................ .. 41 2 4 Nitrogen stable isotope 15 N) of soil, periphyton, and plants at the three sites (mean SE). ................................ ................................ .................... 42 2 5 Nutrient resorption efficiency and nutrient use efficiency of nitrogen and phosphorus (NRE N, NRE P, NUE N, and NUE P) for the community level vegetation (composite biomass). ................................ ................................ ....... 43 3 1 Geographical coordinates and the approxim ate elevation of the sampling sites used in this study. ................................ ................................ ...................... 63 3 2 Mean water chemistry values at all sites in the October 200 9 when the sites was flooded ................................ ................................ ................................ ........ 64 4 1 S ummary of three way ANOVA tests for main effects and interactions of time, site, and elevation on periphyton parameters ................................ ............ 89 4 2 Mean water chemistry values observ ed at the two restored wetlands and the native reference wetland in different elevation during the wet flooded sampling time ................................ ................................ ................................ ... 90 4 3 Correlation between the N 2 fixation measured by acetylene reduction (AR) rates and total nitrogen (TN), total phosphorus (TP) a nd TN:TP molar ratio in the wet and dry season ................................ ................................ ..................... 91 5 1 Summary of three way ANOVA tests for main effects and interactions of time, site, and elevation on soil parameters ................................ .................... 114 6 1 Changes of basic soil properties and selected microbial activities 2 days, 1month and 1 year after the fire in the burn and control plots in the rest ored and reference sites ................................ ................................ .......................... 135 6 2 Changes of selected microbial activities 2 days, 1month and 1 year after the fire in the burn and contr ol plots in the restored and reference sites. .............. 136

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9 LIST OF FIGURES Figure page 2 1 Location of the study sites Hole in the Donut in the Florida Everglades National Park. ................................ ................................ ................................ ..... 44 2 2 Relationship between plant TN and TP concentration for the Res00, Res03, and Reference sites(a); plant TN:TP mass ratios sorted by different species at the three sites (b ) ................................ ................................ ........................... 45 2 3 Activities of N related enzymes and P related enzymes at the Res00, Res03, and the reference sites i n different seasons. ................................ ..................... 46 2 4 Periphyton acetylene reduction (AR) rates under dark (a), light (b) incubation, and the ratio of AR rates (c) determined in each of the th ree Everglades sites in the dry a nd wet seasons ................................ ................................ ................ 47 2 5 Score plots of principal components analysis on the biogeochemical properties of soil and periphyton in the wet and dry season. .............................. 48 3 1 Map showing the general area of Everglades National Park and the locations of restored and reference marl prairie sites used in this study. ........................... 65 3 2 Areal estimates of N 2 fixation in the restored and reference sites .................... 66 3 3 Relationships between periphyton acetylene reduction (AR) under light conditions and periphyton TP (a) and TN:TP ratio (b) in the dry and wet seasons. ................................ ................................ ................................ ............. 67 3 4 15 15 ................................ ..... 68 3 5 15 TP and TN:TP ratio ................................ ................................ ............................. 69 4 1 Location of sampling sit es in the two restored wetlands and a native reference wetland of Hole in the Donut in the Florida Everglades, US. ............. 92 4 2 Mean maximum and minimum daily tempe rature and mean maximum daily photosythetically active radiation mean water depth and the daily mean and maximum precipitation for the study site in 2010. ................................ ............... 93 4 3 Seasonal patterns of periphyton nutrient in the two restored wetlands a nd the native reference wetland during 2010 and 2011 ................................ ................ 94 4 4 15 N ( a nd the native reference wetland in different e levation. ................................ ............ 95

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10 4 5 Seasonal changes of periphyton acetylene reduction (AR) rates under light condition in the two restored wetlands a nd the native reference wetland in different elevation. ................................ ................................ .............................. 96 4 6 Nitrogen fixation measured estimated fixed nitrogen per day nitrogen storage and nitrogen stable isotopic signature of periphyton in the dry and wet season ................................ ................................ ................................ ........ 97 5 1 pH, soil mo isture content, TN TP TOC:TN and T N:TP over two years (2010 2011) in the restored and reference sites in the Hole in the Donut ................. 115 5 2 Extractable NOx N and NH 4 N with temperature and rainfall events over two years (2010 2011) in the restored a nd reference sit es in the Hole in the Donut ................................ ................................ ................................ ............... 116 5 3 Microbial biomass c arbon, nitrogen and their ratio ratios with temperature and rainfall events over two ye ars (2010 2011) in the restored and reference sites in the Hole in the Donut. ................................ ................................ ......... 117 5 4 Potentially mineralized nitrogen (PMN) with rainfall events (light grey line) over two years (2010 2011) in the restored and reference sites in the Hole in the Donut ................................ ................................ ................................ .......... 118 5 5 Leucine aminopeptidase a nd N acetyl D glucosaminidase activities with rainfall events and temperature over two years (2010 2011) in the restored and reference sites in the Hole in the Donut. ................................ .................. 119 5 6 Denitrified enzyme activi ties (DEA) with rainfall events over two ye ars (2010 2011) in the restored and reference sit es in the Hole in the Donut ................. 120 6 1 Sampling locations in the restored and reference wetland in the Hole in the Donut of Florida Everglades. ................................ ................................ ............ 137 6 2 Changes of soil extractable NO X N in the burn an d control plots in the restored and reference wetlands 2 days, 1 month and 1year after the fire. ..... 138 6 3 Changes of the extractable NH 4 N of the soil in the burn and control plots in the restored and ref erence wetlands 2 days, 1 month and 1year after the fire. ................................ ................................ ................................ .................. 139 6 4 Changes of denitrification enzyme activities (DEA) in the burn and control plots at the restored and ref erence wetlands 2 days, 1 month and 1year after the fire ................................ ................................ ................................ ............. 140 6 5 Changes of LAP and NAG in the burn an d control plots in the restored and ref erence wetlands 2 days, 1 month and 1year a fter the fire .......................... 141

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11 6 6 Changes of periphyton N 2 fixation under light condition in the burn and control plots in the restored and ref erence wetlands 2 days, 1 month and 1year af ter the fire ................................ ................................ ........................... 142 6 7 Correlation between nitrogen mineralization a nd Leucine aminopeptidase (a) betwe en extractable nitr ite/nitrate and denitrification (b) in both burn and control plots in the reference a nd restored sites ................................ ............... 143

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12 LIST OF ABBREVIATION S AR Acetylene reducation rates DEA Denitrification enzyme activity HID Hole in the Donut LAP Leucine aminopeptidase MBC Microbial biomass carbon MBN Microbial biomass nitrogen MBP Microbial biomass phosphorus NAG N acetyl D glucosaminidase PMN Potentially mineralizable nitrogen Res00 Wetlands restored in 2000 Res03 Wetlands restored in 2003

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of th e Requirements for the Degree of Doctor of Philosophy NITROGEN DYNAMICS DURING THE RESTORATION OF CALCAREOUS WETLANDS IN THE FLORIDA EVERGLADES By Xiaolin Liao August 2012 Chair: Patrick W. Inglett Major: Soil and Water Science In wetlands, the nitroge n (N) cycle is dynamically controlled by hydrology, fire and phosphorus (P), but despite its role as both a limiting nutrient and pollutant, N cycling has received little attention in most of the Everglades system. This dissertation investigated the spati al and temporal patterns of N cycling in the context of ecosystem restoration using the short hydroperiod, marl prairies of the Hole in the Donut (HID) region of Everglades National Park. Specifically, two restored sites with high P and a native reference site with low P were followed seasonally and in response to a prescribed fire to assess the role of N in this landscape and in the recovery of these systems following restoration. N 2 fi xation by periphyton was significantly higher in the more N limited r estored sites, and most of this occurred during only 3 months in the early wet season. For the soil, NH 4 + and NO X dominated the wet and dry seasons, respectively, and accumulated NO X levels were quickly consumed by denitrification at the onset of the we t season. N mineralization and enzyme activity also revealed dramatic shifts in the abundance of bacteria and fungal communities during these hydrologic transitions. F ire resulted in immediate increases in soil N, and at the restored sites, increased rate s of periphyton

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14 N 2 fixation suggesting either a direct input of N through charcoal or an indirect stimulation of mineralization/nitrification by P in ash. Overall, these findings illustrate the importance of hydrology and fire in maintaining N balance and fate in the Southern Everglades, and an increased need for consideration of N cycle processes during the management of restoration of P impacted sites.

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15 CHAPTER 1 INTRODUCTION Background Nitrogen (N) cycle includes a variety of potential inputs, numerous forms ranging from particulate and dissolved organic to dissolved inorganic (e.g., NO 3 and NH 4 + ) and gaseous species (e.g., N 2 NH 3 and N 2 O), and a diversity of transformations (e.g., ammonification, N mineralization, nitrification and denitrification). These complexities first involve understanding, describing, and quantifying the natural N cycle at different landscapes. N has ecological significances, for example, it is the limiting nutrient for most plant growth typically in temperate forests, grasslan ds and the coastal ocean (Vitousek and Howarth 1991) N 2 fixation plays as an important N source especially in a N limited ecosystem; and the N 2 O gas is 298 times more powerful than carbon dioxide as a greenhouse gas (IPCC 2007). There is a large variabili ty on the magnitude of the natural fluxes of N and the storage of N in the N reservoirs. For instance, the estimates of marine N fixation range from 40 200 million metric tons per year, atmosipheric deposition of organic N 10 100 million metric tons per ye ar, terrestrial ammonia/ammonium (NH 3 /NH 4 + ) emissions 91 186 million metric tons N per year ( http://www.isws.illinois.edu/nitro/hinfl.asp ). It is difficult to precisely quantify the magnitude of the major components of the N cycle since the natural world is large and heterogeneous and moreover, has been widely influenced by a variety of human disturbance (Vitousek and Farrington 1997). Intensive agricultural activities are believed to be a major factor contributing to the degradation of ecosystems ( Hausman 2007; Bending et al. 2000; Kalinina et al. 2009), which input excess nutrients into the native ecosystems, lead to nutrient problems and

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16 invasion of exotic species (Hamilton and Landman 2011). N and phosphorus (P) are responsible for most of the nutrient problems (Conley et al. 2009). Accordingly e cological restoration has attracted increasing attention from the general public, scientists and the government. Many theories, specific cases, mechani sms and techniques of ecosystem restoration have been proposed and studied to restore the degraded ecosystems (Fraser and Kindscher 2001; Mitsch and Day Jr. 2004; Hausman et al. 2007; Kiehl and Pfadenhauer 2007; Smith et al. 2011). The restoration process es modify the N and P cycle, and the nutrient status would change during the restoration (Davidson et al. 2007). Therefore, a diversity of indicators has been used to demonstrate different nutrient status. For example, field fertilizations are the standard approach to testing nutrient limitation, but these require a long time (Craine and Jackson 2010). TN:TP ratio of plant tissues was then found to be a more simple indicator (van den Driessche 1974; Powers 1980; Koerselman and Meuleman 1996; Vitousek et al. 2010). Microbial parameters recently were paid more attention on its utility as a dynamic indicator of the restoration and nutrient limitation (Olander and Vitousek 2000; Harris 2003, 2009). N 2 fixation (i.e. nitrogenase activity), for example, was repor ted to negatively related with N availability (Tyler et al. 2003). Extracellular enzyme activities can rapidly respond to different nutrient status (Sinsabaugh et al. 1993, 2009; Garcia Ruiz et al. 2008; Geisseler and Horwath 2009; Hernndez and Hobbie 201 0; Tabatabai et al. 2010) and were even suggested as a functional measurement, which can vary independently of microbial community structure (Waldrop et al. 2004; Cusack et al. 2011). Most recently, N stable isotope ratio ( 15 N) is being

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17 used as an indicator of plant N demand and N 2 fixation (Davidson et al. 2007; Inglett and Reddy 2006). Numerous factors regulate different N transformation and N species such as soil physical properties, nutrient supply, and climate conditi on (e.g., temperature, permafrost and freeze, and precipitation) (Harrison et al. 2005; Heffernan et al. 2010; Hefting et al. 2004). Spatial and temporal variations of these factors and their interactions often lead to the high variability of soil N cycle (Clment et al. 2002). Hydrology is the major driver of the N cycle in wetland ecosystems (Christense et al. 1990; Heffernan et al. 2010; Hefting et al. 2004). It was reported that water table could enhance nitrification and N 2 O fluxes on peatlands (Regina et al. 1996; Elmi et al. 2005). The frequency of dry rewetting period and the intensity of the wetting pulse have significant effects on microbial biomass N, N mineralization, nitrification, denitrification, and N 2 fixation (Olfs et al. 2004; Borken and M atzner 2009; Muhr et al. 2008; Jeffries et al. 1992). Especially when a globally increasing likelihood of severe drought has been predicted during this century (Meehl et al. 2007), it is emergent to investigate the seasonal changes or hydroperiod effects o n the N flux which would further correlate with the primary production of plants, greenhouse gas emission (i.e., N 2 O). Fire is considered as another important disturbance both natural and anthropogenic that can modify the N cycle (Wan et al. 2001). During the combustion, heat releases, transports to the soil and elevates the soil temperature, leading to N loss through volatilization, the transformation of organic N to inorganic forms, and the further biochemical reactions like nitrification and denitrificat ion (Raison 1979). Similarly, the combustion converts the organic P to available forms and deposes as a form of ash.

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18 The P was reported to stimulate the N cycling especially in P limited ecosystems (Howarth et al. 1988a; White and Reddy 1999, 2000; Inglett et al. 2004). Charcoal as another fire residue, would have the potential of increasing nitrification and N mineralization that has been found in forest ecosystem (Zackrisson et al. 1996; DeLuca et al. 2002; DeLuca and Sala 2006). Compared to the terrestri al ecosystems, little is known about fire effects on nutrient cycle or specifically N cycle in a wetland ecosystem where the interaction between fire and water makes the N cycle more complex. However, fire is widely used as a restoration and management too l in wetlands to reestablish the native vegetation. More is needed to know the underlying mechanisms that how fire could work better. Everglades Restoration and Nitrogen Cycle The Everglades is the second largest wetland on the planet and to save it from was early performed as the Comprehensive Everglades Restoration Plan (CERP) in which the water including both water quality and quantity is the key issue. The divers ity of hydrology across the Everglades featured a variety of spatial and temporal patterns of N cycle. Most existing researches on N cycle focused on the long hydroperiod wetlands (e.g., northern marshes, Shark River and Taylor River Sloughs) that remain f looded for more than 8 months of the year (Rudnick et al. 1999; Sutula et al. 2001, 2003; White and Reddy 1999, 2000, 2003; Inglett et al. 2004, 2011). For example, Sutula et al. (2001) made rough N budgets in the Taylor Slough/C 111 basin wetlands during the wet and dry season and pointed out that high N 2 fixation rates or an underestimation of groundwater N flux may explain the difference between estimates of hydrologic N import and sediment N burier rates. In contrast, N cycle in the short

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19 hydroperiod we tlands that remain inundated for less than 8 months of the year (e.g., southern marl and calcareous wetlands) has rarely received any attention. However, those ecosystems undergo more pronounced dry rewetting periodic cycles driven by seasonal patterns of rainfall (Ewe et al. 200 6 ), which would lead to distinctive seasonal patterns of N cycle. Besides reestablishing a more natural hydrology, t he establishment of natural fire regime is also a guarantee for the successful Everglades restoration (Lockwood et a l. 2003). The Everglades is a fire dependent landscape during the large scale and long term restoration (Beckage et al. 2005). Numerous fire projects have been conducted or been going on in a variety of landscapes across Everglades, such as the River of G rass Prescribed Fire Plan for the wet prairie and sawgrass marsh ecosystems, the Pineland burn plan, the mangrove marsh ecotone fire, and the fire project in Water Conservation Area 2A (WCA 2A) of the northern Everglades (Spier and Snyder 1998; Qian et al. 2009; Beckage et al. 2005). However, most of the projects focused on restoring native plant species, little is known about the fire effects on nutrient biogeochemistry, let alone on the N cycle that has been ignored across the whole Everglades ecosystem ( Miao and Sklar 1998; Miao and Carstenn 2006; Qian et al. 2009). In addition, with the human disturbance, many areas of Everglades accumulated excess P, forming two contrasting wetlands that is P enriched versus P limited ecosystems (Noe et al. 2001) In h ighly P impacted areas, N can even be the limiting previously discussed that N cycle can be affected by P, the study on N cycle between the high and low P would become more interesting.

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20 Case Study and Objectives Base on the literature review and those to be explored questions in the Everglades, a case study on the N cycle was chosen in a unique ecosystem, the Hole in the Donut (HID), which combined the major features of wetland restoration, contrasting P impacts, short hydroperiod, and fire disturbance together. The HID is located in the Miami Dade County of Miami, Florida. As early as the Everglades National Park (ENP) was established in 1948, farming was allowed in the park. Historic farming enriched the P; and after the farming ceased in 1975, Brazilian pepper ( Schinus terebinthifolius ) was invaded and dominated (Li and Norland 2001). To restore HID to the natural sawgrass ( Cladium jamaicense Crantz) ecosystem, various methods were tried, and the complete soil r emoval (CSR) technique that removed all the plants and soils down to the bedrock were proved to be the most efficient (Dalrym ple et al. 2003). Then the ENP initiated a large scale restoration program in spring1997: during each dry season (December to May), a new area was restored through CSR method. By 2010, about 4,100 acres have been restored ( http://www.nps.gov/ever/naturescience/hidprogram.htm ). During the restoration to a native mar l/calcareous prairie wetland ecosystem, the N and P status would shift from more N limited immediately after the CSR to more P limited status ( Inglett et al. 2011; Smith et al. 2011). At the very beginning of the restoration, periphyton mats colonize the b edrock, cause the precipitation of the calcite to form soils and simultaneously fix N 2 With the accumulation of N and the co nsumption of P, the TN:TP ratio would accordingly vary with time, and become closer to the native calcareous wetlands with low N an d P. The comparison of N cycle between the restored and native reference sites could thus help to trace the trajectory of the restoration

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21 In addition, the hydrology modified by the shallow soil at the restored sites could differ from that at the native r eference site, which would affect the seasonal patterns of N cycle when different flooding occurred Therefore, t he specific objectives of the proposed study were to (1) demonstrate the different nutrient status of the restored and native wetlands in the H ID using various biogeochemical indicators; (2) investigate the spatial and temporal patterns of N 2 fixation of periphyton in the restored and native wetlands in the HID; (3) determine the seasonal pattern of the soil N dynamics in the restored and native wetlands in the HID; and lastly, (4) explore the effects of fire on the N cycle in the restored and native wetlands in the HID Dissertation Format Chapter 1 provides an introduction for the overall dissertation, beginning with a review of the N cycle, it s large variability and the relative controlling factors then narrowing down to the Florida Everglades discussing the lack of attention on the N research across the Everglades ecosystems, interpreting the need of the dissertation work and finally statin g the reason why the case study in the HID was selected. Chapter 2 6 are formatted as complete manuscript works intended for subsequent publication with each containing appropriate literature citations for the specified processes discussed. Chapter 2 integ rates all the components of the ecosystem (plants, soil, and the periphyton), discussing the potential indicators for nutrient status including not only the commonly reported foliar TN:TP ratio and nutrient use and adsorption efficiency, but also the newly reported soil enzyme activities, N stable isotopes, and the N 2 fixation of periphyton. Chapters 3 and 4 focus on the component of periphyton, presenting the spatial and temporal patterns of the N 2 fixation of periphyton. Chapter 5 focuses on the component of soil, investigating the temporal pattern of the main N

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22 processes ( e.g., denitrification and N mineralization). Chapter 6 explores the effects of fire on the N cycle. Finally, a summary of these studies with concluding remarks as to the limitation and f urther efforts of the current research is presented in Chapter 7.

