Methane emissions from freshwater cypress (Taxodium distichum) swamp soils with different

hydroperiods in Southwest Florida

A Thesis Presented to

The Faculty of the College of Arts and Sciences

Florida Gulf Coast University

In partial Fulfullment of the Requirement for the Degree of

Master of Science

By

Andrea Pereyra

2015

APPROVAL SHEET

This thesis is submitted in partial fulfillment of the

requirements for the degree of

Master of Science

Andrea Pereyra

Approved: December 9, 2015

William J. Mitsch, Ph.D., Advisor

Brian Bovard, Ph.D.

Shawn E. Clem, Ph.D.

The final copy of this thesis [dissertation] has been examined by the signatories, and we find that both the content and the form meet acceptable presentation standards of scholarly work in the above mentioned discipline.. iii

Abstract

Wetlands are natural sources of methane (CH4) emissions, with the majority of those releases in tropical and subtropical regions. Land-use modifications can change a wetland’s hydroperiod and hydrologic connectivity, among two important factors controlling methanogenesis. I measured

CH4 fluxes from soils in two southwestern Florida cypress (Taxodium) swamps. Three research sites were in a highly protected strand of cypress in Corkscrew Swamp Sanctuary and three were on the campus of Florida Gulf Coast University. Campus sites had experienced modifications in land use well before the campus was constructed in the 1990s. Net methane emissions were measured twice daily 10 times from January through December 2014. Mean ± standard error net

-2 -1 methane fluxes were 25.9 ± 15.6, 22 ± 21.8, and 49.5 ± 24.7 (mg CH4-C m d ) for the reference bald cypress slough, pond cypress slough and cypress dome, respectively, and 4.0 ±

-2 -1 3.8, -1.4 ± 0.8, and 0.5 ± 0.5 (mg CH4-C m d ) for the disturbed pond cypress slough, cypress dome 1 and cypress dome 2, respectively. The only median flux different than 0 was at the

-2 -1 reference cypress dome (12.9 mg CH4-C m d ). Fluxes from the reference sites were significantly higher than fluxes from the disturbed sites. Deeper water and higher soil temperatures at the time of sampling, by themselves, did not necessarily explain higher CH4 fluxes. More continuous surface flooding at the reference sites compared to seasonal flooding at the disturbed sites appear to be the main cause for higher methane emissions at the reference sites.

iv

Dedication

I dedicate my research work to my parents. All that I am is because of you. You have taught me to believe in myself and to never lose hope or surrender in the face of adversity. You have raised me to be kind, fair and optimistic. Thanks to you I am a happy decent human being. Thanks for giving me everything and more.

If you fail, never give up because F.A.I.L. means "first Attempt In Learning", End is not the end, in fact E.N.D. means "Effort Never Dies" and if you get No as an answer, remember N.O. means "Next Opportunity". So Let's be positive. "

Dr. A.P.J. Abdul Kalam

v

Acknowledgments

I would like to start by thanking my advisor and mentor, Dr. William J. Mitsch, for his constant guidance and support during these years. You have taught me to challenge myself and that we all have a great capacity to overcome our own limits if we believe in ourselves. I am also very grateful for my committee members, Dr. Brian Bovard and Dr. Shawn Clem. The interesting discussions we have had throughout these years have kept me motivated and your inputs have considerably improved this paper. Thanks to all of you, for helping me to think outside the box, challenging me to see the big picture and making my graduate research experience a memorable one.

This research would not have been possible without the help of my peers and close friends: Li

Zhang, Jorge Villa, Alvaro Cabezas, Xiaoyu Li, Connor MacDonnell, Daniel Marchio, Darryl

Marois and Frank Bydalek. Thank you for your assistance during the long days in the field and lab, but, foremost for your friendship and love. You welcomed me to this country with open arms, making me feel home. I will not forget our camping trips, beach days and cheese and wine nights after the MOM lectures. Special thanks to Jorge Villa and Alvaro Cabezas for your tutoring while designing my experiment and analyzing my results. Without your help I would probably still be working on my thesis. Also, thanks to Daniel Marchio and Darryl Marois for your help during the writing and for proof-editing some of my chapters.

My family back in Lima, Peru has played a substantial role in these past 2.5 years. My success as a graduate student is because of them. Mom and Dad, I am deeply thankful for always believing in me, for never cutting my wings to go after my dreams and for giving me a big smile even when you missed me like crazy. My siblings, Claudia and Gabriel, gave me strength when I was home sick and helped me keep motivated. Thanks for always being there for me, in an

vi unconditional manner. Thank you family for coming to visit me and for sending me Peruvian delicacies to ease my cravings.