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23 CHAPTER 2 INDICATORS OF NUTRIENT STATUS DURING THE RESTORATION OF CALCAREOUS WETLANDS IN THE FLORIDA EVERGLADES Background Due to the increase of human disturbance, degradation of various ec osystems has become a serious global issue. E cological restoration has thus attracted increasing attention from the general public, scientists and government s Many theories, specific cases, mechanisms and techniques of ecosystem restoration have been prop osed an d studied (Zedler 2000; Fraser and Kindscher 20 01; Hausman et al. 2007; Kiehl and Pfadenhauer 2007). Around the world, there are several ecosystems that are characterized as low nutrient and species rich ecosystems. The plants in these nutrient poor ecosystems are very sensitive even to small changes in nutrient availability ( Fischer and Stcklin 1997 ). Heathland with acidic soils and calcareous ecosystems with alkaline soils are two typical examples (Gibson et al. 1991; Mitchell et al. 2000; Niineme ts and Kull 2005; Diaz et al. 2008; Piqueray et al. 2011). When historical agricultural activities were abandoned in these ecosystems, residual nutrients were left, leading to nutrient problems and invasion of exotic species (Bending et al. 2000; Hausman 2 007; Kalinina et al. 2009; Hamilton and Landman 2011). One of the key objectives for ecological restoration of these ecosystems is to reduce the nutrient l evel s. Nitrogen (N) and phosphorus (P) are responsible for most of the nutrient problems. During the restoration processes, the ecosystem would exhibit different N and P cycling patterns and a specific case is that N could be more limited on young soils, while P could become the limiting element for the ecosystem with old soils (Walker and Syers 1976; V itousek and Farrington 1997; Davidson et al. 2007 )

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24 Field fertilization is the standard approach to testing nutrient limitation, but this approach is time and labor intensive (Craine and Jackson 2010). As a result, many indirect indicators have been discu ssed and recommended (van den Driessche 1974; Powers 1980; Koerselman and Meuleman 1996; Vitousek et al. 2010; Piqueray et al. 2011). The TN:TP ratio of plant tissues is widely used to determine the nutrient limitation to primary production in terrestrial ecosystem s (van den Driessche 1974; Koerselman and Meuleman 1996; Niinemets and Kull 2005). McGroddy et al. (2004) estimated global nutrient ratios for limitation of N and P in terrestrial ecosystems based on molar basis, and reported that global C:N:P rat ios for limitations of N and P were 1212:28:1 for foliage B y reviewing data on fertilization studies in a variety of European freshwater wetland ecosystems (bogs, fens, wet heathlands, dune slacks, wet grasslands) Koerselman and Meuleman (1996) generaliz ed that at N:P mass ratios >16 community biomass production is P limited; at N:P values <14, N limits plant growth; at N:P ratios between 14 and 16, either N or P may limit plant growth or both elements are equally limiting (co limitation) but they also p ointed out that t he method can only be used under conditions where eithe r N or P controls plant growth. It is also argued that the critical ratios have large spatial and temporal variations (Reich and Oleksyn 2004). In addition to foliar critical values, the Diagnosis and Recommendation Integrated System (DRIS) index is another way to determine nutrient limitations of different ecosystems (Comerford and Fisher 1984). The DRIS method ranks automatically excesses or deficiencies of nutrients in order of impo rta nce (Walworth and Sumner 1987) based on a DRIS norm that needs to be established for each ecosystem. Since a norm is usually determined easier for agronomic crops it has been successfully used to

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25 interpret results of nutrient sufficiency or def iciency of many different crops (Bangroo et al. 2010). Only one DRIS norm has been established for a plantation forest of Loblolly pine in Georgia and the Carolinas ( Nguyen 2011 ). Other nutrient indicators of plant nutrient status include nutrient use efficiency (NUE) and nutrient resorption use efficiency (NRE). The underlying theory behind these two indicators is that plants themselves develop a physiological strategy to deal with the nutrient limitation or deficiency. They can either use the limiting nutrients more efficiently or withdraw the nutrients from senescing parts to the young tissues. Compared to the plants, n o agreement has been made in using soil TN:TP ratio to assess the nutrient status in terrestrial ecosystem, but it somehow indicates a general pa ttern of nutrient limitation in the sites. For example, Fenn et al. (1998) used both foliar C:N and soil C:N ratios to evaluate N saturation of the mixed forest (Pine, Oak, Fern) in the San Gabriel Mountains, Northeast Los Angeles, CA and concluded that i ndications for N saturation in the mixed forest were from 24 to 48 for foliar C:N and from 18.8 to 26.6 for soil C:N. Carlyle and Nambiar (2001) showed that soil C:N ratios for N saturation in the Yellow podsolic, Krasnozem, Siliceous sand soil were 31.3, 34.1, 37.7 ; Soil N:P ratios for P limitation ranged from 13 to 18.8 and did not differ much among three types of soils Cleveland and Liptzin (2007) found that similar to the marine phytoplankton, there was a consistent atomic C:N:P ratios in the soil (186 :13:1) at the global scale. Microbial indicators recently were paid more attention on its utility as a dynamic indicator of the restoration and nutrient limitation ( Burke et al. 2011; Olander and Vitousek 2000; Harris 2003, 2009). N 2 fixation (i.e. nitrog enase activity), for example, was reported to negatively related with N availability (Tyler et al. 2003). Extracellular

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26 enzyme activities can rapidly respond to different nutrient status (Sinsabaugh et al. 1993; Sinsabaugh et al. 2009; Geisseler and Horwat h 2009; Hernndez and Hobbie 2010; Tabatabai et al. 2010) and were even suggested as a functional measurement, which can vary independently of microbial community structure (Waldrop et al. 2004; Cusack et al. 2011). Most recently, N stable isotop ic ratios ( 15 N) is being used as an indicator of plant N demand (Davidson et al. 2007; Inglett and Reddy 2006). Sundareshwar et al. (2003) further observed that in the same ecosystem, individual trophic groups c ould show different nutrient limitation. For example, p rimary production of plants in coastal wetlands was limited by N; but the bacterial community in the soil was limited by P. To test those indicators of nutrient status, two restored wetlands with young soils and high P, and a native reference wetland with old soils and low P were selected in the Florida Everglades National Park, USA. The total nutrients of the major components, extractable nutrients, microbial activities, and plant nutrient use and resorption efficiency were measured. The objectives of thi s study are to 1) describe the nutrient status during the restoration, and 2) to see if different nutrient indicators could give the consistent results. Methods and Materials Study S ite T wo wetlands restored in 2000 and 2003 as well as an unfarmed refer ence site adjacent to the restored areas were selected in the HID region of Everglades National Park (Fig. 2 1). In each site, five sampling stations were identified Soil depth varied among the three sites, with deeper marl soils (Biscayne and Perrine ser ies) in the

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27 reference areas (10 cm) and shallower soils (2 3 cm) which have developed after site clearing in the restored areas (Smith et al. 2011). Sampling M ethods Unlike most areas of the United States, the Everglades experiences only two seasons: dry and wet, corresponding to winter and summer. The dry winter season begins from November through April, with cooler and scant rainfall. The wet summer season accounts for approximately 80% of the region's average annual rainfall of 137 cm (54 inches). Rainf all within the Everglades system can vary dramatically from year to year. Historically, some wet years peaked at over 254 cm (100 inches) of rainfall, whereas some dry years received less than 76 cm (30 inches) ( http://www.waterencyclopedia.com/En Ge/Everglades.html ). S amples were collected in October 2009 (wet season) and February 2010 (dry season). To estimate soil bulk density, soil cores were collected with three replicates and each depth recorded by driving 3.6 cm diameter aluminum pipe into the soil. T he soil depth was measured every 0.5 m along a 10 m line northwards at each transect in each site and then averaged them as the soil depth. At each of the 15 transect location s thre e composite samples of surface soil and periphyton were collected. Live healthy leaves of the dominant vegetation were collected by hand at the 15 locations in both wet and dry season. Not all species were sampled, but an effort was made to include species that were present at the most sites. The targeted species included representatives of the genera Muhlenbergia Cladium Typha Andropogon and Schinus (Dalrymple et al. 2003) All samples were sealed in plastic bags and kept on ice until their return to t he laboratory where the samples were refrigerated at 4C until subsequent analysis. Soil samples were sieved to remove roots

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28 and rock fragments greater than 2 mm diameter. Sieved soil samples were used in determination of all microbial and enzyme related p arameters, while a subsample of sieved soil was oven dried at 105C for 3 days and ground using a mortar and pestle for moisture content and total nutrient determinations. Plant tissues were oven dried at 65C for 3 days and ball milled for total nutrient and isotopic ratio determination. Plant Biomass and Nutrient U se/Resorption E fficiency In April, 2010, four replicates of aboveground plant biomass were collected in four 1 m 2 square plots at two locations in the three sites (Fig. 2 1). After bringing th em to the lab, dead and live parts were separated; then oven dried at 65C for 3 days and ball milled for total nutrient and isotopic ratio determination. The nutrient use efficiency (NUE) and nutrient resorption efficiency (NRE) of N and phosphorus were determined following methods outlined by Berendse and Aerts (1987), Aerts et al. (1999), and Feller et al. (2002). The live and senescent fractions of each biomass collection were used in the following calculations. The NRE was calculated as the percentage of N (or P) recovered from senescing leaves before stem fall: NRE = (N live N senescent )/(N live ) 100 (%) The NUE was calculated as: NUE = A/L n (g biomass mg 1 N) where A is the N productivity, dry matter production per unit of N in the plant and is c alculated as: A = biomass production (g dry wt m 2 yr 1 )/biomass N (mg N m 2 yr 1 ), (g dry wt mg 1 N) and L n is the N requirement per unit of N in the plant and is calculated as: L n = N live (mg m 2 ) / N senescent (mg m 2 ), (unitless)

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29 The NRE is the fracti on of N that is remaining in the biomass during a given time period. The NUE at the ecosystem level was taken as biomass production per unit of N in senescent leaves (g dry wt biomass mg 1 N). Biogeochemical A nalysis Total C and N contents were measured us ing a Thermo Flash EA 1112 elemental analyzer (CE Elantech, Inc.). Total P was measured colorimetrically using a Shimadzu UV 160 spectrometer (method 365.1 U.S. EPA 1993) following ashing and dissolution in 6M HCl (Anderson 1976). Loss on ignition (LOI) w as estimated by weight loss after 4 5h combustion in the muffle furnace at 550C. Extractable NH 4 N was determined by 0.5M K 2 SO 4 extraction and analyzed colorimetrically for NH 4 N using a Technicon TM Autoanalyzer ( method 350.1 EPA 1993). Microbial biomass C and N (MBC and MBN) were determined using the chloroform fumigation extraction technique (Brookes et al. 1985). The extraction efficiency factors for MBC and MBN in the study were 0.37 and 0.42 respectively These parameters were performed by the Wetlan d biogeochemistry Lab oratory in the Soil and Water Science Department, University of Florida. Four extracelluar enzymes related to the N and P cycle s i.e., alkaline phosphatase (AP, EC 3.1.3.1), phosphodiesterase (BisP, EC 3.1.4.1), N acetyl D glucosami nidase (NAG, EC 3.2.1.30), and Leucine aminopeptidase (LAP, EC 3.4.11.1), were measured using fluorogenic enzyme substrate s (Hoppe 1983). Methods were modified from Sinsabaugh et al. (1997) to optimize the substrate concentrations in soil samples with fluo rogenic substrates methylumbellifheryl for phosphatase and NAG, and L leucine 7 amido 4 methyl coumarin (AMC) for LAP. F luorescence was measured at

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30 excitation of 350nm and emission of 450nm using a Bio Tek Model FL600 fluorometric plate reader (Bio Tek In struments, Inc. Winooski, VT). The measurement of nitrogenase activity (potential N 2 fixation) was using the acetylene (C 2 H 2 ) reduction (AR) assay and the experiment details were according to those of Inglett et al. (2004). Statistical A nalysis Data were analyzed with JMP v.8 statistical software (SAS Institute Inc., Cary, NC). Comparisons of means between different sites and seasons were determined using a one way univariate analysis of variance (ANOVA) with Tukey Kramer test. Two way ANOVA was applied to test for difference of the means of various soil properties among sites, seasons and their interaction. Simple regression analysis was applied to evaluate the relationship between different properties. Where applicable variables were log transformed to improve normality. Princip a l component analysis (PCA) was used to ordinate sites on the basis of the multiple nutrients and microbial parameters. Significances and correlation coefficients are significant at the P <0.05 level, unless otherwise noted Result s Basic Properties of the Soil, Periphyton, and the P lants The two way ANOVA showed the TC TN TP and TN:TP ratios did not show significant seasonal difference s (Table 2 1). For the soil, the pH, TC, and TN at the restored sites and reference site were s imilar (Table 2 2). However, significantly higher TP values were observed at the restored sites compared to those at the reference site ( P <0.001, n =15) (Table 2 2). Accordingly, the TN:TP molar ratio at the reference site was markedly higher than those in the two restored site. Moreover, significant ly higher

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31 extractable inorganic P and N (i.e., NH 4 N) were observed in the restored sites compared to the reference site and the magnitudes varied with season (Table 2 1 and 2 3 ). For the periphyton, TP concentra tion at the restored sites were approximately three fold s higher than that at the reference site; and accordingly the TN:TP molar ratio of the periphyton was significantly higher at the reference site ( P <0.001, n =15, Table 2 2). The f oliar TN and TP values were plotted with the two critical TN:TP thresholds reported by Koerselman and Meuleman (1996) across the three sites and species (Fig. 2 2 ). F oliar TN:TP ratios varied with different plant species, seasons, and sites. 15 N values we re measured for the soil at the reference site in both wet and dry seasons (Table 2 4 ). Similar trends were also found for the periphyton 15 N values but were only significan tly different in the dry season (Table 2 4 ). In 15 N values of plant s at the species level were much lower at the reference site relative to the younger restored site (i.e., Res03) except for the Cladium A t the 15 N values for the dead fraction of plant aboveground biomass were significantly lower at the reference site compared to the Res 03 site but no significant difference s were observed for the live tissue s between the three sites (Table 2 4 ). Plant Nutrient Use and Resorption E fficiency The community level nutrient resoprtion efficiency of N (NRE N) was significantly lower for the reference site (approximately 23%) relative to the two restored sites with values above 40%. A significantly low er nutrient use efficiency of N (NUE N) was also observed at the reference site with the average of 0.25 g bi omass mg 1 N compared to the two restored sites with the NUE N values above 0.4 g biomass mg 1 N (Table 2 5 ).

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32 However, the NRE P and NUE P did not show consistent pattern between the three sites (Table 2 5 ). Extracellular Enzyme A ctivities In general, th e activities of the enzymes involved in the N cycle, i.e., LAP and NAG, were significantly higher at the restored sites compared to the reference site (Fig. 2 3ab). In contrast, activities of the enzyme s related to the P cycle, i.e., AP and BisP, were sign ificantly higher at the reference site ( P <0.05, n =15) (Fig. 2 3cd). Significantly higher N 2 fixation rates (measured by the acetylene reduction method) were found in periphyton mats of the sites cleared in 2000 and 2003 (3 10 nmol g 1 dw h 1 ) compared to the reference marl prairie wetland site (< 1 nmol g 1 dw h 1) (Fig. 2 4, Liao and Inglett 2012). Multivariate Analyses T he multivariate statistical technique of principal components analysis (PCA) was used to characterize the sites on the basis of the bioge ochemical parameters of soil and periphyton (Fig. 2 5 ). The first two components explained approximately 60% information, and were used to categorize the three sites. In the wet and dry season, restored sites were separated from the reference site predomin antly on the basis of PC 1 with major factor loadings of soil and periphyton TP, TC:TP, TN:TP, soil extractable inorganic and organic P Principal component 2 also contributed to this separation on the basis of soil TN, TC, TC:TN ratio, and MBC. Discussion TN:TP Ratio and Nutrient L imitation Based on the threshold of N:P ratio given by Koerselman and Meuleman (1996), the TN:TP mass ratio for the plant foliar in this study could not tell any solid information

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33 about nutrient limitation. In the wet season, it seemed that most of the TN:TP ratios at the Res03 site fell in the N limitation zone, and reference site fell in the P limitation zone. However, all the TN:TP values fell in the P limitation zone in the dry season (Fig. 2 2a). Moreover, the plant species a lso have large effects on the TN:TP rations as shown in the Fig. 2 2b. All of these results suggested that the critical values of TN:TP ratios for diagnosing nutrient limitation would have large temporal and seasonal variation and should be used with cauti on (Gsewell 2004; von Oheimb et al. 2010). Comparing with foliar N:P ratio, no agreement has been made in using soil TN:TP ratio to assess the nutrient status in terrestrial ecosystem, but the TN:TP ratios for both periphyton and soil in this study showe d consistent trends between the restored sites and the reference site that is significantly higher TN:TP ratios were observed at the reference sites compared to the restored sites (Table 2 2), which attributed to the significantly higher P in the restored sites. As Cleveland and Liptzin (2007) found that similar to the marine phytoplankton, there was a consistent atomic C:N:P ratios in the soil (186:13:1) at the global scale it was likely to expect that there could potentially be a critical values for diff erent nutrient limitation Nutrient Use and R esorption E fficiency Nutrient use and resorption efficiency (NUE and NRE) have been commonly used to evaluate the response of vegetation to different levels of nutrient availability (Vitousek 1982; Eckstein et al. 1999). It has been suggested that in nutrient limited systems the NRE of senescing leaves will be greater than in nutrient rich environments (Gsewell 2005). In this study, the NRE and NUE of N at the restored sites were significantly higher relative t o that at the reference site, which would indicate that the restored sites were more likely to be limited by N However, the NUE and NRE of P did not show the

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34 patterns as expected and interestingly showed higher numbers in both high P restored sites and lo w P reference site (Table 2 5 ). The inefficiency of NUE and NRE in supporting the nutrient limitation was discussed by earlier reviews of the literature (Aerts 1996; Eckstein et al. 1999), and a study on wetland graminoids by Gsewell (2005) found that NRE of both N and P were on average higher in P limited systems. More NRE and NUE data were needed for individual species since the index varied widely among and within species (Aerts 1996; Vitousek 1998). Extracellular Enzyme A ctivities In this study, the e nzyme activities were reported based on MBC. Many studies have found significant correlations between soil enzyme activities, soil organic matter and microbial biomass (Ajwa et al. 1999). Therefore, sometimes enzyme activities based on dry weight cannot gi ve us the real picture of the microbial activities; the enzyme activities normalized by MBC on the other hand would be a better indicator. There are two categories of enzymes: constitutive enzymes which are always present and active; inducible enzymes whic h are synthesized or activated when needed. Thus the inducible enzymes related to N and P cycling will be produced differently under different N and P availability. Chrst (1991) found out that the activities of aminopeptidases were induced at limited N availability. NAG involved in the degradation of chitin which is a relevant source of N in soils (Hanzlikova and Jandera 1993; Gooday 1994) Sinsabaugh et al. (1993) a ssum ed NAG activity to be induced by low N conditions. The phosphatase was widely discussed as an indicator of P limitation (Olander and Vitousek 2000). In this study, higher NAG and LAP activities were observed in the restored sites, indicating that the resto red sites were lack of available N Similarly, significantly higher

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35 AP and BisP enzyme activities were observed in the reference site, suggesting that the reference sites were more likely to be limited by P. N 2 Fixation of P eriphyton Periphyton at the you ng restored site (Res03) was found to have high er N 2 fixation rates ( Res 03 area) compared to the reference sites. This agreed with the assumption that the young soils tend to be N limitation. There was a significantly linear positive relationship between N 2 fixation rates and TP, accordingly, significantly negative relationship between N 2 fixation rates and periphyton TN:TP were found in both dry and wet season (Liao and Inglett 2012 also see the details in the Chapter 3 ), indicating that P may limit the N 2 fixation. Rejmnkov and Komrkov (2000, 2001) have already demonstrated that addition of P increases N 2 fixation of periphyton in the P limited Belizean marshes. Inglett et al. (2004, 2009) also found similar results that periphyton N 2 fixation rates w ere much higher in the areas with high levels of P and low TN:TP ratios in the Everglades. Stable I sotope Ratios 15 N) of Soil, Periphyton, and Pl ants 15 N value can be an indicator of N 2 fixation. N 2 fixing cyanobacteria should 15 N close to th 2 fixing organisms generally show slight isotope fractionation against 15 N which results in 15 N (Peterson and Fry 1987; Gu and Alexander 1993). In this study, the stable N isotope signature of the periphyton at the three sites all fell within the range of 2 1) as observed by Nadelhoffer and Fry (1994). 15 N values were more positive than periphyton, suggesting that other processes except atmospheric N 2 fixation affect 15 N values. For example coupled nitrification/denitrification and ammonia volatilization or atmospheric deposition

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36 of 15 15 N within the soil. Thus, natural 15 N abundance of soils is presume d to be an indicator of N cycling (Dawson et al. 2002). In this 15 N and the TN: TP ratio for both the soil and the periphyton was found (Fig. 2 5a b ) though the points were in two clusters along the regressi 15 N as the indicator of nutrient limitation. Along a chronosequence spanning 3 to 3000 Ky in a California 15 N values of the soil increased by several 15 N values whereas the 20 Ky or older sites and Farrington 1997). As 15 N values would be expected early in soil development since biota are strongly limited by N, microbial immobilization of 15 N enriched N is rapid, and nitrification is very slow. Later in primary succession, N is not limiting, elevated N losses would enrich the remaining N in 15 N. In 15 N will serve as a proxy of the nutrient status or restoration. Most non nitrogen 15 N values, reflecting the fact that most soils are en riched in 15 N compared to atmospheric N 2 (Vitousek et al. 1989). In this study, the soil is indeed enriched 15 N compared to the periphyton that fixe s atmospheric N, while the native species Cladium 15 N values (Table 2 4 ). However, str 15 N values have been observed in other non nitrogen fixing plants (Table 2 4 ). It could be caused by the inputs of 15 N depleted N from precipitation coupled with very low N outputs as supposed for the Hawaii forests

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37 (Vitousek et al.1989). H owever, in contrast with the results of Hawaii an (Vitousek et al.1989) and Amazonian (Davidson et al. 2007) forest s most of the species with 15 N values in this study were found at the old site (i.e., reference site) rather than the early success ional sites (i.e., two restored sites). Isotope discrimination can occur during the N uptake, assimilation, and allocation, and different plant parts can 15 N values (Dawson et al. 2002). Yoneyama et al. (2001) proposed that there may be lar ge whole plant depletion in 15 N at high N concentrations. Therefore, it is 15 N could be a indicator of nutrient status. Overall, the PCA separated the three sites into two groups based on all the nutrient parameters (Fi g. 2 5), i.e., the restored sites and reference site, indicating that the nutrient status between the two groups is different. The results from PCA also showed that the soil physical properties and 15 N had week correlation with the two principal component s, suggesting the soil fertility changed more dramatically and rapidly during the restoration. Conclusions The restoration in the HID of Florida Everglades through complete soil removal enables a primary succession in the ecosystem. Periphyton primarily co lonizes the habitat and facilitates the soil development, followed by pioneer plants expansion At the same time, the restoration aims in gradually shifting the P enriched ecosystem to low nutrient status. In this study, the biogeochemical properties (e.g. total nutrients, available nutrients, microbial activities, and biomass) of the three major components (soil, plants, and periphyton) were measured at two restored and a native reference calcareous wetlands in the wet (October, 2009) and dry season (Febr uary, 2010) in the Florida Everglades National Park. The results showed that (1) foliar TN:TP ratio varying

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38 with species and seasons should be carefully used as a indicator of nutrient limitation; (2) the N use efficiency and N resorption efficiency were s ignificantly higher at the restored sites than the reference site suggesting that restored sites were more likely to be limited by N; (3) N enzyme (i.e., N AG and LAP ) activities were significantly higher at the restored sites and P related enzymes (Alkali n e phosphatase and phosphodiesterase) were significantly higher at the reference site, suggesting different nutrient demands between the restored and reference wetlands; (4) periphyton at the restored sites fixed more N 2 compared to that at the reference si te, especially in the wet 15 N values could be indicators of the restoration and 15 N patterns during restoration still requires more explicit testing. These results indicated that the microbial a ctivities could be more sensitive to the nutrient status, and should be paid more attention during the restoration process.