The National Audubon Society’s Corkscrew Swamp Sanctuary and its staff, especially Jason

Lauritsen granted the permission to sample and assisted me with logistics at the Sanctuary.

Thanks to all the friendly volunteers that always made my sampling days more interesting by letting me know about the location of wild animals. It has been a privilege to work in such a beautiful swamp.

The Everglades Wetland Research Park (EWRP) at the Florida Gulf Coast University (FGCU) embraced me as a member of an efficient working team. Being part of the EWRP has given me access to field and lab equipment, but more importantly, to interact with renowned scientists that visited us periodically and during the MOM lectures.

I am grateful for my professors at FGCU, especially Dr. Edwin Everham. Thanks for always being available to clarify my doubts and for enjoying a good scientific discussion. To my friends at FGCU and Lima, thanks for the laughs, the long talks and all the good moments shared.

The EWRP provided support through the Juliet C. Sproul Endowed Chair for Southwest Florida

Habitat Restoration and Management. This research was also partially supported by the National

Science Foundation, Award CBET 1033451. Thanks to all the people involved in managing these funds.

vii

Vita

2006…………………………………………B. S. Biology, Universidad Nacional Agraria La

Molina, Lima, Peru

2006 - 2008…………………………………Field Researcher in Herpetology, Daimi Peru, Lima,

Peru

2007…………………………………………Certificate in Quality Management and

Environmental Auditing, Universidad Nacional

Agraria La Molina, Lima, Peru

2008 - 2013…………………………………Scientist II, Knight Piésold Consulting, Lima, Peru

2013 - 2015…………………………………Research Assistant, Everglades Wetland Research

Park, Florida Gulf Coast University, Naples

December, 2015…………………………… M. S. Environmental Science, Florida Gulf Coast

University

Publications

Pereyra, A., and W. J. Mitsch. In review. Methane emissions from freshwater cypress

(Taxodium distichum) swamp soils with different hydroperiods in Southwest Florida.

Limnology and Oceanography.

viii

Chavez, G., Medina-Muller, M., Pereyra, A. 2008. Amphibia, Anura, Hylidae, Osteocephalus

leoniae: Distribution extension. Checklist Journal 4(4): 403.

Published abstracts

Pereyra, A., and W. J. Mitsch. 2015. Methane emissions from freshwater swamp soils with

different hydroperiods. Abstract at Society of Wetland Scientists annual conference.

Pereyra, A., and W. J. Mitsch. 2015. Methane emissions from freshwater swamp soils with

different hydroperiods. Abstract at Florida Lake Management Society annual symposium.

Field of study

Major field: Environmental Science

Area of specialization: Wetland ecology

ix

Table of contents

Abstract iii

Dedication iv

Acknowledgments v

Vita vii

1. Introduction 1

2. Materials and Methods 5

2.1 Study sites 5

2.2 Methane sampling 7

2.3 Laboratory analysis 9

2.4 Data analysis 10

3. Results 12

3.1 Precipitation, water levels and soil temperature 12

3.2 Methane fluxes 13

3.3 Relationship of CH4 fluxes to physiochemistry 14

4. Discussion 16

4.1 Methane fluxes 16

4.2 Methane oxidation 18

4.3 Discontinuities in methane emissions and ebullition 18

4.4 Methane fluxes and hydrology 19

4.5. Comparison within swamps 20

4. 6 Methane fluxes and soil temperature 21

4. 7 Study significance 21

5. Conclusions 23

x

References 25

Figures and Tables 33

Appendix A 44

Appendix B 46

1

Chapter 1

Introduction

Methane is a greenhouse gas (GHG) that, once in the atmosphere, affects the radiation balance of the Earth. Each molecule has a global warming potential (GWP) of 28 times that of carbon dioxide molecules after 100 years (Pachauri et al. 2015). Methane has been estimated to have more than doubled from 720 ppb in preindustrial times to current levels over 1800 ppb; most of the sources for this increase (68 - 70%) are anthropogenic (Mitsch and Gosselink 2015).

Several studies have demonstrated the atmospheric concentration of CH4 increased by about 13% between 1978 and 1999 (Khalil and Rasmussen 1990; Dlugokencky et al. 2001; Cunnold et al.