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39 Table 2 1. Summary of two way ANOVA tests for effects of season (seas.) site, and their interaction on basic properties of soil and periphyton. *** P<0.001, ** P<0.01, P<0.05, NS no t significan t Parameter Soil Periphyton s ite s eas s ite s eas s ite s eas s ite s eas Loss on ignition (LOI) *** NS NS ** ** NS Total phosphorus (TP) *** NS NS *** NS NS Total nitrogen (TN) ** NS NS *** ** ** Total carbon (TC) ** NS NS NS ** NS TN:TP ratio *** NS NS *** NS NS Extractable inorganic P(P i ) *** *** Extractable organic P *** *** Extractable NH 4 + N *** *** NS Extractable organic N NS *** ***

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40 Table 2 2. Basic properties of soil and periphyton at Res00, Res03, and Reference sites (mean SE, n =90). Different lowercase letters denotes significant difference, P <0.05. Parameter Unit Site Res00 Res03 Reference Soil pH 7. 6 1 0.02 7.55 0.03 7.98 0.03 bulk density g cm 3 0.50 0.03 0.57 0.05 0.52 0.03 soil depth cm 2.2 0.2 1.8 0.2 11.7 1.2 Loss on Ignition (LOI) % 18.5 0.6 a 18.1 0.5 a 12.9 0.5 b Total Phosphorus (TP) mg kg 1 630 32 b 983 4 7 a 140 6 c Total Nitrogen (TN) g kg 1 7.5 0.3 a 6.3 0.2 b 6.7 0.2 b Total Carbon (TC) g kg 1 155 2 a 143 3 b 149 1 ab TN:TP molar ratio mol:mol 28 1 b 15 1 c 109 3 a Periphyton Loss on Ignition (LOI) % 30.0 1.6 b 38.7 1.9 b 35.3 2.2 ab Total Phosphorus (TP) mg kg 1 313 30 a 314 34 a 99 7 b Total Nitrogen (TN) g kg 1 12.9 0.4 b 13.2 22.6 b 16.2 0.6 a Total Carbon (TC) g kg 1 244 4 a 246 3 a 250 6 a TN:TP molar ratio mol:mol 121 12 b 102 8 b 393 23 a

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41 Table 2 3. Soil extractable nutrients at Res00, Res03, and Reference sites in the dry and wet season (mean SE, n =15) Different lowercase letters denotes significant difference, P <0.05 Ext. ON extractable organic nitrogen; Ext. Pi extrac table inorganic phosphorus; Ext. Po extractable organic phosphorus. Parameter Unit season Res00 Res03 Reference Ext.NH 3 N, mg kg 1 wet 18.7 1.1 b 29.4 2.4 a 14.3 1.7 b dry 54.2 5.3 ab 59.0 3.8 a 43.1 2.7 b Ext O N mg kg 1 wet 54.1 2.5 b 6 7.2 3.8 a 56.6 4.1 ab dry 39.1 1.8 b 32.7 1.3 b 51.2 3.5 a Ext Pi mg kg 1 wet 8.6 0.6 b 14.4 1.3 a 1.5 0.1 c dry 10.9 0.9 b 19.3 0.7 a 2.6 0.2 c Ext Po mg kg 1 wet 32.0 3.0 a 39.2 3.3 a 19.8 1.1 b dry 11.3 0.9 b 13.9 0 .6 a 4.5 0.2 c

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42 Table 2 4. 15 N) of soil, periphyton, and plants at the Different lowercase letters denotes significant difference, P <0.05. For soil, periphyton, and plants at species level, samples were collected in the wet season (October, 2009) and dry season (February, 2010), n =15 at each site. For plants at community level, samples were collected in April, 2010, n =8 for each site. Component season Res00 Res03 Reference -----------------------15 N ( ) ------------------------Soil wet 1.71 0.07 b 1.85 0.16 b 2.35 0.06 a dry 1.23 0.10 b 1.42 0.13 b 2.47 0.11 a Periphyton wet 0.49 0.08 0.61 0.19 0.02 0.14 dry 0.75 0.08 b 0.97 0.09 b 0.21 0.10 a Pla nts dead 3.05 0.27 ab 2.26 0.23 a 3.10 0.17 b Plants l ive 1.92 0.32 1.94 0.28 1.45 0.33 Plant Species Andropogon wet 3.13 0.13 b 1.85 0.22 a 4.11 0.14 c dry 3.88 0.42 3.38 0.12 3.86 0.26 Muhlenbergia wet 2. 51 0.20 a NA 3.28 0.16 b dry 3.17 0.20 b 2.13 0.69 a 4.66 0.13 c Cladium wet 2.43 0.18 a 0.78 0.07 b 2.4 0 0.17 a dry 1.92 0.09 a 0.70 0.34 b 1.12 0.13 b Typha wet 1.33 0.18 1.77 0.14 NA dry 0.46 0.07 a 0.21 0.21 b 0.22 0.08 ab Schinus wet 1.82 0.29 b 2.11 0.33 a 1.67 0.44 b dry 2.48 0.27 b 0.85 0.15 a 1.77 0.30 ab

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43 Table 2 5. Nutrient resorption efficiency and nutrient use efficiency of nitrogen and phosphorus (NRE N, NRE P, NUE N, and NUE P) for the commun ity level vegetation (composite biomass). The different letters denote significance amo ng the three sites, mean SE. Site NRE_N NUE_N NRE_P NUE_P % g biomass mg 1 N % g biomass mg 1 P Res00 41.3 1.3 a 0.41 0.04 ab 63.1 6.6 7.2 1.4 b Res03 42.5 1.5 a 0.58 0.08 a 71.0 3.9 14.0 2.2 a Reference 22.9 2.6 b 0.25 0.02 b 58.6 8.0 14.0 4.3 a

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44 Figure 2 1 Location of the study sites Hole in the Donut in the Florida Everglades National Park. Miami Homestead Hole in the Donut

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45 Figure 2 2. Relationship between plant TN and TP concentration for the Res00, Res03, and Reference sites (a); p lant TN:TP mass ratios sorted by different species at the three sites (b) The critical TN:TP ratios were from Koerselman and Meuleman (1996).

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46 Figure 2 3 Activiti es of N related enzymes (a) Leucine aminopeptidase, LAP, (b) N acetyl D glucosaminidase, NAG (only measured in the dry season), and P related enzymes (c) Alkaline phosphotase, AP, (d) phosphodiesterase, BisP (only measured in the dry season) at the Res00 Res03, and the reference sites in different seasons. The different lowercase and uppercase letters denote significance in the wet (grey bars) and dry season (white bars), respectively.

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47 Figure 2 4 Periphyton ace tylene reduction (AR) rates under dark (a), light (b) incubation, and the ratio of AR rates under light and dark conditions (c) determined in each of the three Everglades sites in the dry (white bars) and wet seasons (shaded bars). The different lowercase and uppercase letters denote significant differences between the sites (P<0.05).

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48 (a) wet season (b) dry season Figure 2 5. Score plots of principal components analysis on the biogeochemical properties of soil and periphyton in the wet and dry season.

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49 CHAPTER 3 SPATIAL PATTERNS OF PERIPHYTON NITROGENASE ACTIVITY IN THE CALCAREOUS WETLANDS Background Periphyton mats consist of a complex mixture of algae, heterotrophic microbes and particles (mineral and detritus) and play an important ecological role in shallow aquatic ecosystems. Periphyton can serve as a major contributor to primary productivity (Dodds et al. 2002 ), a regulator of water column nutrient levels (Gaiser et al. 2004 ; Thomas et al. 2006 ; Rejmnkov and Komrkov 2005 ) and benthic fluxes, and based on species composition (McCormick and Stevenson 1998 ) and enzyme expression (Sharma et al. 2005 ), can also be a sensitive indicator of water quality (Vis et al. 1998 ; McCormick 2011 ). Periphyton is often ab undant in shallow wetland systems, including marine tidal flats (Pinckney et al. 2011 ) and freshwater portions of the limestone based Caribbean ( Rejmnkov et al. 2000, 2004 ) Similarly, the Florida Everglades (USA) wetland system also maintains an abundan ce of periphytic assemblages, with biomass estimates ranging between 3 and 6235 g AFDW m 2 (Hagerthey et al. 2011 ). Periphyton is well described in the Everglades, varying both temporally and spatially in terms of biomass, productivity, and species richnes s and diversity in relation to macrophyte abundance and nutrients ( Browder et al. 1994 ; McCormick and O'Dell 1996 ; McCormick et al. 1996, 1998 ; Gaiser et al. 2006, 2011 ; Hagerthey et al. 2011 ). In particular, phosphorus (P) levels are a key regulator of a variety of processes in the McCormick et al. 1998 ), production and respiration (Iwaniec et al. 2006 ). Everglades Reprinted with permission from Liao X, Inglett PW (2012) Biological nitrogen fixation in periphyton of native and restored everglades marl prairies. Wetlands, 32(1):137 148

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50 periphyton also show the ability to fix atmospheric N 2 (biologi cal N 2 fixation) (Inglett et al. 2004, 2009). This process was estimated to contribute 10 g N m 2 yr 1 to a northern o ligotrophic Everglades system. Periphyton is abundant throughout much of the Everglades, however, there are few studies to document the sig nificance of biological fixation in other areas of the Everglades, especially in the southern systems (Inglett et al. 2011 ). One Everglades system where periphyton plays a crucial ecological role is the Hole in the Donut (HID) region of Everglades Nationa l Park (ENP) (Fig. 3 1). This area had a history of farming which disturbed and added excess P to the native pine rockland and marl prairie ecosystems (Smith et al. 2011 ). Disturbance and excess nutrients led to the invasion of Brazilian pepper ( Schinus te rebinthifolius ) after farming ceased (Smith et al. 2011). To restore the HID to marl prairie ecosystem, the technique of complete soil removal, in which all the vegetation and underlying rock plowed substrate was removed down to bedrock was adopted (Dalry mple et al. 2003 ). In this process soils are mechanically cleared to bedrock and allowed to naturally reestablish biotic communities such as periphyton and macrophytes. During the HID restoration processes, periphyton plays an important role in soil format ion, as a source of organic matter and calcium carbonate (CaCO 3 ) both main components of marl soils ( Gaiser et al. 2011 ). N 2 fixation also provides an important N source particularly in the recently cleared sites where N is limiting (Smith et al. 2011; In glett et al. 2011). Though studied intensely in other parts of Everglades, no study has been focused on periphyton in this unique restored calcareous ecosystem where this component is a key target and evaluation metric for restoration (Gaiser 2009).

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51 Furthe rmore, few studie s ha ve been done on the N 2 fixation in P limited ecosystem (Inglett et al. 2004, 2009; Rejmnkov and Komrkov 2000; Rejmnkov 2001) It is important and deserving of attention to understand the role of periphyton in the marl prairie (Da vis et al. 2005). For these reasons, the following study was conducted to (1) characterize periphyton N 2 fixation between the restored and reference wetlands; (2) relate the N 2 fixation with nutrient limitation (i.e., N and P limitation); and (3) assess th e potential of N stable isotopes as an indicator of N 2 fixation and nutrient limitation. Materials and M ethods Study S ite The Everglades experiences two primary seasons including a mostly dry winter (November through May ) and a wet summer ( June to October ). The wet summer season accounts for approximately 80 percent of the region's average annual rainfall of 137 cm. Rainfall within the Everglades system can vary dramatically from year to year. Historically, some wet years peaked at over 254 cm of rainfall, whereas some dry years received less than 76 cm ( http://www.waterencyclopedia.com/En Ge/Everglades.html ). T wo wetlands restored in 2000 and 2003 (referred to as Res00 and Res03) as we ll as an unfarmed reference site adjacent to the restored areas were selected in the HID region of Everglades National Park (Fig. 3 1). In each site, five sampling stations (A, B, C, D, E) were identified along a elevation gradient from 0.5 m to 1.0m AMSL (Table 3 1 ). Soil depth varied among the three sites, with deeper marl soils (Biscayne and Perrine series) in the reference areas (10 cm) and shallower soils (2 3 cm) which have developed after site clearing in the restored areas (Smith et al. 2011). The p rimary vegetation in the reference site is a mixture of grasses ( Muhlenbergia sp., Andropogon sp.) and sedges ( Cladium jamaicense Crantz Shoenus sp.) while the restored sites are

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52 dominated by pioneer species such as Ludwigia spp Baccharis spp. and Andr opogon spp. (Dalrymple et al. 2003 ). The periphyton in the three sites are all calcitic epilithon or calcareous epiphytic periphyton mat (Gaiser et al. 2006, 2011). The mats in the restored site were very thin and greenish and the periphyton in the Res 03 s ite is more disaggregated compared to that in Res 00 site; while the mats in the reference sites were 1 cm thick with dark color. Sampling M ethods Samplings were conducted in October 2009 (wet season) and February 2010 (dry season). At each of the 15 transe ct locations, three composite samples of surface soil and periphyton were collected. Surface soils were collected using sharpened metal tubes (3.75 cm ID) inserted to bedrock (restored sites) or to a d epth of 5 cm (reference area). Periphyton was collected by randomly placing a plastic ring (8 cm ID) and since the study sites were not well flooded, it was easy to remove the periphyton biomass contained within the ring area by hand. This was repeated (up to 5 times) until sufficient biomass had been collecte d for each composite sample which could then be used to determine periphyton biomass per unit area (g m 2 ). Samples were stored on ice until their return to the laboratory where the samples were refrigerated at 4C until subsequent analysis. Periphyton sa mples were kept intact (periphyton mat) and inspected to remove large organic d ebris (plant litter) and soil. Soil samples were sieved to remove roots and rock fragments greater than 2 mm diameter. Sieved soil samples were oven dried at 105C for 3 days an d ground using a mortar and pestle for moisture content and total nutrient determinations.

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53 Nitrogenase Analysis Nitrogenase activity (N 2 fixation) was measured using the acetylene (C 2 H 2 ) reduction (AR) assay described by Inglett et al. ( 2004 ). Wet periphy ton (5 g) were placed into 42 mL, screw capped culture tubes (Kimax TM ) with an open top cap containing a teflon Acetylene gas (generated by adding water to CaC 2 in an evacuated serum bottle) was added to each tube (4 ml, approximately 10% headspace) and the tubes were shaken to make sure the gas evenly distributed in the whole space. Tubes containing samples and blanks containing only injected acetylene were incubated at constant temperature (27C) for up to 3 hours un der either light (~900 mol m 2 s 1 PAR) or dark conditions. After incubation, gas samples (4 ml) were taken from each tube and stored in evacuated 3.5 ml exetainers. Gas samples were analyzed for ethylene using a Shimadzu GC 8A gas chromatograph equipped with a flame ionization detector (110C) and a Poropak N column (80C). Two standard gases (1 and 10ppm; Scott Specialty Gases, Inc., Plumsteadville, PA) were used to calibrate the measurement being expressed as nmol C 2 H 4 g dw 1 h 1 The gas dissolved in t he liquid phase was ignored since there was no accumulated water in the tube. Blank corrected AR values were used to estimate actual rates of N 2 fixation using a theoretical conversion ratio of 3 moles of C 2 H 2 reduced to 1 moles of N 2 fixed (Howarth et al. 1988a). When estimating the annual fixed N the following assumption s were made : (1) there were two seasons with six months of wet season represented by the October and 6 months of dry season by February; (2) the whole day was divided by 12 hours light co ndition and 12 hours dark condition; (3) the biomass was constant with the season.

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54 Following the incubation, periphyton contained in each tube was dried at 70C for 3 days to determine dry weight of periphyton biomass. The dried sample was then ground usi ng a ball mill for nutrient and isotopic analysis. Chemical and Isotopic A nalysis Total C and N content were measured using Thermo Flash EA 1112 elemental analyzer (CE Elantech, Inc.). Total P of periphyton was measured colorimetrically using a Shimadzu UV 160 spectrometer (method 365.1 U.S. EPA 1993) following ashing and dissolution in 6M HCl (Anderson 1976). Total organic C was estimated by loss on ignition (LOI) at 550C for 4 h after conversion to organic C with a coefficient factor of 0.51 ( Goldin 1987 ; Wright et al. 2008 ). Stable N isotopic ratios were determined using a Finnigan MAT Delta Plus isotopic ratio mass spectrometer (Finnigan Corp. San Jose, CA) (Inglett and Reddy 2006 standard isotopic ratio of atmospheric N 2 Water C hemistry Water chemistry samples were collected in acid washed polyethylene bottles in October 2009 when the sites were flooded. The water samples were filtered through brane filter and acidified to preserve on ice until their return to the laboratory. T he total dissolved phosphorus (TDP), soluble reactive phosphorus (SRP, PO 4 3 ), ammonia nitrogen (NH 4 N), nitrite nitrogen plus nitrate nitrogen (NO x N), total dissolved Kj eldahl Nitrogen (TKN) were analyzed for the filtered water samples with standard methods (EPA, US 1983).

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55 Statistical A nalysis All statistical analyses were performed using JMP v.8 statistical software ( SAS Institute Inc., Cary, NC). Two way ANOVA was app lied to test for differences of various soil properties between sites, seasons and the interaction of site and season. Comparisons of means for significant effects were determined using Tukey HSD tests. Regression analysis with Pearson correlation coeffici ent was used to evaluate the relationship between various nutrient properties, isotopic composition and nitrogenase activity. Differences among means and correlation coefficients were deemed significant at the P <0.05 level, unless otherwise noted. Results and D iscussion Water C hemistry Results of water chemistry sampling are provided in Table 3 2. NH 4 N, dissolved inorganic nitrogen (DIN=NO X N+NH 4 N), TKN, TDP and SRP were significantly higher in the Res 00 site comparing to the reference site and Res 03 si te ( P <0.05). The water 1 (Thomas et al. 2002). However, the ratio of DIN: SRP and TDKN: TDP were significantly lower in the restored site compared to the reference site. Nutri ent C omposition A main difference between the reference and restored sites is the nutrient status, in particular the levels of N and P (Smith et al. 2011). For the periphyton, the TN content in the reference site was significantly higher ( P <0.05) with the average of 16 mg g 1 comparing to the restored sites with the average of 13 mg g 1 Periphyton TP contents in the restored sites with the average of 313 mg kg 1 were approximately three times of that in the reference site with the average of 99 mg kg 1 (Tab le 2 2 ). Accordingly, the

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56 periphyton molar TN:TP ratios in the reference site were approximately three times higher than those in the restored sites, with average values of 393 23 (Table 2 2 ). Compared to the periphyton in other parts of Everglades, the TP in the restored sites were closer to that of the Water conservation Area 1 (WCA 1) with the average of 423 mg kg 1 and the TP in the reference site was comparable to the values in the Taylor slough (TS) that has similar calcareous mats with a average of 124 mg kg 1 TP (Gaiser et al. 2006). Periphyton TN was significant higher in the dry season for the Res 00 site (with the average of 15mg g 1 ) and Res 03 site (with the average of 14 mg g 1 ) compared to the wet season with the average of 11 mg g 1 in the Re s 00 site and 12 mg g 1 in the Res 03 site. No significant seasonal difference of periphyton TN was observed in the reference site; neither was periphyton TP and TN:TP ratio in all of the three sites. The ratio of N:P is often used as an indicator of nutrien t limitation, with the value of 16:1 (molar) being the theoretical threshold between N and P limitation for oceanic phytoplankton (Redfield 1973 ). Other studies have reported optimal N: P of 20 (Hecky and Kilham 1988) or 30 (Smith 1983) for freshwater phyto plankton. However, other There were not significant differences in the nutrient parameters between the two restored wet l ands at different age ( Res 03 vs Res 00) as expected; the possible explanation is that the gaps between the two restored sites were small (Table 2 3). However, the soil TP and TN:TP ratio somehow distinguished the two sites Nitrogenase A ctivity Acetylene r eduction (AR) rates under both light and dark conditions were sig nificantly higher ( P <0.05) in the restored sites (Fig. 2 4 ab ). For the Res 00 and reference sites, there were not significant differences in the AR rates between the two

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57 seasons; while for the Res 03 site, the AR rates were significantly higher in the dry se ason. Rejmnkov et al. ( 2004 ) reported that the N 2 fixation of periphyton in the tropic marsh of Belize was higher in the wet season (i.e., July and September), and they speculated that the warmer temperature and higher solar radiation in the wet season w ould facilitate N 2 fixation. Vargas and Novelo (2007) also found that the highest AR rates of periphyton appeared during the rainy season in the Yucatan peninsula. In this study, the co ntrasting seasonal patterns of periphyton N 2 fixation between Res 00, r eference sites and Res 03 site could be the result of the sampling time at the end of the wet season (i.e., October) and the dry season (i.e., February), when the environmental factors may not have been dramatically different. Actually, precipitation in Oc tober 2009 was even lower than that in February 2010 ( NOAA Daily Surface Meteorologic Data ). A number of other factors could contribute to observed patterns of N 2 fixation including temperature, solar intensity, different vegetation in different seasons (r esulting in shading), but without experimentally testing these effects we can only speculate about the possible factors for the difference. In this study, the periphyton biomass was found to be 155 16 g AFDW m 2 in the Res 00 area, 208 28 g AFDW m 2 in the Res 03 area and 295 33 g AFDW m 2 in the reference site, which fell in the range of those reported for the marl prairies of Florida Everglades (Gottlieb et al. 2005; Iwaniec et al. 2006; Gaiser et al. 2011). P eriphyton production in marl prairies, es pecially in the wet season, was much higher than the long hydroperiod mats of periphyton, such as the periphyton mats in the water conservation area (McCormick et al. 1998; Hage rthey et al. 2011).

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58 Using the theoretical ratio of 3 moles of C 2 H 4 produced per mole of N 2 fixed, it is possible to estimate the amount of N 2 fixed for a given AR rates. Although the empirical ratio vary among different ecosystems, the theoretical ratio of 1:3 has proven reasonable for cyanobacterial mats (Howarth et al. 1988a; Doyle and Fisher 1994). In this study, significantly higher N 2 fixation rates of periphyton were observed in the Res 03 site with average of 0.2 0.03 g N m 2 yr 1 compared to the reference sites with average of 0.05 0.01 g N m 2 yr 1 ( P <0.05) (Fig. 3 2 b ). Th ese values are much smaller i n comparison with the unimpacted Water Conservation Area 2A (WCA 2A) in Florida Everglades, where the N 2 fixation rates ranged from1.8 18 g N m 2 yr 1 (Inglett et al. 2004 ). In this regard, a rough estimate of the time for the R es 03 site to reach the same level of N storage as the reference site was ma de The N storage for the Res 03 and reference site is 35 g m 2 and 125 g m 2 respectively ( Inglett et al. 2011, in review ). Assumed that all the N fixed by the periphyton in the Re s 0 3 site was absorbed by the soil, the time when the TN storage in the Res 0 3 site was close to the reference site could be roughly calculated, which is (125 35) g N m 2 / 0.2 g m 2 yr 1 =450yr. It was a long time even without considering any pathways of N lo ss. There was no significant difference in the light:dark AR ratio among the three sites (Fig. 2 4 c) with the average of 2 and 3. Overall, the ratio was similar to that of periphyton at unimpacted interior of WCA 2A with the ratio of 3.3 0.5 (Inglett et al. 2004). The response of nitrogenase activity to light intensity can give us information of the N 2 fixing species in a microbial community (Fay 1992). The light: dark AR ratios in this study were greater than 1, suggesting that photosynthetically driven microbes such

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59 as heterocystous cyanobacteria were largely responsible for the observed rates. The light:dark AR ratio was significantly higher in the dry season for the Res 0 3 and reference sites ( P <0.05), likely reflecting seasonal changes in the species composition of the periphyton communities. McCormick et al. (1998) observed that oligotrophic periphyton assemblages exhibited strong seasonal shifts in species composition and were dominated by cyanobacteria during the wet season and diatoms during the dr y season. Periphyton and S 15 N N 2 fixation by cyanobacteria is accompanied by relatively little isotopic fractionation and as a result, N 2 15 (Goericke et al. 1994 ; Kline and Lewin 1999 ). In this study, th 15 N of periphyton over all three sites all fell within the range of 2 3 ), which agrees with observations from other studies (Nadelhoffer and Fry 1994; Gu and Alexander 1993 ; Rejmnkov et al. 2004 ), but is on average lower than the rang e reported for northern Everglades marshes (1.1 ) and the freshwater metaphyton (1 2007 ). Differences in periphyt 15 N also correspond to differences in N 2 fixation measured between the three sites, with the high 15 N corresponding to the lowest nitrogenase activity observed in the reference site. Stable isotope 15 N signatures are often used to characterize N source and the mechanism of algal and plant N metabolism (Handley and Raven 1992 ; Nadelhoffer and Fry 1994 ; Inglett and Reddy 2006 ). Differences in 15 N natural abundance among freshwater N 2 fixing cyanobacteria and non fixing green algae were reported by Gu and Alexander ( 1993 ) as a reliable indicator of N 2 fixation. They found that the natural abundance o f 15 N of six N 2 fixing blue green algae was 1.0 15 N

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60 of six green algae showed an average of 6.6 The higher 15 N of non N 2 fixing algae reflected the utilization of dissolved inorganic N. 15 N of periphyton and soil were s ignificantly correlated with each other (Fig. 3 4 ), suggesting the N source in soil is derived from that fixed by the periphyton. At 15 N than the periphyton, suggesting that other processes in addition to atmospheric N f ixation affected their 15 N composition. For example coupled nitrification/denitrification, ammonia volatilization, or atmospheric deposition of 15 15 N within the soil (Aranibar et al. 200 3 ; Evans and Ehleringer 199 3 ). Relationships between Nutrients, Nitrogenase A 15 N Various factors affect N 2 fixation, including physical factors such as light intensity and temperature, and nutrient status (Howarth et al. 1988b). N and P concentrations and loadings ar e two mostly discussed factors. Scott et al. (2007) showed that periphyton N 2 fixation decreased as reactive N accumulated in the periphyton matrix. In this study the N 2 fixation of periphyton was negatively and weakly correlated with the TN content of per iphyton (R 2 =0.2, P <0.05); however significantly higher TN content in the reference site corresponded with significantly lower AR rates. In contrast with the results of Scott et al. (2005) that decreasing N 2 fixation was related to the increase in metaphyto n N content, this study showed that increase TN content in February corresponded to increase AR rates for the Res 0 3 site. Ammonium concentrations can be quite important in regulating fixation rates in sediments since they can inhibit the synthesis of new nitrogenase (reviewed by Howarth et al. 1988b). The ammonium concentrations of the water samples collected in October 2009 in HID 1 (Table 3 2), which was much higher

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61 1 for the WCA 2A of northern Florida Everglades). This would explain why the AR rates of periphyton in this study were extre mely lower than their values, even though the TN:TP ratio in the restored sites was similar with the northern Everglades by Inglett et al. (2004). There was a significantly positive linear relationship between N 2 fixation rates and TP; accordingly, signif icantly negative relationship between N 2 fixation rates and periphyton TN:TP were found in both dry and wet season (Fig. 3 3 ), indicating that P likely controls N 2 fixation. P controlled N 2 fixation has been reported by many researchers (Howarth 1988b; Smi th 1990; Rejmnkov and Komrkov 2000, 2001 ; Inglett et al. 2004, 2009). However, TN:TP ratio is regarded as more reasonable factor than TP in regulating the N 2 fixation since it integrates the N information, another controlling factor of N 2 fixation and would further indicate nutrient limitation (Howarth 1990). In this study, the restored sites with lower TN:TP ratio were more likely to be N limited, which then induced higher nitrogenase activities; while the reference site with higher TN:TP ratio was P l imited and suppressed N 2 fixation. There was a significantly negative correlation between N 2 fixation rates and the 15 N in both the wet and dry season (Fig. 3 5 a ), which was similar to the finding of Rejmnkov et al. ( 2004) and Inglett et al. (2004). Sc ott et al. (2007) also found that in 15 N decreased with increasing N 2 fixation during May through September sampling. Like the correlation of nitrogenase activity with TN:TP ratio and 15 N, significant relationship s bet ween 15 N and the periphyton TP, TN:TP ratio were also found (Fig. 3 5 b,c).

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62 Conclusions Periphyton plays a significant role in the Everglades, especially in the restoration of the calcareous Hole in the Donut wetland ecosystem. This study documented the p resence of nitrogenase activity in calcareous periphyton mats of the Southern Everglades (marl prairie) and estimated its annual contribution to the N budget of these wetlands. Overall rates of the reference marl prairie system were low compared to publis hed estimates of northern Everglades systems. Periphyton in restored sites exhibited significantly higher AR rates than the reference marl prairie system, serving as evidence of N limitation in the developing systems following soil removal. Like other repo rts of calcareous periphyton mats, nitrogenase activity in this study was stimulated by light indicating the primary N 2 fixing community was cyanobacterial in nature. Periphyton N 2 fixation is considered as a major part in the N budget in the oligotrophic Everglades that has received less attention. For the young restored ecosystems, N 2 fixation would be an important N source and thus a key target or evaluation metric for restoration. The seasonal pattern of N 2 fixation and 15 N were not 15 N has a potential of characterizing the N 2 fixation and tracing the N cycle, more information such as the 15 N signature for other inorganic nitrogen form, N 2 fixing species composition were needed to e xplain the differences. The periphyton community is sensitive to nutrient status, and in this study, N 2 fixation, 15 N were correlated in both wet and dry season. These findings suggest that N 2 15 N could also be employe d as indicators of nutrient limitation and patterns of nitrogenase activity.

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63 Table 3 1. Geographical coordinates and the approxim ate elevation of the sampling sites used in this study. Sampling site Station Latitude Longitude Elevation (m) Res 00 A N2 5.38283 W80.67442 0.7 0.8 B N25.38150 W80.67452 0.7 0.8 C N25.37856 W80.67477 0.6 0.7 D N25.37408 W80.67515 0.6 0.7 E N25.37019 W80.67652 0.5 0.6 Res 03 A N25.38865 W80.69132 0.8 0.9 B N25.38618 W80.69414 0.6 0.7 C N25.38260 W80.70003 0.6 0.7 D N25.37903 W80.69982 0.6 0.7 E N25.37678 W80.69946 0.5 0.6 Ref erence A N25.38117 W80.67275 0.8 0.9 B N25.37916 W80.67246 0.7 0.8 C N25.37635 W80.67224 0.7 0.8 D N25.37293 W80.67229 0.6 0.7 E N25.36985 W80.67223 0.6 0.7 Note: The ground eleva tion data extracted from the EDEN DEM model ( http://sofia.usgs.gov/eden/models/groundelevmod.php ).

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64 Table 3 2. Mean water chemistry values at all sites in the October 2009 when the sites was flooded (mean SE, n=6 for Res00 site; n=12 for Res03 site and n=12 for the reference site) Site SRP NH 4 N NO x N TDP TDKN DIN:SRP TDKN:TDP 1 1 1 1 mg L 1 mol:mol mol:mol Res 00 6.6 (2.1) 250 (62) 5.4 (0.4) 9.9 (1.4) 1.6 (0.2) 1 03 (29) 364 (81) Res03 3.0 (0.1) 47 (6) 3.3 (0.4) 4.4 (0.7) 0.8 (0.1) 37 (5) 456 (40) Reference 2.7 (0.1) 88 (12) 4.2 (0.9) 2.6 (0.6) 0.6 (0.0) 78 (10) 914 (228)

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65 Figure 3 1 Map showing the general area of Everglades National P ar k and the locations of restored and reference marl prairie sites used in this study. Miami Homestead Hole in the Donut Florida, USA

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66 Figure 3 2 Areal estimates of N 2 fixation in the restored and reference sites (see text for calculation details). The white bars represent the data in the dry seaso n, the shaded bars represent the data in the wet season; and the dash bars represent the whole year estimate. The different lowercase letters denote significant differences ( P <0.05) between the three sites.

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67 Figure 3 3. Relationshi ps between periphyton acetylene reduction (AR) under light conditions and periphyton TP (a) and TN:TP ratio (b) in the dry and wet seasons.

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68 Figure 3 4 15 15 N

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69 Figure 3 5 15 a cetylene r eduction rates (AR), TP and TN:TP ratio

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70 CHAPTER 4 SEASONAL PATTERN OF PERIPHYTO N NITROGENASE IN THE SHORT HYDROPERIOD, CALCAREOUS WETLANDS Background Periphyton are complex assemblages of cyanobacteria, algae, heterotrophic microbes, microfauna and detritus growing attached to a substrate, and can als o be called cyanobacteria mats. P eriphyton are widely distributed in aquatic systems, limestone based tropical wetlands in the Caribbean, Florida Everglades, Yucatan Peninsula of Mexico and Belize alkaline marshes (Rejmnkov and Komrkov 2000; Gaiser et al. 2011; Vargas and Novelo, 2007 ; Rejmnkov et al. 2004). As an important component of these ecosystems, periphyton perform a range of vital functions including: facilitating and stabilizing soil formation through calcium carbonate precipitation (Hagerthey et al. 2011); serving as a mai n contributor to primary productivity (McCormic 2011); regulating water column nutrient levels and benthic fluxes; and based on species composition and microbial activities, serving as a sensitive indicator of water quality and nutrient status (McCormick et al. 1996; McCormick and Stevenson 1998; Sharma et al. 2005; Jasrotia and Ogram 2008; Scott et al. 2007, 2008, 2009). In addition, periphyton can be an important nitrogen (N) source as diazotrophs in the periphyton convert atmospheric N 2 to bioavailable N fo rms However, not many studies have focused on the contribution of N 2 fixation by these organisms, especially in tro pical wetlands (Vargas and Novelo 2007). Periphytic mats are a ubiquitous feature of the Florida Everglad es, USA, and can be categorized into four distinct habitats including the marl prairies and rocky glades, the ridge and slough of the central Everglades, the soft water marshes (such as Water Conservation Area 1, WCA 1), and the coastal mangroves along Flo rida and Biscayne

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71 Bays (Gaiser et al. 2011). Periphyton can be further classified into two habitats, based on hydroperiod including long (flooded for more than 8 months of the year) and short (inundated for less than 8 months of the year) hydroperiod marsh es (Acosta and Perry 2000). Accordingly, spatial patterns of periphyton species composition, abundance, and appearance with water quality and hydrologic conditions have been well described (Brow der et al. 1994; Gaiser et al. 2011; Gottli eb et al. 2005; Iwa niec et al. 2006). In comparison, the temporal dynamics of periphyton has received less attention. McCormick et al. (1998) described the changes of the periphyton biomass and species composition during the wet and dry season in the long hydroperiod northe rn Everglades, finding that epiphyton and metaphyton biomass was seasonal and peaked during the wet season. In contrast, seasonal variation of periphyton has been rarely studied in shor t hydroperiod marshes (Gottlieb 2003). These short hydroperiod periphyt on have a rapid response to the dry rewetting cycle similar to cyanobacterial crust communities found i n desert environments (Gottlieb 2003; Belnap 20 04; Aranibar et al. 2003; Pcs 2009). Gottlieb (2003) found that the dry short hydroperiod mats became net primary producers within a short t ime after hydration. Similarly, respiration and photosynthesis of soil biological crusts in the southern Utah begin almost immediately upon rewetting; but there is generally a lag phase between wetting and the initiation of N 2 fixation, with a further lag time before maximal fix ation rates are reached (Belnap 2001). The longer they are desiccated, the more time it takes for N 2 fixation to begin and to reach maxima l rates after rewetting (Belnap 2001). Therefore, sampling s trategy with finer time span is needed to know more details about the temporal pattern of periphyton especially in the short hydroperiod landscape.

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72 It is well known that periphyton can be used as a indicator of restoration in the Florida Everglades since it responds very rapidly to the changes in the two dominant drivers of wetland structure and function, i.e., hydrology and water quality (McCormick et al. 19 96; Gaiser et al. 2004; Gaiser 2009). Specifically, phosphorus (P) the limiting nutrient in most ar eas of the Everglades controls the abundance and community of t he periphyton (McCormick et al. 1996). However, few people have investigated the response of periphyton N 2 fixation to the nutrient status in Everglades (Inglett et al., 2004, 2009, 2011; Jasro tia and Ogram 2008) though in other ecosystems like lakes, numerous evidences have shown that P regulates N 2 fixation. Moreover, some fractions of Everglades have been experiencing a shift from P limitation to N limitation, such as the downstream areas tow ards Florida Bay where P derive from the Gulf of Mexico is much more abundant (Childers et al. 2006; Inglett et al. 2011). In those areas under the N limitation, periphyton N 2 fixation will function as an important N source and serves as another attribute of periphyton except biomass and species composition in indicating the restoration process. The Hole in the Donut (HID) located in the Everglades National Park is such a case that P was accumulated from the historical agricultural activities (Smith et al 2011, Fig. 4 1). To restore the HID to its originally low nutrient status, the technique of complete soil removal was adopted (Li and Norland 2001; Dalrymple et al. 2003). In this process soils are mechanically cleared to bedrock and allowed to naturally reestablish biotic communities such as periphyton and macrophytes. During the HID restoration processes, periphyton plays an important role in soil formation, as a source of organic matter and calcium carbonate (CaCO 3 ) both main componen ts of marl soils ( Gaiser et

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73 al. 2011). N 2 fixation of periphyton also provides an important N source particularly in the recently cleared sites wh ere N is limiting (Smith et al. 2011; Inglett et al. 2011). Liao and Inglett (2012) described the spatial pattern of periphyton N 2 fixation in two restored and one reference wetlands in this area. However, temporal dynamics of periphyton N 2 fixation i s still lack of understanding. Therefore, the following questions were proposed in this study: (1) Does N 2 fixation vary with time in the sub tropical, oligotrophic, calcareous prairie wetlands? (2) Are the seasonal patterns for the restored and native wetlands the same? and (3) What nutrient parameters control seasonal changes of periphyton N 2 fixation in these wetlands. Materials and M ethods Study S ite T wo restored wetlands (cleared in 2000 and 2003, and referred to as Res2000 and Res2003, respectively) as well as an unfarmed reference site adjacent to the restored areas were selected in the HID region of Everglades National Park (Fig. 4 1). In each site, two sampling stations with different elevation were identified (Fig. 4 1). The soil depth, vegetation composition, and the periphyton description were the same as the previous chapter. Precipitation, water depth, and solar radiation f or this location are shown in Fig. 4 2. The mean daily precipitation and temperature were obtained from the Royal Palm Ranger Station of South Florida published by National Oceanic and Atmospheric Administration (NOAA). Water depth was estimated by subtrac ting the water level from site elevation. The water level was provided by the Everglades National Park from a nearby station and corrected based on a common datum (NAD88). Seasonal levels of 1 s 1 ) were esti mated by multiplying the

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74 solar radiation (W m 2 ) observed at the weather station in the Florida International University with conversion factor 1.93 in the winter and 1 .95 in the summer (Lee and Downum 1991) (Fig. 4 2). Field S ampling The study sites were visited in the wet and dry season during 2010 and 2011 (April, May, June, July, and September in 2010 and January, May in 2011). Based on water levels and rainfall patterns, the seven sampling dates were classified into wet and dry season, where the dry se ason included the April, May, June 2010 and January in 2011, and the wet season included the July and September samplings. A grid sampling system was used in this study as follows. Three 1030 m plots were designated in each sampling station. Each of thes e plots was further divided into seventy five 2 m2 m sampling squares. At each sampling, three 2 m2 m grid squares were randomly sampled within each 1030 m plot and composited to form one replicate. Thus, for each sampling, nine 2 m2 m sampling squares were compos ited to form three replicates. All samples were sealed in plastic bags and kept on ice until their return to the laboratory where the samples were refrigerated at 4C until subsequent analysis. Nitrogenase A nalysis Nitrogenase activity (N 2 fix ation) was measured using the acetylene (C 2 H 2 ) reduction (AR) assay described by Inglett et al. (2004). The details are the same as the previous chapter, also could referred to Liao and Inglett (2012). When estimating the annual fixed N I (1) used the pe riphyton biomass data from the previous study (February 2010, Liao and Inglett 2012 ) and assumed the biomass was constant with the season ; (2) averaged the N 2 fixation rates from April, May,

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75 January and June sampling as the N 2 fixation rate in the dry seas on (7 months), the rates from July and September sampling as the N 2 fixation rate in the wet season (5 months); ( 3 ) day length was considered as 12 hours of light and 12 hours dark Following the incubation, periphyton contained in each tube was dried at 7 0C for 3 days to determine their moisture contents. The dr ied sample was then ground using a ball mill for chemical and isotopic analysis. Chemical and Isotopic A nalysis When surface water was present (e.g., in July and September), triplicate water sam ples from the sites were randomly collected which were filtered through the 0.45 Ammonium (NH 4 + ) and soluble reactive phosphorus (SRP ) were measured by flow injection with a Bran+Luebbe Auto Analyzer 3 Digital Colorime ter (Bran+Luebbe, Norderstedt, Germany) for NH 4 + (EPA Method 350.1) and nitrate (NO 3 ) was measured on a Alpkem Rapid Flow Analyzer 300 Series (Alpkem Corp., Clackamas, Oregon) using EPA Method 353.3. Total Kjeldahl nitrogen (TKN) was determined via Kjelda hl block digestion and analyzed by flow injection with a Bran+Luebbe Auto Analyzer 3 Digital Colorimeter for NH 4 + (EPA Method 350.1). Total dissolved phosphorus (TDP) was measured as SRP on a Bran+Luebbe Auto Analyzer 3 Digital Colorimeter after digestion with H 2 SO 4 and potassium persulfate (EPA Method 365.1). Periphyton total carbon (C) and N content were measured using Thermo Flash EA 1112 elemental analyzer (CE Elantech Inc.,USA). Total P of periphyton was measured colorimetrically on a Shimadzu UV 160 spectrometer (Shimadzu Cor., Kyoto, Japan) using EPA method 365.1 following ashing and dissolution in 6M HCl (Anderson 1976) Loss on ignition (LOI) was obtained by combustion in the mu ffle furnace at 550C for 4

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76 h. Stable N isotopic ratios were determine d using a Finnigan MAT Delta Plus isotopic ratio mass spectrometer (Finnigan Corp., San Jose, CA) (Inglett and Reddy 2006) and 2 ows: Statistical A nalysis Data were analyzed with JMP v.8 statistical software (SAS Institute Inc., Cary, NC). Shapiro Wilk test was used to test for normalit y, and data were log transformed when necessary. A three way ANOVA was applied to analyze the main effects of site, tests ( P <0.05) for means comparisons. Regressions be tween the nitrogenase activity and the nutrients of periphyton were performed using the linear least squares method for the wet and dry season. Results Periphyton P roperties Results of the three way ANOVA demonstrated that the factors of time and site h ad significant effects on almost all the periphyton parameters (Table 4 1), while 15 N and the N 2 fixation rates (Table 4 1). For some parameters, such as TP, TN:TP, and the N 2 fixation rates, there w as a significant interaction between time, site and elevation (Table 4 1). In all the samplings, TN of periphyton was significantly higher ( P <0.05) in the reference site, followed by the Res2000 site and the lowest TN values in the Res2003 site (Fig. 4 3) Converting concentration to an areal basis, TN storage in periphyton was

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77 even higher at the reference site (mean of 15.6 g N m 2 ) compared to either the Res2000 or Res2003 wetlands with an average of 8.0 g N m 2 and 6.4 g N m 2 respectively (Fig. 4 6). Both site and elevation were significant factors affecting TP and TN:TP ratio (Table 4 1). TP was 2 3 times higher in the restored sites than the reference sites, but the specific magnitudes and orders among the three sites were variable between the high a nd low elevation (Fig. 4 3). In the Res2000 site, significantly higher TP values were observed in the high (181 532 mg kg 1 ) compared to the low elevation (75 171 mg kg 1 ). However, no significant differences of TP were found between the high and low eleva tion in either of the Res2003 or reference sites. Accordingly, significantly lower TN:TP ratios of periphyton were found in the restored areas (88 336 in the 2003 restored site) compared to the reference site (199 840)( P <0.05) (Fig. 4 3). The effect of ele vation was significant only for the TN:TP molar ratio of the Res2000 site where significantly higher ratios were observed in the low elevation (163 382) than in the high elevation (24 154) ( P <0.05). The stable N 15 N) fell in the range of sites (Fig. 4 4). There were not consistent order s in magnitude among the three sites. 15 N was higher in the low elevat ion than the high elevation (Fig. 4 4). Ni trogen F ixation Nitrogen fixation rates measured by acetylene reduction (AR) for the periphyton in the reference site were significantly lower than the restored sites ( P <0.05); especially during the wet season (e.g ., July), where periphyton AR rates under light condition were approximately 10 times that in the reference site (Fig. 4 5). Using the biomass