2002). Currently on the planet, wetlands are described as the most important natural source of

CH4 emissions releasing 170 Tg CH4 annually to the atmosphere (Whalen 2005; Bloom et al.

2010; Bridgham et al. 2013). Recent studies have estimated that half or more of wetland CH4 emissions originate from tropical wetlands (Bloom et al. 2010).

In wetlands, CH4 is formed under anaerobic conditions by microbial decomposition of organic matter (van Amstel and Swart 1994), and it is consumed by CH4 oxidizing microbes in aerobic environments (Whalen 2005). The net budget between production and consumption determines the rate of methane release into the atmosphere. Methane can be transported from a wetland’s soil to the atmosphere via molecular diffusion, ebullition (i.e., gas bubbling up from the soils through the water) or through the vascular system of emergent plants (Villa and Mitsch

2014; Mitsch and Gosselink 2015).

2

From the several factors that determine wetland processes (e.g., hydrology, soil temperature, soil properties, microbial and vegetation community, etc.), hydrology is one of the most important (Moore and Knowles 1989; Segers 1998; Whalen 2005; Kayranli et al. 2010).

Seasonal variability of water table can explain much of the variability of annual patterns of CH4 emissions (Whalen 2005). More specifically, flooding duration (i.e., the amount of time that water stands at or above ground surface) and flooding frequency (i.e., the average number of times that a wetland has standing water), regulate the exchange of electrons during the redox reactions that can lead to the production of methane (Brandt and Ewel 1989; Mitsch and

Gosselink 2015). Another important factor controlling gas emissions is soil temperature, although, it may be more important at higher latitudes, where seasonal variation is well marked

(Bartlett and Harris 1993; Bloom et al. 2010).

Tropical and subtropical cypress swamps often have seasonal hydroperiods; that is, they have a period or periods when they are inundated and periods when there is little or no standing water (Mitsch et al. 2009). Water flow through south Florida cypress wetlands during the dry season is not apparent, while over the wet season water flow occurs but is slow (Bondavalli et al.

2000). Happell and Chanton (1993) argued that water level controls methane gas emissions from

Florida swamp forests.

Wetlands play an important role in climate regulation because they constantly cycle carbon through its abiotic and biotic components (i.e. water, soils, microbes, plants and animals).

Therefore, wetlands have the potential to be sources or sinks of atmospheric carbon in the form of CO2 or CH4. A wetland’s anoxic wet-conditions and potential for peat formation favor the sequestration and storage of atmospheric carbon (Bondavalli et al. 2000). Carbon fluxes models

3 suggest that wetlands are acting as net carbon sinks, especially when analyzed in the long-term

(Whitting and Chanton, 2001; Mitsch et al. 2013).

Land-cover modifications due to activities such as agriculture, urban development and roads, canals and ditches construction have a great impact on wetland function (Zedler 2003;

Torbick et al. 2006; Kettlewell et al. 2008; Sullivan and Fisher 2011). These activities usually occur in association with filling, dredging and draining wetlands, all of which can alter hydroperiods, reduce water levels and change the course of water-flow. Some of the reported consequences of these alterations in Florida freshwater wetlands are reduction in cypress growth, expansion of invasive species and excessive wildfires (Tabb et al. 1976; Duever et al. 1978).

These changes also have the potential to alter methane production and consumption.

According to Bondavalli et al. (2000), relying on the carbon sink potential of wetlands is dependent upon the patterns of land use. When land-use modifications are proposed, it is necessary to consider carbon emission and uptake from wetlands as part of the decision-making process. Doing so would help making responsible decisions towards climate change (Torbick et al. 2006; Erwin 2009).

There are relatively few studies of methane emissions and carbon sequestration in subtropical and tropical wetlands despite their overall dominance of the global carbon fluxes from wetlands (Mitsch et al. 2010, 2013; Bloom et al. 2010). The goal of this research was to determine and compare CH4 fluxes from two subtropical cypress swamps in southwest Florida, each represented by three research sites. Each regional swamp has different hydroperiods and noticeably different land-use conditions (i.e., one is a reference area and the other is hydrologically disturbed). I explored the influence of water levels and soil temperatures on CH4

4 fluxes by analyzing the fluxes from each site relative to the environmental variables. Two research hypotheses were addressed:

1. Cypress swamps with longer flooded phases and higher water levels will

result in higher CH4 emissions than those having shorter and lower flooded

phases and water levels, respectively.