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78 estimated from a previous study (Liao and Inglett, 2012) and the theoretical ratio of three moles of C 2 H 4 produce d per mole of N 2 fixed, it was estimated that approximately 0.4 g N m 2 yr 1 was fixed by the periphyton in the Res2003 site compared to 0.2 g N m 2 yr 1 in the reference site (Fig. 4 6). Seasonal P atterns In all sites, concentrations of major nutrients s howed contrasting seasonal patterns during the study (Fig. 4 3). TN showed a general decrease throughout the summer from May to July followed by an increase from September to the next May in the Res2000 site and reference sites (Fig. 4 3). The seasonal pa tterns of TP and TN:TP molar ratio in the Res2000 and reference sites were similar and more apparent than the pattern observed in the Res2003 site (Fig. 4 3). The TP peak and minimum for the reference site appeared in September (147 6 mg kg 1 ) and April (63 5 mg kg 1 ). According ly, the TN:TP ratio showed in a inverse way with the peak in April (595 56) and the minimum in September (221 6). Similarly, for the Res2000 site, the TP varied from 165 4 (September) to 91 5 mg kg 1 (April) in the low el evation station while from 523 9 (September) to 263 82 mg kg 1 (July) in the high elevation station. Inversely, the peak and minimum of TN:TP ratio appeared in April (352 11 and 112 21 for the low and high elevation, respectively) and September (17 1 6 for the low elevation and 43 10 for the high elevation), respectively. In contrast, the TP and TN:TP ratio in the Res2003 site did not vary as much with season as in the other two sites, with the peak and minimum of TP in July (192 31 mg kg 1 ) an d May (118 11 mg kg 1 ); and TN:TP ratios in April (228 35) and July (144 25). Despite the appearance of similar trends, there was not a clear seasonal pattern of 15 N in any of the three sites (Fig. 4 4). Overall, there was an increase fr om

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79 the end of the dry period (i.e., April and May) into the beginning o f the wet season (i.e., June). Values remained stable during the flooded time period of July and September, but did not return back to the initial level as a cycle in the next May 2011. The seasonal patterns of N 2 fixation rates for the periphyton under both light and dark conditions (the dark condition is not shown) in all the three sites appeared to follow a unimodal curve (Fig. 4 5). The peak of periphyton AR rates in the Res2000 an d Res2003 areas appeared in July within the range of 20 79 nmol g 1 dw h 1 and 31 53 nmol g 1 dw h 1 respectively. In contrast, the peak of periphyton AR rate at the reference site was observed in September with the range of 2 5 nmol g 1 dw h 1 Relationship between N 2 F 15 N Throughout the seasonal sampling, there were strong correlations between nitrogenase activity and measured physical, chemical, and isotopic variables (Table 4 2, Fig.4 6). Periphyton moisture contents displayed a significant ly positive relationship with the AR rates in the dry season ( R 2 =0.5 P<0.0001 ) but not in the wet season ( R 2 =0.002 P=0.78 ). Overall, nitrogenase activity was negatively related with TN:TP ratio, while correlations with other parameters were seasonal. For example, in the dry season there was no significant correlation between the TN content, but rates of AR were positively correlated with TP (R 2 = 0.25, P <0.05) and negatively correlated with TN:TP molar ratio (R 2 = 0.27, P <0.05) (Table 4 2). In contrast, for the wet season, TN had a significantly negative correlation with the N 2 fixation rates (R 2 = 0.42, P <0.05), while TP did not (R 2 = 0.14, P =0.23) and TN:TP molar ratio again showed a significant negative correlation with AR rate (R 2 = 0.62, P <0.05) (Table 4 2 ). 15 N had an inverse pattern of the AR rates through the three sites with higher AR rates and corresponding ly 15 N

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80 values in the restored sites (Fig. 4 15 N an approximately 10 fold change in AR rates. Discussion Spatial and Seasonal Patterns of Periphyton N 2 Fixation The site differences in nitrogenase activity agreed with previous studies conducted in this area (Liao and Inglett 2012) where higher N 2 fixat ion rates were found in the restored sites than in the reference site. I also found in this study that AR rates were often greater in the high elevation sites, but in July when the sites were flooded, the AR rates were higher in the low elevations sites (F ig. 4 5). Higher P levels in the high elevations (Fig. 4 3) could explain the greater AR rates in that location; however, in these shallow wetland systems, elevation is also a surro gate of hydrology (Smith et al. 2011) which could regulate periphyton produ ctivity and nitrogenase activity through patterns of flooding and drought. The low elevation is more frequently inundated and the water depth tends to be higher than the high elevation station. In this regard, the light availability for the periphyton in t he low elevation would decline more quickly with the deeper water depth than the h igh elevation (Hagerthey et al. 2011; Kahn and Wetzel 1999), leading to more flood tolerant N 2 fixers dominated in the low elevation stations which could fix N 2 more efficien tly in the wet season. I observed very pronounced seasonal patterns of N 2 fixation with overall higher N 2 fixation in the wet season (e.g., July and September) (Fig. 4 5). These patterns in the restored sites (Fig. 4 5) were similar with those observed in Water Conservation Area 2A (WCA 2A) of the nort hern Everglades (Inglett et al. 2004), in which the AR rates of the floating mats showed a peak in July and then dropped dramatically in September and continued the decline to a minimum in November. In contra st, for the reference site,

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81 the nitrogenase pattern was much more gradual increasing to a later peak in September. Considering the seasonality of N 2 fixation, the estimated areal annual N fixed by periphyton in this study was much higher with the average of 0.4 and 0.2 g N m 2 yr 1 compared to the previous estimate with the average of 0.1 and 0.05 g N m 2 yr 1 in the restored and reference sites, respectively (Liao and Inglett 2012). This study resulted in a larger estimate primarily because the previous s tudy missed the dramatic high peak of nitrogenase activities observed in the early wet season. A variety of factors have been used to explain patterns of nitrogenase activity in algal systems. Rejmnkov et al. (2004) reported that N 2 fixation of periphyto n in a tropical marsh of Belize was higher in the wet season (i.e., July and September), and they speculated that warmer temperatures and higher solar radiation facilitate N 2 fixation. Vargas and Novelo (2007) also found that the highest AR rates of periph yton appeared during the rainy season in the Yucatan peninsula. In the northern Everglades, Inglett et al. (2004, 2009) suggested that nutrient levels in combination with elevated water temperatures and patterns of exposure to UV light at the water surface could explain relative rates of N 2 fixation in floating periphyton mats. In the extreme dry season (e.g., May and January), the periphyton in my study sites was more li ke the soil biological crusts in arid or semiarid ecosystems where w ater availability i s highly influential on periphyton activity (Garcia Pichel and Pringault 2001; Stradling et al. 2002; Belnap et al. 2004). In this study, moisture content explained approximately 50% of the variation in N 2 fixation rates (R 2 =0.51, P <0.0001). Nitrogenase ac tivity is absent during dry periods but recovers rapidly following

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82 rehydration in the arid ecosystems ( Evans and Johansen 1999 ; Jeffries et al. 1992; Rychert and Skujin 1974; Skujin and Klubek 1978). It was similar in this study with a dramatic pulse of AR rates in July after the dry season for the restored sites (Fig. 4 5). In addition, light intensity could exert an influence in N 2 fixation by photosythetically driven N 2 fixers (Howarth et al. 1988b; Rejmnkov and Komrkov 2000). In contrast with oth er parts of the Everglades, the periphyton of this marl prairies was benthic. In September, light available for the inundated periphyton was decreased both as incident solar radiation and by the increase of water depth (more flooded than July) (Fig. 4 2). Less light penetrating to the benthos could explain the decline of AR r ates after July. Relationship o f N 2 Fixation w ith Nutrient Status The spatial pattern of periphyton TN and TP between the restored and reference sites agreed with a previous study condu cted in this system (Liao and Inglett 2012). The significant effects of elevation on the TP and TN:TP ratios in the Res2000d site were unclear, however the enrichment of P in the Res2000 high station may be the result of this site being farmed more frequen tly throughout its history prior to re storation (Smith et al. 2011). In this study, periphyton TN:TP ratios were in a wide range from 24 to 840 and the values for the reference site were consistently higher than those in the restored sites, which may sugge st that reference site would be strongly limited by P, while restored areas would be either N limited or co limited by N and P. In this regard, the result that periphyton in the restored sites with lower N content tend to fix more N would be an internal me chanism of overcoming N limitation (S cott et al. 2007). If ignored other N sources and the N loss from periphyton, to accumulate the similar amount N in the

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83 reference site (15.6 g N m 2 ), it would take 38.8 years for the Res2000 wetland and 37.7 years for the Res2003 wetland. The seasonal pattern of TN was in contrast with the results reported by Scott et al. (2007). In their study, they found that TN was increasing throughout the summer (from May to July) before falli ng down in September. It was found that there was a gradual increase of TP after the dry period (i.e., April and May) with peaks appearing at different times for different sites (Fig. 4 3). This could be explained by the dry rewetting theory summarized by Gottlieb et al. (2005) and Thomas et al (2006) where it was found that absorption of 90% of released TP fro m periphyton mats. In all of the three sites, an increase of TP in the wet season but time delayed in the reference site and Res2000 site were observed which would be the different recovery time and P uptake and release rates in the different sites. Furthermore, since the increase of TP could elevate the N 2 fixation as discussed before, the dry rewetting regime could explain the increa se of AR rates in the flooded July and September. Nitrogenase activity will be stimulated by higher P and inhibited by excess N ( Howarth et al. 1988a, 1990; Smith 1990; Rejmnkov and Komrkov 2000, 2001; Inglett et al. 2004, 2009). For this study, in the dry season with lower P and higher N content, periphyton nitrogenase activity was only significantly positively correlated with TP. In the wet season when P increased and N content declined, nitrogenase activity significantly correlated with N content ins tead (Table 4 2). Through both seasons, TN:TP ratio was significantly negatively correlated with nitrogenase activity (Table 4 2). It seems to indicate that during the development of periphyton, P would become the

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84 primary control factor of N 2 fixation at t he very beginning with lower nitrogenase activity; then with the time when fixed N is high (i.e., higher nitrogenase activity), the N 2 fixation tends to be inhibited by the availability of an alternative N source (Doyle and Fisher 1994; Crews et al. 2001 ab ), such as extractable inorganic N from attached soil mineralization. Water column NO 3 N in this study wa 1 ) while dissolved NH 4 1 1 ). However, Horne et al. (1972, 1979) found that planktonic N 2 fixation was regulated in part by NO 3 and NH 4 + concentrations; heterocyst formation by cyanobacteria was s uppressed at nitrate concentrations of 2.0 1 and NH 4 N concentrations of 20 1 (Horne et al. 1979). This may explain why N 2 fixation rates for the reference site did not reach the peak in July where the dissolved NO 3 was significantly hig 1 in the high and low elevation, respectively, compared to the two restored sites (Table 4 3). Relationship o f N 2 Fixation w ith 15 N In general, N 2 fixation by cyanobacteria is accompanied by relatively litt le isotopic fractionation and as a result, N 15 (Goericke et al. 1994; Kline and Lewin 1999). In this 15 N of periphyton over all three sites fell within the range of 4), which ag rees with observations from other studies (Nadelhoffer and Fry 1994; Gu and Alexander 1993; Rejm nkov et al. 2004). One source of evidence to substantiate a seasonal N 2 fixation pattern could be the seasonal trend of periphyton 15 N where it is widely re garded that relative increases in N 2 15 N (Rejm nkov et al. 2004, Inglett et al.

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85 2004, Scott et al. 15 N did not agree with the results from the northern Everglades (Inglett et al. 15 N had a significant seasonal 15 15 15 N depletion also did not correspond with the seasonal peak in 15 N c oincided with the highest nitrogenase activity (Fig. 4 4). 15 N had an obvious inverse pattern with the N 2 fixation across the sites (Fig. 4 6), suggesting it is a good indicator of N 2 fixation for the different sites; however, it may not be as a reliable indicator through different season. These 15 N decreased with increasing N 2 fixation during the May, July and September sampling at the Lake Waco Wetland complex, near Waco, Texas, US, but the correlation between these variables did not appear robust among months. The possible explanation for this contradicted observation is that the N isotopic signatures would depend on other environmental factors. For example, MacLeo d and Barton (1998) found that the isotopic signatures for N in samples of periphyton in a small headwater stream varied with light intensity and season. Kendall et al. (2001) observed a strong negative correlation between periphyton N isotopic compositio n and 15 N 15 N values of inorganic N in the rainwater and the surroundings could also affect the final N stable isotopic signature of the periphyton (S cott et al. 2007), which would weaken the correlation with nitrogenase activity (Rolff et al. 2008).

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86 15 N values of periphyton (Kendall and McDonnell 1998 ; Triska and Oremland 1981 ). Independent measurements of soil at these sites showed high denitrification potential in the July and September (unpublished data), which could enrich the N stable isotope signature of available N and weaken the 15 N with nitrogenase activity. Hagerthey et al. (2011) also emphasized that although nitrification and denitrification has not been directly measured, they cannot be easily discounted because anoxia does occur in periphyton. 15 N of periphyton is an integration of N isotopic signatures of al l N sources, thus the seasonal changes of other components attached with periphyton, 15 N natural abundance (Handley and Raven 1992; Nadelhoffer and Fry 1994; Inglett and Reddy 2006). Conclusions Periphyton has important ecological functions in the Everglades, especially through N 2 fixation which is considered as a major part in the N budget (Wozniak et al. 2008; Inglett et al. 2011) Of the two main periphyton habitats in the Florida Everglades, few records on the spatial and temporal pattern of periphyton N 2 fixation exist in the short hydroperiod wetland ecosystem, which responds more rapidly to the dry rewetting regime and is therefore highly seasonal (Gaiser et al. 2011). This study focused on the seasona l pattern of nitrogenase activity of periphyton in native and restored marl prairie wetlands of the Hole in the Donut region of the Southern Everglades. Similar seasonal patterns of N 2 fixation were observed among the natural and restored sites, with highe r rates in the wet season, especially in the flooded period from July through September. The peak of N 2 fixation differed between native and sites restored by complete soil removal. In native reference areas, the seasonal pattern was

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87 a gradual increase and decrease throughout the year with a peak observed in September, while most N 2 fixation occurred during a brief 2 3 month period at the onset of flooding and a peak in July for the two restored sites. Calculated annual rates from the seasonal patterns were higher compared to the previous results ( Liao and Inglett 2012 ) when real peaks were included in this study I n this study, N 2 fixation was controlled by both physical (moisture) and chemical (N and P) parameters. Dry season rates were a function of both moisture content (hydrating periphyton) and P content, while during flooded months excess N levels appeared to suppress nitrogenase activity. Unlike other studies, seasonal patterns of periphyton 15 N did not strictly correlate with N 2 fixation. In genera l, 15 N in the three sites showed inverse pattern of the N 2 fixation rates, but the negative relationship was much weaker in the wet season (e.g., July and September). It is suggested that 15 N could be a good indicator of the relative N 2 fixation between sites, but would not be as robust in determining seasonal N 2 fixation since t 15 N values of periphyton integrate a variety of N sources and processes (e.g., denitrification) Results of this study have important implications for the N budget of t he HID wetlands and other wetlands with extreme seasonal dry/wet cycles. The dynamic interaction between flooding, nutrient availability, and periphyton N 2 fixation warrant further study in light of N cycling in both native and restoring systems. More work is also needed to relate seasonal patterns of species composition to those observed for nutrients and nitrogenase activity in this study. Understanding the seasonal dynamics of these parameters can help improve the models for N dynamics and our understand ing

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88 of the role of periphyton in Southern Everglades marshes and other similar wet prairie wetlands.

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89 Table 4 1. Summary of three way ANOVA tests for main effects and interactions of time, site, and elevation on periphyton parameters: loss of ignition (LO I, %), total nitrogen (TN, g kg 1 ), total phosphorus (TP, mg kg 1 ), TN:TP molar ratio, N stable isotop 15 2 fixation measured by acetylene reduction rates under light condition (AR_light, nmol C 2 H 4 g 1 dw 1 hr 1 ) and the ratios of AR under light and dark conditions (AR_L:D). P <0.05; ** P <0.01, *** P <0.001, NS no significant differenc e. Source LOI TN TP TN:TP 15 N AR_light AR_L:D time ** ** ** ** NS site ** ** ** ** ** ** ** elevation NS NS ** ** NS timesite ** NS NS NS ** NS timeelevation NS NS NS NS NS ** elevationsite NS NS ** ** NS NS NS timesiteelevation NS NS NS NS NS NS

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90 Table 4 2. Mean water chemistry values observed at the two restored wetlands (Res2000 and Res2003) and the native reference wetland (Reference) in different elevation. during the wet flooded sampling time (July and September, 2010). D ata were represented as the mean (standard error). DOC dissolved organic carbon, TDP total dissolved phosphorus, TDKN total dissolved Kjeldahl nitrogen. N=3. Site Elevation DOC NH 4 N NO 3 N TDP TDKN TDKN:TDP mg L 1 1 1 1 mg L 1 mol:mol July, 2010 Res2000 High 9.5 (1.9) 41 (7) 4.2 (0.1) 4.4 (0.4) 0.7 (0.1) 437 (43) Low 14 (0.4) 42 (2) 5.1 (0.3) 4.4 (0.3) 1.4 (0.0) 696 (40) Res2003 High 5.3 (0.5) 19 (0) 5.4 (0.9) 3.9 (2.0) 0.5 (0.1) 541 (26) Low 7.8 (0.2) 25 (1) 5.9 (0.2) 4.4 (1.8) 0.6 (0.0) 542 (104) Reference High 5.8 (2.0) 46 (9) 15 (4.3) 4.0 (1.0) 0.9 (0.3) 464 (134) Low 7.4 (0.9) 34 (4) 20 (4.1) 1.8 (0.2) 0.9 (0.1) 1124 (206) Sep., 2010 Res2000 High 4.2 (0.2) 12 (1) 1.0 ( ) 3 .4 (0.4) 0.8 (0.0) 516 (27) Low 3.6 (0.2) 13 (1) 2.1 ( ) 5.9 (2.5) 0.8 (0.1) 506 (8) Res2003 High 2.9 (0.1) 19 (5) 4.8 (0.3) 2.9 (0.1) 0.6 (0.1) 475 (36) Low 4.4 (0.6) 20 (9) 4.8 (0.4) 4.4 (0.9) 0.8 (0.0) 436 (73) Reference High 5.3 (0.2) 16 (0) 1.2 (0.2) 7.1 (0.4) 0.9 (0.0) 267 (17) Low 4.4 (0.5) 21 (0) 1.8 (0.4) 5.9 (0.9) 1.0 (0.2) 386 (44)

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9 1 Table 4 3 Correlation between the N 2 fixation measured by acetylene reduction (AR) rates and total nitrogen (TN), total phosphorus (TP) and TN:TP molar rat io in the wet (right side, N=22) and dry season (left side, N=12). The AR rates satisfied the normalization after the log transformation (Y=log (AR)). The wet season included the July and September sampling results and the dry season included the April, Ma y, June and January sampling results. Parameters (X) Dry season Wet season TN Y=0.03X+0.8, R 2 =0.01, P=0.59 Y= 0.7X+10, R 2 =0.42, P=0.02 TP Y=0.002X+0.1, R 2 =0.25, P=0.02 Y=0.005X+1.3, R 2 =0.14, P=0.23 TN:TP molar ratio Y=0.001X+0.9, R 2 =0.27, P=0.01 Y= 0.0 2X+4.2, R 2 =0.62, P=0.003

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92 Figure 4 1 Location of sampling sites in the two restored wetlands (restored in 2000 and 2003) and a native reference wetland of Hole in the Donut in the Florida Everglades, US. Miami Homestead Hole in the Donut Florida, USA

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93 Figure 4 2 Mean maximum (max.) and minimum (min.) daily temperature (temp, o C) 2 s 1 ), mean water depth (WD, mm) (from Jan. to Jun., Nov. to Dec., the water depth was negative values), and the da ily mean and maximum precipitation for the study site in 2010. Date source in details seen in the text.

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94 Figure 4 3 Seasonal patterns of periphyton nutrient in the two restored wetlands (Res2000 and Res2003) and the native reference wetland (Ref erence) during 2010 and 2011. Elevation factor has a significant effects on the total phosphorus (TP) and the TN:TP molar ratio, so the seasonal patterns of TP and TN:TP were separate from the high and low elevation.

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95 Figure 4 4. Seasonal change 15 (Res2000 and Res2003) and the native reference wetland (Reference) in different elevation. (* denotes significant elevation difference, P <0.05).

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96 Figure 4 5 Seasonal changes of periphyton acetylene reduction (AR) rates under light condition in the two restored wetlands (Res2000 and Res2003) and the native reference wetland (Reference) in different elevation. (* denotes significant elevation difference, P <0. 05)

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97 Figure 4 6 Nitrogen fixation measured by acetylene reduction rates (AR) (nmol C 2 H 4 g 1 dw h 1 ), estimated fixed nitrogen per day using the 3:1 ratio and assuming 12 hours light condition and 12h dark condition (mg N m 2 day 1 ), nitrogen storage (g N m 2 ), and nitrogen stable isotopic signature of periphyton in the dry (including the April, May, June and January, on the left side) and wet (including the July and September, on the right side) season for the Res2000, Res2003 and reference site. Erro r bars represent standard error (S.E.).

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98 CHAPTER 5 SEASONAL DYNAMICS OF SOIL NITROGEN PROCESSING DURING THE RESTORATION IN SHORT HYDROPERIOD, CALCAREOUS WETLANDS Background Nitrogen (N) and phosphorus (P) are critical for primary production (Hecky and Kil ham 1988; Elser et al. 1990; Vitousek and Howarth 1991). Compared to P cycle, the N cycle is more complex especially in wetlands including a diversity of transformations (e.g., ammonification, N mineralization, nitrification and denitrification) and numero us forms ranging from particulate and dissolved organic to dissolved inorganic and gaseous species. The extractable ammonium (NH 4 + ) and nitrate (NO 3 ) as the products of N mineralization can be adsorbed by the plants and thus enhance their primary producti on. Microbial groups actively involve in these processes and can decompose complex and refractory organic matters into available N by releasing enzymes. Denitrification is the major pathway leading to the N loss and potentially contributes to the greenhous e gas emission (i.e., the intermediate product N 2 O). Accordingly, a variety of factors regulate N cycling such as temperature, moisture content, plant growth. Spatial and temporal variations of these factors and their interactions often lead to the high v ariability of soil N cycle. For example, available N to plants (NH 4 + and NO 3 ) tend to pulse after a rain event (Cain et al. 1999), and are taken up and depleted by plants (Evans et al. 2001; Giese et al. 2011). Temporal pattern of soil N cycle can simulta neously reflect the effects of temperature, moisture contents and plant growth (Parker and Schimel 2011), thus it is important and necessary to understand the temporal or seasonal patterns of N cycle. In addition, the temporal patterns of N cycle vary wit h different climate regime, soil types and landscapes. The seasonal pattern in Mediterranean type climate of California

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99 grasslands with wet and cool winter and hot and dry summer would be expected to greatly differ from that found in other grassland system s (Parker and Schimel 2011). The seasonal pattern in the tropic or subtropical wetlands would be driven more by precipitation rather than temperature in the northern peat wetlands. The acidic and calcareous soil would also show a distinct seasonal trend of availability of N (Taylor et al. 1982; Van Hoewyk et al. 2000). Many of the N processes are governed by oxidation reduction reactions, which in wetlands are mainly caused by hydrologic fluctuations. Specifically for the Florida Everglades wetland ecosyst em, little has been done on the N cycle compared to P (White and Reddy 1999, 2003; Sutula et al. 2001; Inglett et al. 2004, 2006, 2009). However, an understanding of N dynamics could be crucial to understanding the spread of P impacts in the Everglades sys tems (Inglett et al. 2011). For example, White and Reddy (2000) found that the increase of potentially mineralized nitrogen (PMN) was linked to the P enrichment in the northern Everglades. Newman et al. (2001) further confirmed their results and concluded P enrichment could enhance soil porewater NH 4 + regeneration. N 2 fixation, an important function of periphyton, is a significant N source in Everglades (Craft et al. 1995) and was reported to be stimulated by P (Inglett et a l. 2004, 2009). Moreover, the div ersity of hydrology across the Everglades featured a variety of spatial and temporal patterns of N cycle. Most existing researches on N cycle has focused on the long hydroperiod wetlands (e.g., northern marshes) that remain flooded for more than 8 mont hs o f the year (White and Reddy 2000, 2003; Inglett et al. 2004, 2006). Comparatively less attention has been paid on the short hydroperiod wetlands that remain inundated for less than 8 months of the year (e.g., southern marl and

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100 calcareous wetlands) (Gottlie b et al. 2005; Gaiser et al. 2011 ; Sutula et al. 2001 ). Those short hydroperiod, marl/calcareous wetlands undergo more pronounced dry rewetting periodic cycles that is directly pulsed by seasonal changes of rainfall (Ewe et al. 200 6 ), which would lead to d istinctive seasonal patterns of N cycle. However, little is known about importance of these processes. For those reasons, three calcareous wetlands with short hydroperiod, in southern Everglades of Florida, i.e., two restored wetlands with high P and one native reference wetlands with low P were selected to investigate the seasonal patterns of soil N cycling The following questions were proposed: (1) how do soil N availability, the internal processes and microbial activities vary with the time? (2) Do th e temporal patterns of N cycle differ between the restored and reference wetlands? I measured extractable inorganic N pools, potentially mineralizable nitrogen (PMN), potential rates of denitrification, microbial biomass carbon and nitrogen (MBC and MBN), and N related enzymes seven times during 2010 to 2011. I expected that during the wet season when soils are moist and microbes are active, N processing rates and microbial activities would be higher Under the influence of different P concentration, the re stored and reference sites would show some differences in the seasonal patterns. Methods and M aterial Study S ite The climate of the south Florida mainland is sub tropical with a rainy season that extends from the end of May to October followed by a pronoun ced dry season from November to most of May (Ross et al. 2006). Plant growth also exhibit different seasonal patterns. The study site was located in the Hole in the Donut (HID) in the Everglades National Park (ENP) which had a long history of farming (Smit h et al. 2011).

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101 Disturbance and excess nutrients by the agricultural activities led to the invasion of Brazilian pepper ( Schinus terebinthifolius ) after farming ceased (Li and Norland 2001). To restore the HID to marl prairie ecosystem, complete soil remov al that is removing all the vegetation and soil down to the bedrocks was adopted (Dalrymple et al. 2003). During the soil development, the young restored sites would be more N limited with high P while the reference site tend to limited by P (Smith et al. 2011). I selected two wetlands restored in 2000 and 2003 (referred to as Res00 and Res03) as well as an unfarmed reference site adjacent to the restored areas in the Hole in the Donut region of Everglades National Park (Fig. 4 1). The description of the st udy sites including the vegetation, and soil depth were the same as the Chapter 2. Field S ampling This section is the same as the Chapter 4 Soil Physical and Chemical A nalysis Soil samples were sieved to remove roots and rock fragments greater than 2 mm d iameter. Sieved soil samples were used in determination of all microbial and enzyme related parameters, while a subsample of sieved soil was oven dried at 105C for 3 days and ground using a mortar and pestle for moisture content and total nutrient determ inations. The measurement of Loss on ignition (LOI), total carbon (TC), total nitrogen (TN), total phosphorus (TP), e xtractable ammonium (NH 4 N) and nitrite/nitrate (NO x N) m icrobial biomass C and N (MBC and MBN) follow the same methods as described in t he Chapter 2. To measure soil potential mineralizable nitrogen (PMN), a procedure modified from White and Reddy (2000) was used. The soil was incubated under anaerobic

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102 condition at 40C for 10 days, followed by KCl extraction, and then the NH 4 N was deter mined colorimetrically using a Technicon TM Autoanalyzer (EPA 350.1, 1993). On the same day as the preparation of the incubated samples, time zero control samples were extracted and the NH 4 N was measured in the same way. The PMN was calculated as the diffe rence between the final and time zero NH 4 N content. Denitrification Enzyme A ctivit i es Denitrification enzyme assay (DEA) was modified from Smith and Tiedje (1979), using the acetylene block technique (yielding N 2 O production) and adding sufficient NO 3 an d carbon source. Triplicate, 5 g wet soil samples were sealed with rubber septa stoppers in a 30ml tube; 3ml distilled deionized (DDI) water were added to the tube and purged with N 2 to maintain anaerobic condition s 2 ml acetylene gas (C 2 H 2 approximately 10% headspace) was injected to the tubes to block the conversion from N 2 O to N 2 and the tubes were then shaken for 1h to disperse the gas. After shaking, DEA solution (0.202 g KNO 3 L 1 0.25 g chloramphenicol L 1 and 0.360 g C 6 H 12 O 6 L 1 ) purged with oxy gen free N 2 gas was added to the tubes. Samples were shaken and incubated at room temperature. Headspace gas was collected at 1h interval for about 5 hours. The potential denitrification rate was calculated from the steepest portion of curve produced when cumulative N 2 O evolution was plotted against time. The sampled gas was injected in a Shimadzu GC 14 A ECD gas chromatograph equipped with an electron capture detector (ECD) and Porapak Q packed column The operation temperatures for the column, injection p ort, and detector were set at 70, 120, and 230 C respectively. A 10 ppm standard concentration gas (Scott Specialty Gases, Inc., Plumsteadville, PA) w as used to calibrate the measurement and results were reported as N 2 O N produced per gram of dry weight soil per hour.

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103 Extracellu l a r Enzyme A ctivities Two N related enzymes, i.e., N acetyl D glucosaminidase (NAG, EC 3.2.1.30) and Leucine aminopeptidase (LAP, EC 3.4.11.1), were measured using fluorogenic enzyme substrate (Hoppe 1983). Methods were modified from Sinsabaugh et al. (1997) to optimize the s ubstrate concentrations in soil samples with fluorogenic substrates 4 Methylumbelliferyl N acetyl D glucosaminide (MUF N) for NAG, and L leucine 7 amido 4 methyl coumarin (AMC) for LAP. Enzyme substrates MUB N and AMC produce fluorochrome methylumbellif erone (MUF) and aminomethylcoumarin (AMC) once they are hydrolyzed by the enzyme NAGase and LAPase respectively. The fluorescence of samples and standards was measured at excitation of 350nm and emission of 450nm using a Bio Tek Model FL600 fluorometric pl ate reader (Bio Tek MUF or AMC released g 1 dw h 1 Statistical A nalysis Data were analyzed with JMP v.8 statistical software (SAS Institute Inc., Cary, NC). A Three way ANOVA was applied to analyze the main effects of site, elevation and P <0.05). When way analyses for the different site P < 0.05. Multivariate analysis with Pearson product moment correlation coefficients was used to evaluate the relationship between different soil properties. Data were log transformed w hen necessary to improve normality.

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104 Results Spatial Patterns of Soil P roperties All the three sites have alkaline soil with pH ranging from 7.4 to 8.5 (Fig. 5 1 ). In general, pH at the reference site was significantly higher than the restored sites. The soil moisture contents were significantly lower at the reference site compared to the restored sites (Fig. 5 1 P <0.05). Interestingly, the total nitrogen (TN) was higher at the Res 00 site compared to the reference and Res 03 sites. During all the sampling s, the total phosphorus (TP) at the reference site was significantly lower within the range from 93 to 199 mg kg 1 compared to the Res 00 site from 256 to 888 mg kg 1 and the Res 03 site from 377 to 905 mg kg 1 ( P <0.05). Accordingly, the TN:TP molar ratios a t the reference site were significantly higher than the values in the restored sites ( P <0.05). In addition, the TOC:TN molar ratios at the three sites were all below 25 with significantly higher values at the restored sites compared to the reference site ( P <0.05). In general, the extractable NO x N and extractable NH 4 N was significantly higher at the restored sites compared to the reference site ( P <0.05 Fig. 5 2 ). Patterns of the MBC and MBN at the three sites was consistent with Res 00 site the highest and the reference site lowest; while the MBC:MBN ratio was significantly higher at the reference site than those at the restored sites ( P <0.05, Fig. 5 3 ). Overall significantly higher PMN was found at the restored sites than the reference site ( P <0.05 Fig. 5 4 ). Significantly higher potential denitrification enzyme activity rates were found at the restored sites compared to the reference site ( P <0.05) during all the sampling times (Fig. 5 5 ). T he two N related enzymes (LAP and NAG) were significantly higher at the restored sites than the values at the reference site during most of the sampling times ( P <0.05 Fig. 5 6 ).

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105 Seasonal P atterns of Soil P roperties The total nutrients did not vary significantly with time (Fig. 5 1 ). S oil moisture contents showed a pro nounced seasonal pattern at all the three sites with higher values in the wet season (i.e., June, July and September) than those in the dry season, especially in the May 2011, the soil was extremely dry falling down to 10% 20% moisture content (Fig. 5 1 ). The seasonal pattern of extractable NO x N was inverse with NH 4 N that is higher values of NO x N appeared in the dry season while higher values of the extractable NH 4 N occur red in the wet season (Fig. 5 2 ). The dominant inorganic N form in the HID was NH 4 N indicated by high NH 4 N:NO X N ratio in most sampling times. However, in the extreme dry sampling time (May 2011), the extractable NO x N exceeded NH 4 N concentration and h igher PMN was observed in the dry season or the spring rather than the wet season ( Fig. 5 4 ). The two restored sites demonstrated more similar seasonal patterns and different from the pattern at the reference site. Interestingly, the peaks of the MBC and MBN for the reference site both appeared in the September while for the restored sit e, the peaks appeared in June or July and January. The ratio of MBC:MBN dropped to the minimum values in July. The LAP at the three sites showed similar seasonal patterns with significantly higher values in September ( P <0.05, Fig. 5 5 ). There were no con sistent seasonal trends of NAG among the three sites. For the restored sites, the NAG activities dropped to the lowest point in July and then increased to the highest point in September; but there w ere no t apparent seasonal changes for the reference site c ompared to the restored sites.

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106 There was a pronounced seasonal pattern for DEA at all the three sites with higher denitrification rate in the wet season and the peak appearing in July. The DEA in the May, 2011 was unexpectedly higher comparing with other sampling time in the dry season. Discussion Seasonal Pattern of Available Nitrogen In this study, NH 4 N concentration was consistently higher than the NO x N in most of the sampling dates. The NH 4 N showed inverse seasonal patterns as NO x N, which may cont ribute to the different properties of the two ions. NH 4 + tends negatively charged cation exchange complex; while NO x do es not bind to the soil solids because of its negative charges. NO x is usually more subject to loss than is NH 4 + Significant loss mechanisms include leaching and denitrification. The NO x is so soluble that it leaches easily when excess water percolates through the soil. In this regard, very low NO x N was measured in the wet season with abundant rainfall in this stu dy (Fig. 5 2 ). High NO x N accumulations during the extreme dry May 2011 was explained by reduced ion mobility in thin water films of dry soils (Giese et al. 2011) or as a result of high net nitrification (Hefting et al. 2004). Different seasonal patterns of available inorganic N and N mineralization rates were observed in different ecosystems. Jackson et al. (1998) observed that since plant accumulation of N was mainly confined to two short periods of the year: fall and early spring in California annual gr assland, the soil nitrate was low or depleted. However, peak values of available N and mineralization rates were reported in spring and autumn in different British grasslands (Morecroft et al. 1992). Taylor et al. (1982) showed mineralization rates and ins tant inorganic N concentrations were maximum in late winter

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107 or early spring in an acidic and calcareous soil; and further concluded from other researches that overall, the spring peak in N mineralization has been observed by many workers. They at tribute th e high N of the winter climate where protein substrate accumulates from the death of soil organisms and can be utilized when warmer conditions allow the development of the appropriate microbial p opulations T he decline in the N mineralizaton rates during late spring and early summer may be due to the lowest soil water contents. In this study, N mineralization did not exhibit a pronounced seasonal pattern but lower rates were observ ed in the summe r period (wet season) and increased in the end of the winter (January) and the spring (April). However, in the summer, the moisture explain the pattern in this stu dy since the Florida winter was still warm with the mean daily temperature dropping to 15 C ( grey line at the bottom of Fig. 5 3 ). The results here contradict the results reported by Sing and Kashyap (2007) that during an annual cycle the maximum N mineral ization rates were recorded in the rainy season in a seasonally dry tropical forest and savanna ecosystems in Vindhyan region, India. However, as they suggested, the variations in rates are related to differences in soil moisture content, nutrient status a nd vegetation cover in combination with other environmental factors. In addition, since TP and TN:TP ratio did not change significantly with the time, it is unlikely that the TP or TN:TP ratio would indirectly affect the seasonal patterns of potentially m ineralization even though White and Reddy (2000) pointed out that high P could enhance the N mineralization in a northern E verglades ecosystem.

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108 Seasonal Patterns of Microbial Biomass Carbon and N itrogen Microbial biomass is the labile portion of the orga nic fraction in soils and serves as both an important source of and sink for plant available nutrients (Jenkinson and Ladd 1981; Garcia and Rice 1994). Moisture condition is a major factor controlling survival and activity of microorganisms in the soil. T he results showed different seasonal patterns between the restored sites and reference site. Generally, at the reference site the seasonal patterns of MBC and MBN corresponded very closely with the patterns of soil moisture content increasing across the we t season from April to September with the peak appearing in the September (Fig. 5 1 and Fig. 5 3 ). However, for the restored sites, when the soil moisture content increased over 60% in July and September, the MBC and MBN showed a sudden drop especially for the Res 03 site (Fig. 5 3 ). This contrasting results between the reference and restored sites would suggest that adequate soil moisture increases microbial biomass and activity whereas beyond some point (e.g., field capacity), microbial activity decreases with increasing moisture, due to limited oxygen availability (Killham 1994). My results also supported the review by Wardle (1998) who selected 58 published studies on the temporal patterns of soil MB C and MBN to generalize the controls of the temporal var iability. In the review, it was concluded that the microbial biomass peaks appeared in different seasons and showed both positive and negative responses to temporal patterns of soil moisture. The MBC:MBN ratio is considered to be an indicator of the micr obial composition that is bacteria ha ve low MBC:MBN ratio than fungi ( Anderson and Domsch 1980 ). Enhanced root growth and increased root exudates are thought to st imulate bacterial growth ( Clarholm 1985 ), which have a low C:N ratio. In this study, a decreasing

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109 microbial C:N ratio from spring to summer was observed (Fig. 5 2 ) which agree with the results reported by Corre et al. (2002). As vegetation expanded from spring towards summer, root exudates might have also increased and favored the growth of bacteria. Seasonal Pattern of Extracellular Enzyme A ctivities Soil enzymes play an essential role in catalyzing reactions necessary for organic matter decomposition and nutrient cycling and are often used as indices of microbial activity and soil fertility (Dick 1994; Tabatabai 1994; Ajwa et al. 1999 ). The two N acquisition enzym es measured in this study, i.e., LAP and NAG, are inducible enzymes synthes ized or activated when needed. Chrst (1991) found the activities of LAP were induced at limited N availability. NAG involved in the degradation of chitin which is a relevant source of N in soils. The results in this study showed that significantly higher N related enzyme activities were found at the restored sites, indicating more N limitation at those sites (Smith et al. 2011). Various factors affect the enzyme activities such as the temperature, moisture, and nutrient demand and availability (Tabatabai 1994). Seasonality of enzyme activity can be understood as a driver that integrates simultaneous variance in those factors (Weedon et al. 2011). Studies that measured seasonal varia tion in potential enzyme activities have typically found large intra annual differences (Saiya Cork et al. 2002; Boerner et al. 2005; Wallenstein et al. 2009; Kluber et al. 2011). For example, in the Arctic and sub Arctic environments, the highest enzyme a ctivities were observed around the spring thaw which would be contributed to their temperature sensitivity (Wallenstein et al. 2009 ). However, in this study sites, the temperature seems not play that significant role in controlling the enzyme activities

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110 T he key to understanding seasonality in enzyme activity may be in the factors that regulate various systems (Boerner et al. 2005; Weedon et al. 2011). C hitinase (NAG) and acid phosphatase were reported to be regulated by primarily microclimate and soil chem ical factors, whereas lignocellulose degrading enzymes such as glucosidase and phenol oxidase are more regulated by substrate availability (Sinsabaugh et al. 1992, 1993). In this study, LAP was significantly correlated with moisture content ( r =0.64, P <0.00 1) but did not correlate with LOI (a surrogate of organic matter) ( r =0.19). In contrast, the seasonal pattern of NAG did not exactly follow the soil moisture content and even showed lowest v alues in the very wet July (Fig. 5 5 ). In this regard, NAG activit ies were significantly correlated with LOI ( r =0.51, P <0.001), suggesting that NAG would be more regulated by substrate availability. This result was different from other studies in which moisture content primarily controlled the NAG activities. For example Kluber et al. (2011) observed lowest levels of NAG activity in summer with low water content in the coniferous forests of the Pacific Northwest (USA). Seasonal Patterns of D enitrification In this study, the potential denitrification activity ex situ was measured under idealized conditions, i.e. constant room temperature and with enough carbon and nitrate supply. Though the results may overestimate the real activities, it is still considered to be effective to compare the difference between different sites (Hernandez and Mitsch 2007; Kjellin et al. 2007). Despite having less than 5 cm of soil and moisture contents similar to the reference site, relatively higher DEA activities were observed at the two restored sites, which was also reported by Smith and Ogr am (2008). The trend of increasing denitrification rates in the wet season (from May to July) could be explained by the optimum water moisture content that created anaerobic

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111 condition for the denitrification. Most studies ( Blackmer et al. 1980 ; Ruz Jerez et al. 1994; Ashby et al. 1998; Shelton et al. 2000; Machefert and Dise 2004) have shown that with increasing moisture content s the denitrification and the pr oduction of N 2 O increased. In this study, the average precipitation in May 2010 was only 2 cm with the soil moisture contents less than 45% and in July, the rainfall exceeded 8 cm with the soil moisture contents over 60%. Consequently, denitrification was stimulated and a drastic pulse of N 2 O emission occurred through a dry rewetting cycle (Muhr et al. 2008; Davidson et al. 1993). Accordingly, higher denitrification would further deplete the soil NO 3 which was the case in this study (Fig. 5 2 ). The result from Hefting et al. (2007) that higher denitrification reduced the N availability in soils also supported my finding. However, in the last sampling, the moisture content was extremely low (~ 20% gravimetric water content) but unexpectedly higher potentiall y denitrification rates were measured (Fig. 5 5 ). The high temperature could be a reason. Another possible explanation was in the extreme dry condition, the fungi would contribute more to the denitrification since its more tolerance of drought than bacteri a (Shoun et al. 1992; Crenshaw et al. 2008; Yuste et al. 2011). For example, recent work suggests that fungal rather than bacterial pathways dominate denitrification in semiarid and desert soils (McLain and Martens 2005, 2006; Crenshaw et al. 2008). Moreov er, this study showed a marked accumulation of nitrate in the dry season at all the three sites (Fig. 5 2), and followed a increase of de nitrification at the beginning of the wet season (Fig. 5 5). The high amount of nitrate and rapid consumption could mea n a pulse of N 2 O is produced at the transit during the dry rewetting cycle of a short hydroperiod ecosystem.

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112 Conclusions N cycle is involved with a diversity of input, transformation, and export, which is regulated by numerous factors. Temporal pattern s of the soil N cycle can simultaneously reflect the impacts of these variables like temperature, moisture contents and plant growth. In this study, the seasonal dynamics of soil N availability and soil mineralization in the short hydroperiod ecosystem did not follow that of pla n t growth but were more regulated by microclimate (i.e., soil moisture content). In the wet season (June to September), available nitrate/nitrite (NO x ) decreased while available ammonium (NH 4 + ) increased in the wet season, suggest ing th at the NO x may be lost by leaching or consumed by denitrification especially in the rainy seasons. Moreover, the accumulation of nitrate in the dry season closely corresponded with the increasing of denitrification at the beginning of the following wet se ason, which indicated that the short hydroperiod systems would be a potential N 2 O hot spot in particular at the alternating point from dry to wet period. When further correlating this output pathway with the input of N 2 fixation (Chapter 4), we could cons ider more interesting things about the N budget. At the beginning of the wet season, periphyton N 2 fixation also showed a peak. It is possible that the soil could fix N 2 fixation and show the same seasonal pattern. In this regard, the same peak of N 2 fixat ion and denitrification would cancel one another, and it is uncertain what the net effect of these two processes will have on the annual N budget. In addition, the decreasing MBC:MBN ratio from spring to summer would tell us the changes of microbial compo sition with the time that is in the summer, more root exudes would favor the growth of bacteria. More solid evidence from molecular perspective is needed to confirm this inference.

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113 In general, the season patterns of N cycle at the three sites were similar except for some microbial properties like MBC and MBN The different seasonal patterns of MBC and MBN would contribute to the slight hydrological difference between the reference and restored sites or the buffer capability of deeper soil profile at the ref erence site. It would be expected that with the restoration and soil accumulation, the seasonal patterns of the restored sites will be closer to that in the reference site.

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114 Table 5 1. Summary of three way ANOVA tests for main effects and interactions o f time, site, and elevation on soil parameters: soil moisture content (MC),pH, loss of ignition (LOI), total nitrogen (TN), total phosphorus (TP), TN:TP ratio, total organic carbon (TOC): TN ratio, microbial biomass carbon, nitrogen and phosphorus (MBC, M BN,MBP), extractable NH 4 N, extractable NO X N, potentially mineralized nitrogen (PMN), denitrification (DEA), Leucin aminopeptidase (LAP) and N acetly glucosaminidase (NAG). Signi fi cant correlations are mark ed : P <0.05; ** P <0.01 *** P<0.001, NS no signi ficant difference Factors LOI TN TP TOC: TN T N: T P NO X NH 4 NH 4 : NO X DEA PMN LAP NAG MBC MBN MBC: MBN LAP:NAG time *** *** NS *** *** *** *** *** *** *** *** ** *** *** *** *** site *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** NS elev. NS N S *** NS *** ** NS NS NS ** NS NS *** *** NS time*site NS NS NS NS *** *** *** *** *** *** NS *** *** *** NS time*elev. NS NS NS NS NS *** NS *** NS NS *** NS NS NS NS site*elev. NS NS *** NS ** NS NS NS NS NS NS *** NS time*site*elev. NS N S NS NS NS NS NS NS NS NS NS NS NS NS

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115 Figure 5 1. pH, soil moisture content, total nitrogen (TN), total phosphorus (TP), total organic carbon: total nitrogen (TOC:TN) and TN:TP over two years (2010 2011) in the restored (Res00 and Res03) and r eference (Ref.) sites in the Hole in the Donut. Error bars represent 1 standard error of the mean for each sampling date (N=6).

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116 Figure 5 2 Extractable NOx N and NH 4 N with temperature and rainfall events (light grey line) over two years (2010 201 1) in the restored (Res00 and Res03) and reference (Ref.) sites in the Hole in the Donut. Error bars represent 1 standard error of the mean for each sampling date (N=6).

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117 Figure 5 3. Microbial biomass carbon, nitrogen and their ratio (MBC, MBN and MBC:MBN) ratios with temperature and rainfall events (light grey line) over two years (2010 2011) in the restored (Res00 and Res03) and reference (Ref.) sites in the Hole in the Donut. Error bars represent 1 standa rd error of the mean for each sampling date (N=6).

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118 Figure 5 4 Potentially mineralized nitrogen (PMN) with rainfall events (light grey line) over two years (2010 2011) in the restored (Res00 and Res03) and reference (Ref.) sites in the Hole in the Don ut. Error bars represent 1 standard error of the mean for each sampling date (N=6)

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119 Figure 5 5. Leucine aminopeptidase (LAP) and N acetyl D glucosaminidase (NAG) activities with rainfall events and temperature (light grey line) ov er two years (2010 2011) in the restored (Res00 and Res03) and reference (Ref.) sites in the Hole in the Donut. Error bars represent 1 standard error of the mean for each sampling date (N=6).

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120 Figure 5 6. Denitrified enzyme activities (DEA) with rainfa ll events (light grey line) over two years (2010 2011) in the restored (Res00 and Res03) and reference (Ref.) sites in the Hole in the Donut. Error bars represent 1 standard error of the mean for each sampling date (N=6).

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121 CHAPTER 6 FIRE EFFECTS ON THE N ITROGEN CYCLE IN CALCAREOUS WETLANDS Background Fire is widely used as a primary restoration technique in various ecosystems and has ecological significance. It was reported that fire can reduce fuel loads which then reduce the risk caused by the wildfire; prepare sites for seeding and planting and improve wildlife habitat ( Heisler et al. 2004) Moreover, fire also has great influences on nutrient cycles in ecosystem by changing the form, distribution and amount of nutrient as well as by changing species co mposition ( Bissett and Parkinson 1980; Brown et al. 2004; Certini 2005; Dumontet et al. 2006; Grogan et al. 2000; Raison 1979). The effect of fire on the nitrogen (N) cycle is highly important because N is often the limiting nutrient for primary productiv ity. Compared to P the N cycle is more complex and involving various processes, for example, N can be easily lost through denitrification, ammonification and volatili s aiton. Since N is important for plants, many studies have focused on the available N sta tus before and after fire, trying to know the effects of the fire on subsequent primary production During the combustion, huge amounts of heat releases with smoke, directly and immediately lead ing to the volatilization of N and gaseous loss of N as ammoni a (NH 3 ) and nitrogen oxide (NO X ). The transfer of heat to the soil will further increase the soil temperature and affect physical, chemical and biological soil properties (Neary et al. 1999). For example, it can kill soil microbes, decompose organic matter and change the N forms (Raison 1979). The particulate residues left by fire exist in the forms of ash and charcoal, which are the important pathway for nutrients to return back or redistribute in the ecosystem after the fire and have long time effects on nutrient cycles

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122 (Qian et al. 2009; Zackrisson et al. 1996). It was reported that fire residues themselves contain a small amount of inorganic and organic N that directly serve as a N source (Qian et al. 2009; Hogue and Inglett 2012). Indirectly, large amo unts of P carried by fire residues in particular ash, could stimulate some N processes, such as N 2 fixation, nitrification and N mineralization especially in P limited ecosystems ( Eisele et al. 19 89; White and Reddy 1999, 2000) Charcoal with great adsorpti ve surface on the other hand was found to enhance nitrification and then elevate the nitrate level (Zackrisson et al 1996; Wardle et al. 1998; DeLuca et al. 2002, 2006). Both ash and charcoal can also exert an indirect influence on N availability through m odification of the pH and the cation exchange capacity (Raison 1979; Glaser et al. 2002). Studies on prescribed fire were mostly focused on forests, grasslands and prairies (Ojima et al. 1994; Blair 1997; Grogan et al. 2000; Castaldi and Aragosa 2002; Zac krisson et al 2004), but comparatively few discussed on wetland ecosystem s (Battle and Golladay 2003). However, fire together with water is also important element in the wetland management (Lockwood et al. 2003). In fact, fire has widely been used to cont rol the spread of invasive species in both coastal and inland wetlands for a long time but only been intensively discussed in peatlands (Kirby et al. 1988) Florida Everglades for example is regarded as a fire dependent landscape during the long term and l arge scale restoration (Beckage et al. 2005). Besides reestablishing a more natural hydrology, the establishment of natural fire regime is also a guarantee for the successful Everglades restoration (Lockwood et al. 2003). Numerous fire projects have been conducted or been going on in a diversity of landscapes across Everglades, such as the River of Grass Prescribed Fire Plan for the wet prairie and

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123 sawgrass marsh ecosystems, the Pineland burn plan, the mangrove marsh ecotone fire, and the fire project in W ater Conservation Area 2A (WCA 2A) of the northern Everglades (Spier and Snyder 1998; Qian et al. 2009; Beckage et al. 2005). However, most of the projects focused on restoring native plant species, little is known about the fire effects on nutrient biogeo chemistry, let alone on the N cycle that has been ignored across the whole Everglades ecosystem (Miao and Sklar 1998; Miao and Ca rstenn 2006; Qian et al. 2009). To bridge the gap between the knowledge of fire, N cycle, and wetland restoration, a case study on the N responses to fire at restored and reference wetlands was conducted in the Everglades National Park. Based on the literature aforementioned, the following hypotheses are proposed: 1) fire would increase the N availability; and 2) consequently deni trification rates would increase because of the possible increase of nitrate; 3 ) fire would increase N 2 fixation because of the possible elevation of available P after fire. Materials and Methods Study Site a nd Sampling Methods The study site was located i n the Hole in the Donut (HID) of Everglades National Park Florida, USA (Fig. 6 1). This region is subjected to the invasion of Brazilian pepper ( Schinus terebinthifolius ) because of the excess phosphorus introduced by the fertilizer after a long history o f farming (Smith et al. 2011). Since 1975, studies to evaluate the effectiveness of fire as a management option for controlling Schinus has begun (Doren et al. 1991) Dalrymple et al. (2003) recommended the use of prescribed fire on the restored sites afte r complete soil removal at three to five year intervals to mimic fire frequency in natural vegetation, which would not only reduce coverage by

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124 woody shrubs but would also promote expansion of desirable native plant species such as sawgrass ( Cladium jamaic ense ). Specifically, a restored wetland, cleared in 2000 (Res2000) and a native reference wetland were selected for this study. Soil depth varied between the two sites, with deeper marl soils (Biscayne and Perrine series) in the reference area (10cm) and s hallower soils (less than 5 cm) which have developed after site clearing in the restored area (Smith et al. 2011). The primary vegetation in the reference site is a mixture of grasses ( Muhlenbergia sp., Andropogon sp.) and sedges ( Cladium jamaicense Crantz Shoenus sp.) while the restored sites are dominated by pioneer species such as Ludwigia spp Baccharis spp. and Andropogon spp. (Dalrymple et al. 2003). In each site, two sampling stations corresponding to high and low elevation areas were set (Fig. 6 1). At each station, two 30 m30 m plots (i.e., one burn and one control) were set side by side with a 2 m buffer strip in between. Each plot was separated into three 10 m by 30 m subplots which were further divided into seventy five 2 m2 m sampling squar es. At each sampling three squares in each subplot were randomly selected to collect soil and periphyton samples and composite d to form one replicate for each subplot (3 replicates for each burn and control treatment plot) The Res2000 and reference site s were burned on May 4th, 2010. Sampling was conducted before the fire (April 8 10, 2010), then 2 days (May 6 7, 2010), 1 month (June 10 11, 2010) and 1 year (May 25 26, 2011) after the fire. All samples were sealed in plastic bags and kept on ice until th eir return to the laboratory where the samples were refrigerated at 4C until subsequent analysis. Periphyton samples were kept intact (periphyton mat) and inspected to remove large organic debris (plant litter) and soil. Soil

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125 samples were sieved to remove roots and rock fragments greater than 2 mm diameter. Fresh periphyton was used to determine nitrogenase activity, while s ieved soil samples were used in the determination of all soil microbial and enzyme related parameters. A subsample of sieved soil was oven dried at 105C for 3 days and ground using a mortar and pestle for moisture content and total nutrient determinations. P eriphyton moisture content and nutrients were determined using the sample for nitrogenase activity which was oven dried at 65C for 3 days and ball milled. Biogeochemical Analysis The measurement of the Loss on Ignition (LOI), t otal carbon (TC) total nitrogen (TN) t otal phosphorus (TP) extractable ammonium (NH 4 N) and nitrite/nitrate (NO x N) m icrobial biomass C and N (MBC and MBN) potential mineralizable nitrogen (PMN), the extracellu l ar enzymes related to the N cycle, i.e., N acety l D glucosaminidase (NAG, EC 3.2.1.30) and Leucine aminopeptidase (LAP, EC 3.4.11.1) and the d enitrification enzyme activity, followed the same methods described in the Chapter 5. Total organic C was estimated by loss on ignition (LOI) at 550C for 4 h after conversion to organic C with a coefficient factor of 0.51 (Wright et al. 2008). Nitrogenase activity (N 2 fixation) of periphyton was measured using the acetylene (C 2 H 2 ) reduction (AR) assay described by Inglett et al. (2004). The details were seen in the Chapter 3. Statistical Analysis Data were analyzed with JMP v.8 statistical software (SAS Institute Inc., Cary, NC). For the pre fire data, one way analysis of variant (ANOVA) was used to compared the difference in measured parameters between the r estored and reference sites. For the post fire data, t he factors of site and sampling time were fixed and separate one

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126 way analyses for the different sites and dates were performed. Regressions between different parameters were performed using the linear l east squares method. All results P < 0.05. Data were log transformed when necessary to improve normality. Results Fire Effects o n Soil Nitrogen Availability In general, fire did not significantly change the soil total nutrients (Table 6 1 ). F or the reference site, TP content in the burn plots after 2 days of the fire was significantly higher than that in the control plots ( P <0.05). B oth restored and reference sites showed an increase in extractable NOx N and NH 4 N 2 days after the fire (Fig. 6 2, 6 3). The increase of extractable inorganic N after the fire was greater at the reference site (i.e., approximately twice th at of the control plots) compared to the restored site (i.e., only 20 to 40% higher than the control plots); and only at referen ce site was the difference statistically significant ( P <0. 0 5). At longer time scales (1 month to 1 year after the fire), the extractable NO x N even remained higher in the burn plots than the control plots for the reference site (Fig. 6 2) but the extracta ble NH 4 N at both restored and reference sites dropped to levels below the control plot after 1 month of the fire (Fig. 6 3). Fire Effects o n Microbial Activities In this study, there were not significant changes in the MBC and MBN (Table 6 2). The respon ses of N mineralization measured as PMN to the fire did not show discernable differences (Table 6 2). The responses of the two N related enzymes (i.e., LAP and NAG) to the fire were similar with each other (Fig. 6 5). In the restored site, the LAP and NAG did not change appreciably up to 1month after the fire, but increased by 20% and 40% relative to the control plots respectively, after one year of the fire. In

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127 contrast with the restored site, the enzyme activities in the burn plots at the reference site were significantly greater than those in the control plots immediately (2 days) after the fire; but after one month, the enzyme activities returned to or even fell below the control levels. In addition, the LAP:NAG ratio was seen an increase by 80% after 2 days of the fire at the reference site (Fig. 6 5) but did not change significantly at the restored sites. Fire Effects o n Denitrification Both the restored and reference sites showed the similar response of denitrification ( measured as DEA) to the fire ( Fig 6 4). Immediately after the fire, approximately 20% higher DEA rates were observed in the burn plots compared to the control plots at the restored site F or the reference site, the DEA rates at the burn plot were up to twice of the values in the contro l plots. After one year of the fire, the DEA rates in the burn plots return ed to the levels in control plots. Fire Effects o n Periphyton N 2 Fixation The response of periphyton N 2 fixation to the fire differe d between the restored and reference site s At t he restored site, N 2 fixation rates slightly decreased immediately after the fire but then increased to as high as two fold s of those in the control plots one year after the fire In contrast, at the reference site the rates fell below the control levels after one year of the fire (Fig. 6 6). Discussion Responses o f Nitrogen Availability t o t he Fire Previous researches on the effects of fire on N mineralization in prairie and grasslands has resulted in conflicting results with fire either causing a decre ase (Blair 1997; Ojima et al. 1994; Turner et al 1997), an increase (Aranibar et al. 2003; Boerner

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128 and Brinkman 200 3 ), or having no effect on N mineralization ( Raison 1979). The results depend on different ecosystems and fire behavior; accordingly various mechani sms were proposed to explain th e observations For example, Bell and Binkley (1989) pointed out that N mineralization from soil organic matter is enhanced after the fire due to elevated soil temperature; while Vance and Henderson (1984) concluded t hat N mineralization decreased after burn because of the poor substrate quality. Stock and Lewis (1986) also considered that these conflicting results may be attributed to the use of different methods to determine N mineralization. In this study, soil PMN was estimated using a 10 day anaerobic incubation method. There was no significant response of mineralized N to the fire at both sites. Most studies suggest a consistent pattern that fire can increase the availability of soil NH 4 + and NO 3 (Wan et al. 2001 ). NH 4 + is a direct product of the combustion which is adsorbed by the soil, thus increasing the concentration of NH 4 + In this study, higher extractable NH 4 N was observed two days after the fire in the burn plots at both restored and reference sites. The release of NH 4 + from organic matter would further oxidation of NH 4 + to NO 2 and then NO 3 by bacteria ( Hobbs and Schimel 1984 ; Blank and Zamudio 1998 ). Covington et al. (1991) found NO 3 was not immediately affected but 1 year after burning concentrations had become dramatically higher than the pre fire level. In this study, however, both the restored and reference sites saw a n immediate increase in NO x N two days after the fire which i n the reference site, the increase of NO x N even lasted for one year. The elevation of the N availability may contribute to the ash deposition with high concentration of nutrients. Grogan et al. (2000) investigated the effect of natural ash

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129 deposition on p ost fire ecosystem N cycling by removing the surface ash layer from field plots within 1 week of a wildfire in a Californian bishop pine ( Pinus muricata D. Don) forest. They characteri z ed the influence of ash on plant, soil and microbial N pools during the first growing season after fire and found that ash deposition during wildfires can enhance soil N availability to plants and facilitate ecosystem N retention. A recent explanation for the increase of the available N in forest ecosystems was the influence of charcoal on soil N dynamics and in particular, nitrification (DeLuca et al. 2006). In their study DeLuca and Sala (2006) found that charcoal significantly increased nitrification and the NO 3 concentration in the soils. Despite these studies, there ar e few reports on wetland ecosystem s Qian et al. (2009) simulated the fire in the Water Conservation Area (WCA) 2A in the Everglades through muffle furnace combustion but focused only on the effects of ash on the phosphorus. Hogue and Inglett (2012) c harac terized fire residues ( ashes ) created by both muffle furnace and flam e combustion, observing an increase of available N in char residues after the simulated fire. In addition, b urning can increase extractable P through combustion and heating of organic ma tter, and pH increases caused by ash also can cause the release of ortho P bound by iron and aluminum (DeBano and Klopatek 1988 ). H ogue and Inglett (2012) showed that charcoal formed after burning at low temperature in the HID marl prairie ecosystem, conta ined more bicarbonate extractable P. For the low P reference site, the addition of P through ash or charcoal would stimulate the N mineralization and further increase N availability. For example, White and Reddy (1999, 2000) set a series of experiment to t est the P loading effects on N mineralization in Everglades wetland soil,

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130 and found that P enrichment in the P limited soils had the stimulatory effect on microbial activity and led to an increased availability of inorganic N. Fire Effects o n Denitrifica tion F ew studies were focused on the influence of fire on the denitrification. My results showed that there was no significant difference in potential DEA rates, which was similar with the results of Castaldi and Aragosa (2002) that DEA did not change sign ificantly immediately after fire (i.e., 7 days after the fire) in a Mediterranean shrubland. They considered that other factors, such as water content, would affect denitrification more than the fire. In this study, the slightly increase of DEA after 2 day s of the fire c ould be caused by the increase of NO 3 content, especially for the reference site in which significantly positive correlation between NO X N and DEA was observed (Fig. 6 7). Or perhaps the microclimate modified by the fire facilitates the de nitrification, especially for the restored site in which no significant correlation existed between the NO X and DEA (Fig. 6 7). Effects of Fire on Microbial Activities It is important to study the fire effects on the activity of soil microorganisms and enzyme systems because these are responsible for mineralization processes and availability of nutrient (Saa et al. 1993). Moreover, microbial biomass and enzyme activities are affected by management practices and can be used as sensitive indicators of ecol ogical stability (Ajwa et al. 1999). Many studies have focused on physical and chemical changes in the soil after fire, but few have paid attention to the effects on soil microbial communities ( Boerner et al. 2000 ; Fiore tto et al. 2005). In this study, no significant changes in MBC and MBN were observed (Table 6 2), which agree d with the results from the black pine forest in Turkey reported by Kara and

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131 Bolat (2009). The responses of enzyme to the fire were often studied in a comparatively long time with a year interval. For example, Gutknecht et al. (2010) measured the activity of six enzymes for 3 years following wildfire at the Jasper Ridge Global Change Experiment in a California annual grassland. They found that the w ildfire slightly decreased enzyme activities (by 10 20%) in year 1 post fire with a larger decrease (by 25 50%) in the second year after the fire The response was gone by year 3 suggesting that the microbial community was able to recover by 3 years foll owing wildfire. The two N related enzymes measured in this study LAP and NAG, are inducible enzymes which are synthesized or activated when needed. For example, Chrst (1991) found out that the activities of LAP were induced at limited N availability. NAG involved in the degradation of chitin which is a relevant source of N in soils. In this study, LAP activities were significantly positively related with PMN (Fig. 6 7), indicating that LAP activity can be used as a reliable index of N mineralization in soils. NAG activity in this study decreased a month after the fire in the restored and reference sites T his result agreed with Boerner et al. (2008) who observed reductions in chitinase activity in the fire treatme nts in North American forest ecosystems and they considered it may have been the result of the deposition of more labile organic matter following fire. The response of N acquisition enzymes could depend on fire intensity or severity other than nutrient s (B oerner et al. 2000). For example, w ith low severity fire s (as in grassland type systems like the HID) little heat is transferred downward, minimizing the direct effect of heat on microbial death, and thus resulting in no significant short term change in t he enzyme activities.

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132 Fire Effects o n Periphyton N 2 Fixation As an important function of periphyton, N 2 fixation by periphyton at the restored site responded differently from the reference site (Fig. 6 6). For the restored site, periphyton nitrogenase acti vit y became higher in the burn plots compared to the control plots 1 year after the fire. This result was similar to Zackrisson et al. (2004) that in northern boreal forests in Sweden, the N 2 fixation rates increase d linearly with time since fire. They att ributed the increase to the degree of colonization by cyanobacteria and site factors such as presence of available N. In this study, it is likely that ash deposition from the fire elevated P levels in the restored site (Hogue and Inglett 2012). This pulse of new P may have enhanced the N limitation already present in these sites, thus resulting in stimulate d periphyton N 2 fixation. For the reference site, however, the nitrogenase activities only slightly increase one month after the fire and reduced below t heir control levels after one year of the fire. DeLuca et al. (2002) pointed out that since fire increased the availability of N and reduces the presence of P. schreberi and associated N fixing symbionts, it is likely that the N 2 fixation rates by P. schre beri would decrease following the fire. Another research related to the nitrogenase activities response to the fire was done in Mountain shrub and grassland communities during 2 years following burning (Hobbs and Schimel 1984). They found that nitrogenase activity was depressed by fire 1 year after the burn in the mountain shrub community and they considered that the elevation of the soil inorganic N levels in burn plots may contribute to the depression in nitrogenase activity. The mechanism that excess N i nput after fire may explain the observed suppress ion of N 2 fixation in the reference site.

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133 Conclusion s N cycle involves a diversity of input, transformation, and export. Accordingly, lots of uncertainties exist in different ecosystems, such as the factor s regulating denitrification, the quantity of the greenhouse gas (i.e., N 2 O), and the rates of N 2 fixation. The need to understand N dynamics is further highlighted by the potential impacts of restoration and the action of fire again complicates this under standing. For wetlands, fire and water both shape the restoration and nutrient cycle. Thus it is critical to have a good knowledge of fire effects on nutrient cycle before consideration of using fire as a management tool especially in those ecosystems that limited by certain element. For these reason, a restored wetland with high P and a native wetlands with low P in the HID of Florida Everglades were selected to investigate the impacts of fire on N dynamics. It is the first study of the fire impacts on N c ycle in the Everglades. Different responses of N cycle were ob served in an immediate time (2 days), a short time (approximately 1 month) and a longer time (~ 1 year) following the fire. The increase of N availability agreed with my first hypothesis and ot her studies in the terrestrial ecosystems The increase of inorganic N can influence regrowth and seedling establishment of native plant species, invasi on of exotic plant species, and ultimately, site recovery potential (Dalrymple et al. 2003 ; Rau et al. 2 007). One of the most interesting findings in this research is that fire has more stimulant effects on the N availability (i.e., N mineralization, extractable inorganic N, LAP) at the reference site with low P compared to the restored site with high P, i ndicating fire would exert different influences on N cycle at different P status. Several studies have provided evidence that in P limited ecosystems, P addition stimulated the N mineralization and nitrification (White and Reddy 1999, 2000). Hogue and Ingl ett (2012) simulated the

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134 flame and muffle furnace burning after this field fire project and concluded that the fire produced ash did contain available inorganic P. However, another fire residue, i.e., the charcoal, is also reported to have stimulatory effe cts on nitrification and N availability in the forest ecosystems (DeLuca et al. 2002, 2006). Little is known about whether the charcoal from a herbaceous ecosystem could work the same way as forests. Since fire intensity decides the fraction of charcoal an d ash (Qian et al. 2009; Hogue and Inglett 2012), it will be helpful to know which form contributes more to the increase of N availability before deciding a high or low intensity fire plan. Moreover, periphyton N 2 fixation increased only in the restored site after one year of the fire but decreased in the reference site. It is well known that in a N limited ecosystem, P addition can elevate the N 2 fixation (Howarth et al. 1988a; Smith 1990) as a internal N source whereas excess available N (NH 4 + and NO 3 ) could inhibit N 2 fixation (Howarth et al. 1998b). In this regard, fire could ease the N limitation that tends to occur in the very young restored sites in the HID which further enhance the restoration through complete soil removal (Dalrymple et al. 2003 ). Overall, it is important to know the impacts of fire on N cycle in order to make a balance between N and P that could finally lead to the reestablishment of native wetlands with low nutrients. Practically, our understanding of the mechanisms (ash and cha rcoal) is also of critical value in determining which fire regime (low or high fire intensity) should be performed.

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135 Table 6 1. Changes of basic soil properties and selected microbial activities 2 days, 1month and 1 year after the fire in the burn and control plots in the restored (Res.) and reference (Ref.) sites. denotes the significant difference between the burn and control plots ( P <0.05). Site time after fire TOC TN TP TOC:TN TN:TP g kg 1 g kg 1 mg kg 1 molar ratio molar ratio Burn Cont rol Burn Control Burn Control Burn Control Burn Control Res. 2 days 87 (4) 75 (4) 7.3 (0.3) 6.7 (0.3) 592 (66) 480 (47) 14 (0) 13 (1) 29 (4) 33 (4) 1 mo nth 110 (13) 134 (19) 7.3 (0.2) 8.2 (0.7) 618 (73) 537 (78) 18 (2) 20 (3) 28 (4) 37 (6) 1 year 110 (6) 100 (4) 8.2 (0.2) 8.3 (0.3) 646 (67) 609 (51) 16 (1) 14 (0) 30 (3) 31 (3) Ref. 2 days 69 (6) 66 (7) 6.7 (0.3) 5.7 (0.3) 136 (4)* 102 (4) 12 (1) 14 (2) 136 (4) 102 (4) 1 mo nth 65 (5) 71 (5) 6.5 (0.2) 6.0 (0.2) 144 (12) 136 (7) 12 (1) 14 (1) 144 (12) 136 (7) 1 year 65 (2) 63 (3) 6.6 (0.2) 6.6 (0.2) 140 (4) 133 (7) 12 (1) 11 (0) 140 (4) 133 (7)

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136 Table 6 2. Changes of selected microbial activities 2 days, 1month and 1 year after the fire in the burn and control plots in the restored (Res.) and refer ence (Ref.) sites. denotes the significant difference between the burn and control plots ( P <0.05). Site time after fire MBC MBN MBC:MBN PMN mg kg 1 mg g 1 mass ratio mg N kg 1 dw day 1 Burn Control Burn Control Burn Control Burn Control Res. 2 days 3773(123) 3710(79) 421(18) 424(15) 9(0.4) 9(0.3) 15(2) 16(1) 1 mon th 3510(100) 3936(185) 421(15) 448(36) 8(0.4) 9(0.8) 35(3) 43(3) 1 year 4291(174) 3815(271) 417(25) 347(38) 10(0.4) 11(0.8) 13(2) 16(2) Ref. 2 days 2648(153) 2366(175) 234(18) 181(16) 11(0.3) 13(0.6) 7(1) 7(1) 1 mon th 2758(115) 2735(104) 221(15) 204(10) 13(0.5) 13(0.4) 20(1) 17(1) 1 year 2747(418) 2515(127) 211(46) 193(14) 14(1.6) 13(0.6) 5(0) 5(0)

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137 Figure 6 1. Sampling locations in the restored and reference wetla nd in the Hole in the Donut of Florida Everglades. Miami Homestead Hole in the Donut Florida, USA Florida, USA

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138 Figure 6 2. Changes of soil extractable NO X N in the burn and control plots in the restored (Res2000) and reference wetlands 2 days, 1 month (1mon) and 1year after the fire. The black bar represents th e value before fire. The star denotes significant difference between the burn and control plots (P<0.05). Burn: Control ratio Ext. NO X N (mg kg 1 )

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139 Figure 6 3. Changes of the e xtractable N H 4 N of the soil in the burn and control plots in the restored (Res2 000) and reference wetlands 2 days, 1 month (1mon) and 1year after the fire. The black bar represents the value before fire. The star denotes significant difference between the burn and control plots ( P <0.05). Ext. N H 4 N (mg kg 1 ) Burn:Control ratio

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140 Figure 6 4. Changes of denitrification enzyme activities (DEA) in the burn and control plots at the restored (Res2000) and reference wetlands 2 days, 1 month (1mon) and 1year after the fire. The black bar represents the value before fire. DEA (nmol N 2 O g 1 dw h 1 ) Burn:Control ratio

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141 Figure 6 5. Cha nges of LAP and NAG in the burn and control plots in the restored (Res2000) and reference wetlands 2 days, 1 month (1mon) and 1year after the fire. The black bar represents the value before fire.

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142 Figure 6 6. C hanges of periphyton N 2 fixation under light condition in the bur n and control plots in the restored (Res2000) and reference wetlands 2 days, 1 month (1mon) and 1year after the fire. The black bar represents the value before fire. N 2 fiaxation (nmol C 2 H 4 g 1 dw h 1 ) Burn:Control ratio

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143 (a) (b) Figure 6 7. Corre lation between nitrogen mineralization (measured as potentially mineralizable nitrogen PMN) and Leucine aminopeptidase (LAP) (a) between extractable nitrite/nitrate (Ext. NO X N) and denitrification (measured as potentially denitrification rates DEA) (b) in both burn and control plots in the reference and restored sites. Sample numbers N=18.

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144 CHAPTER 7 SUMMARY AND SYNTHESI S In wetlands, the nitrogen (N) cycle is dynamically controlled by hydrology, fire and phosphorus (P). When a globally increasing likelih ood of severe drought has been predicted during this century, it is emergent to investigate the seasonal changes or hydroperiod effects on the N cycle which would further affect the primary production, and the global warming. However, N cycling has receive d little attention in most of the Everglades system where N could be a limiting element and a serious pollutant. This dissertation investigated the spatial and temporal patterns of N cycling, and their responses to a prescribed fire in the context of ecosy stem restoration using the short hydroperiod, marl prairies of the Hole in the Donut (HID) region of Everglades National Park. Summary The nutrient status w as different between the restored and reference wetlands using various indicators (Chapter 2). F oli ar TN:TP ratio varied with species and seasons, and should be carefully used as an indicator of nutrient limitation. A ctivities of N related hydrolytic enzyme s (i.e., N acetyl D glucosaminidase, NAG and Leucine aminopeptidase, LAP) were significantly hig her at the restored sites but P related enzymes (Alkali phosphatase AP and phosphodiesterase BisP ) were significantly higher at the reference site, suggesting different nutrient availability between the restored and reference wetlands. Periphyton at the restored sites fixed more N 2 compared to that at the reference site, especially in the wet season, consistently indicating 15 N values could be indicators of restoration and N availability 15 N patterns during

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145 restoration still requires more explicit testing. These results indicated that the microbial activities could be more sensitive to the nutrient status, and should be paid more attention during the restoration process. Based on the importance of the N 2 fixation of the periphyton, more detailed exploration on periphyton was done (chapter 3 and 4). P eriphyton N 2 fixation in the Hole in the Donut (HID) region of the southern Everglades were quantified. Significantly higher N 2 fixation rates (measured by acetylene reduction) were found in periphyton of the areas cleared in 2000 and 2003 (3 10 nmol g 1 DW h 1 ) compared to the reference wetland site (less than 1 nmol g 1 DW h 1 ). Overall rates were stimulated by light (~2 times the measured dark rates) and a real estimates of fixed N were low compared to other areas of Everglades, ranging from 0.1 0.2 g N m 2 yr 1 in the restored sites to 0.05 g N m 2 yr 1 15 N) ranged from 1.0 15 N could serve as indicators of nutr ient status and restoration success in these systems. However, limitation in quantifying N 2 fixation in the window of only two months was obvious that, some interesting signals of periphyton N 2 fixation caused by the fluctuating hydrology could be easily missed especially in the short hydroperiod wetland ecosystem. Thus, in Chapter 4 much finer sampling strategy was adopted to better quantify the seasonal pattern of periphyton N 2 fixation. Similar seasonal patterns of N 2 fixation were observed among the natural and restored sites, with higher rates in the wet season. At the native reference area, the seasonal pattern was a gradual change throughout the year with a peak observed in September, while most N 2 fixation occurred

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146 during a brief 2 3 month period at the onset of flooding with a peak in Ju ly for the two restored sites. An i nteresting finding is that the N 2 fixation was controlled by both physical (moisture) and chemical (N and P) parameters. Dry season rates were a function of both moisture content s and P content s while during flooded months excess N levels appeared to suppress nitrogenase activity. Moreo ver, seasonal patterns of 15 N did not strictly correlate with N 2 15 N could be a good indic a tor of N 2 fi xation in a spatial dimension but would not be as robust in determining seasonal N 2 15 N values of periphyton integrate a variety of N sources and processes (e.g., atmospheric deposition, denitrification). Periphyton as one compo nent of the ecosystem has its own limitation in characterizing N dynamics; therefore the chapter focusing on soil properties followed Chapter 5 presented the temporal patterns of soil N based on seven sampling across the wet and dry seasons. NH 4 + and NO X dominated the wet and dry seasons, respectively, and accumulated NO X levels were quickly consumed by denitrification at the onset of the wet season. The enzymes LAP and NAG showed different seasonal patterns, suggesting these enzymes were regulated by d ifferent factors (e.g., moisture content and substrate availability). Moreover, the decreasing MBC:MBN ratio from spring to summer would tell us the changes of microbial composition with the time where in the summer, more root exud at es would favor the grow th of bacteria. As in many other systems, in the HID restoration, fire is both a natural process and is considered a technique to further enhance the restoration after the complete soil removal. Therefore it wa s important to investigate the effects of fir e on the N cycle at the restored and reference sites. Different responses were observed immediately ( 2 days

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147 after the fire), after a short term (1 month after the fire) and a long time (~1 year later ) following the fire. A vailable inorganic N (NH 4 N and N O x N) was elevated immediately after the fire in all the three sites. The N mineralization related parameters in the P limited reference site showed a greater respon s e to the fir e compared to the restored site Synthesis Various indicators were used in th is work to demonstrate the nutrient status of restored and native reference wetlands. Though, foliar TN:TP ratio has commonly suggested as a useful indicator of nutrient limitation, this study showed it temporally and spatially varied, and should be used w ith caution. In contrast, microbial activities, especially the enzyme activities, appear to be more sensitive to changes of nutrients and thus, would serve as more robust indicators of nutrient impacts N 2 fixation, as an important function of periphyt on, is a major N source for the restored sites. Based on the rough N budget, it would take approximately 40 years to accumulate the equivalent amount of N as the reference site. The temporal patterns of periphyton N 2 fixation confirmed the importance of th e hydrology It is also the first time that periphyton N 2 fixation has been investigated in detail in the short hyrdroperiod, marl prairie wetlands ecosystems in southern Everglades. Further study is undoubtedly needed to better clarify the relationship of periphyton species composition and its function of N 2 fixation, which would explain the different seasonal pattern of N 2 fixation between the restored and reference sites. T he t emporal pattern of soil N processes reflects the response to a variety of fac tors like temperature, precipitation, and plant growth. This study showed opposite seasonal trends for the extractable NH 4 + and NO X + which would correlate with the two

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148 interconnected processes of denitrification and nitrification. It should be highlighted that NO 3 was accumulated in the dry season and consumed by denitrification at the onset of the wet season, indicating the potential pulse of N 2 O once re wetting after the dry period and its significance in the greenhouse gas emission. The unexpected peak of N 2 O in the extreme dry season (i.e., May 2012) should also be paid attention together with the microbial composition inferred from high MBC:MBN, which would potentially suggest the fungi dominated denitrification pathway. When integrating the temporal patterns of periphyton N 2 fixation and soil N dynamics, we could get more in depth of the N budget. At the restored sites, both periphyton N 2 fixation and denitrification reached their peaks in July, if soil microbes could also fix N 2 and showed same peak in July, then addition and loss of N could potentially cancel one another, it is uncertain what the net effect of the N budget. Moreover, unlike the long hydroperiod wetlands, the dry and wet period alternate more frequently in short hydroperiod ecosystems Accordingly, the seasonal patterns would display differently as shown in this study. It is interesting to know how important the peaks of N 2 fixation and N 2 O emission that narrowed only within 2 3 month in the annual N budget. The results of the f ire effects on the N cycle are not conclusive, and many questions remain to be answered with future research. For example, little is known about the mechanisms of the increase of available N after fire in wetland ecosystems. It could be the stimulatory eff ects of either P addition through ash residue or the adsorptive ability of charcoal on the N cycle. The understanding of the mechanism will facilitate prescribed fire plan since the fire intensity decides the fraction of ash and

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149 charcoal. Moreover, the dif ferent effects of fire on the periphyton N 2 fixation between the restored site with high P and the reference site with low P further provide a clue for the management that different frequency and intensity fire would be performed differently in the P limit ed reference site and N limited restored sites (Hogue and Inglett 2012). Chapter 4 and 5 focused on the temporal patterns of N cycle highlighting the importance of hydrology in regulating the N cycle especially in the short hydroperiod HID; whereas Chapte r 6 emphasized that fire as another management tool also shape the Everglades restoration. After we know the N processes remarkably vary with season, the interaction between fire and hydrology should further be considered. For example, the responses of N c ycle to a fire that occurs in a dry season would differ from the responses to a fire that occurs in a wet season. Overall, this study tried to investigate the spatial and temporal patterns of N cycle in the midst of the large scale and long term restorati on. A unique ecosystem of HID was selected which simultaneously integrates all the elements of restoration, nutrient limitation, short hydroperiod, and fire together. These important features further make the study of N cycle more complex but never less in teresting. It is the first time to document the N dynamics in detail in the southern Everglades. The periphyton N 2 fixation rates were measured, expanding our knowledge of periphyton that was previously discussed mostly in the northern Everglades and in th e biomass and species composition. The seasonal patterns of periphyton N 2 fixation and soil N dynamics require a keen awareness of the different impacts of short hydroperiod on N cycle. The standing out peaks of N 2 fixation and denitrification rates in a 2 3 month window will give us more thoughts on the annual N budget. Fire together with hydrology is the other

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150 important management tools for wetland restoration. The disturbance of fire makes the N cycle more complex but in the HID it highlighted the spread impacts of P on the N cycle, such as N availability and N 2 fixation. To restore the P enrichment wetlands to the original oligotrophic status, it is as important to understand the N cycle as well as the P.

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170 Wardle DA (1998) Controls of t emporal variability of the soil microbial biomass: A global scale synthesis. Soil Biology and Biochemistry 30(13): 1627 1637 Wardle DA, Zackrisson O, Nilsson MC (1998) The charcoal effect in boreal forest: mechanisms and ecological consequences. Oecologia 115: 419 426 Weedon JT, Aerts R, Kowalchuk GA, van Bodegom PM (2011) Enzymology under global change: organic nitrogen turnover in alpine and sub Arctic soils. Biochemical Society Transactions 39: 309 314 White JR, Reddy KR (1999) Influence of nitrate and phosphorus loading on denitrifying enzyme activity in Everglades wetland soils. Soil Science Society of America Journal 63(6): 1945 1954 White JR, Reddy KR (2000) Influence of phosphorus loading on organic nitrogen mineralization of everglades soils. Soil Science Society of America Journal 64(4): 1525 1534 White JR, Reddy KR (2003) Nitrification and denitrification rates of everglades wetland soils along a phosphorus impacted gradient. Journal of Environmental Quality 32(6): 2436 2443 White JR, Reddy KR, Mo ustafa MZ (2004) Influence of hydrologic regime and vegetation on phosphorus retention in Everglades stormwater treatment area wetlands. Hydrological Processes 18(2): 343 355 Wozniak JR, Childers DL, Anderson WT, Rudnick DT, Madden CJ (2008) An in situ mes ocosm method for quantifying nitrogen cycling rates in oligotrophic wetlands using (15)N tracer techniques. Wetlands 28(2): 502 512 Wright AL, Wang Y, Reddy KR (2008) Loss on Ignition Method to Assess Soil Organic Carbon in Calcareous Everglades Wetlands. Communications in Soil Science and Plant Analysis 39(19 20): 3074 3083 Yoneyama T, Matsumaru T, Usui K, Engelaar W (2001) Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sati va L.) plants. Plant Cell and Environment 24(1): 133 139 Yuste JC, Penuelas J, Estiarte M, Garcia Mas J, Mattana S, Ogaya R, Pujol M, Sardans J (2011) Drought resistant fungi control soil organic matter decomposition and its response to temperature. Global Change Biology 17(3): 1475 1486 Zackrisson O, DeLuca TH, Nilsson MC, Sellstedt A, Berglund LM (2004) Nitrogen fixation increases with successional age in boreal forests. Ecology 85(12): 3327 3334

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171 Zackrisson O, Nilsson MC, Wardle DA (1996) Key ecological f unction of charcoal from wildfire in the Boreal forest. Oikos 77: 10 19 Zedler JB (2000) Progress in wetland restoration ecology. Trends in Ecology and Evolution 15(10): 402 407

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172 BIOGRAPHICAL SKETCH Xiaolin Liao was born in a small city of Jingmen, Hubei Province in China. She had her happiest childhood there enjoying the peaceful life and got the basic knowledge from schools. After successfully passing the competitive entrance exam to college in 2002, she chose the University of Science and Technology i n Beijing China, far away from her hometown, to do her undergraduate study. It was her first time to leave home and stay with peers from different places of the country. However, she likes to make friends with diverse background and it came to her that she built a very solid and helpful friendship during the four undergraduate years. At that time, her major was Environmental Engineering, learning a wide range of knowledge on waste water treatment, air pollution control, and waste disposal, and visiting real factories that put those knowledge into reality. She was impressed by a constructed wetland for waste water treatment once visiting in a petrochemical company, and since then her journey for the wetlands began. Based on the well rounded performance durin g the undergraduate, Xiaolin was recommended for admission without exam to the graduate school in the Beijing Normal University in 2006. She chose to study on the wetland ecology in the top ten Environmental School in China. During the three years of gradu ate studies, she got chances to know more about wetlands, e.g., the coastal wetlands in the Yellow River Delta in China, the urban wetlands in Jinnan city of Shandong Province, and the constructed wetland in Beijing. She spent the humid summer and chilly w inter in the Yellow River Delta with other colleagues from different institutes studying the biodiversity conservation of endangered birds. Her passion with wetlands increased which further pushed her to go further to know the wetlands around the world.

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173 W ith the dream of wetlands, Xiaolin was luckily accepted by the Soil and Water Science Department in the University of Florida where she later realized is a center of wetlands studies and for wetlands scientists. She followed her advisor Dr. Patrick Inglett who needs to jump the language barrier, it is really a tough experience to get through. However, Xiaolin finally survived and received his Ph.D. from the University of Florida i n the s ummer of 201 2.