2. Methane emissions from cypress swamps with a steady-flow hydroperiod

will be higher than those from swamps with a pulsed flow.

Estimates of CH4 fluxes, especially from disturbed wetlands, can provide a better understanding on CH4 dynamics during the design of any land-cover change.

5

Chapter 2

Materials and Methods

2.1 Study sites

This study was conducted in Collier and Lee County, Florida, southwest of Lake

Okeechobee and east of the Gulf of Mexico (Fig. 1). This region’s landscape is a mosaic of urban, agricultural and natural patches, but prior to development was mosaic of uplands and wetlands. Within this region, two major wetland areas comprising a total of six research sites were chosen to represent reference and disturbed cypress (Taxodium distichum) swamps. The disturbance category was determined by land-use/land-cover historical changes while the selection of the individual sites was based on the hydrogeomorphology of cypress swamps as described by Odum (1982) and summarized by Mitsch and Gosselink (2015).

National Audubon Society’s Corkscrew Swamp Sanctuary (~5,260 ha) contains the largest and most-preserved stand of mature Taxodium distichum [L.] Rich. (bald cypress) in

Florida (Duever et al. 1984; Gunn 1997; Villa and Mitsch 2014). This old growth cypress forest is considered an ideal location for researching ecological relationships in undisturbed forested wetlands (Duever et al. 1984) and thus, three reference sites were selected to study in Corkscrew

Swamp Sanctuary. These sites were designated as 1) reference bald cypress slough (26° 22’ 45’’

N, 81° 36’ 31’’ W); 2) reference pond cypress (T. distichum var. imbricarium [Nutt.] Croom) slough (26° 22’ 53’’ N, 81° 36’ 18’’ W), and 3) reference cypress dome (26° 23’ 27’’ N, 81° 35’

35’’ W). The Corkscrew cypress slough is a slow-flowing wetland with low erosive power that allows for a build-up of peat. The reference bald cypress slough is located at the center of the strand, in which the water is deepest and peat soils are thickest (> 25 cm). The understory

6 vegetation is sparse with emergent macrophytes like Thalia geniculata and Peltandra virginica, the subcanopy is represented by Annona glabra and Fraxinus caroliniana and the canopy is composed of mature T. distichum (Villa and Mitsch 2014). The reference pond cypress slough is at the edge of the strand, where water depths are lower and peat deposits are shallow. Peat depth in this site was approximately 8 cm, with sandy soils below the peat. Sagittaria graminea and

Ludwigia spp. dominate the understory, while a dense stand of pond cypress T. distichum var. imbricarium forms a closed canopy (Villa and Mitsch 2014). The reference cypress dome has considerable peat deposits (up to 20 cm depth) overlying sandy soils, Pontederia cordata,

Peltandra virginica and Crinum americanum in the understory, Annona glabra and Cephalantus occidentalis in the subcanopy and T. distichum forming the canopy.

Florida Gulf Coast University’s (FGCU) campus (307 ha) is a combination of academic buildings and campus grounds amid conservation areas including several remnant and restored patches of cypress swamps and freshwater marshes. This landscape and its surroundings have undergone several land changes through time, due to agriculture, urbanization, and campus expansion. The initial construction of the FGCU campus in 1995 resulted in the loss of around

38 ha of vegetation cover (current loss is > 61 ha) despite the university’s initial focus on environmental sustainability and the campus design initially including many restored wetlands.

Three sites were selected at FGCU and were designated as: 1) disturbed pond cypress slough

(26° 27’ 49’’ N, 81° 45’ 57’’ W) (a small, fragmented slough with peat deposits of 16 cm depth followed by sandy soils, sparse understory dominated by Blechnum serrulatum, Nephrolepis exaltata and Crinum americanum, a subcanopy of Myrica cerifera and Ilex cassine, and a canopy of T. distichum; 2) disturbed cypress dome 1 (26° 27’ 44’’ N, 81° 46’ 24’’ W), with no apparent peat deposits but a dark-colored surface with little decayed plant material, an understory

7 represented by Phleum sp., Tripsacum floridanum, a subcanopy of Persea palustris, Myrica cerifera and Ilex sp., and an open canopy of T. distichum and Pinus elliottii, and 3) disturbed cypress dome 2 (26° 27’ 55’’ N, 81° 46’ 27’’ W), composed mostly of bare soil, with no apparent peat deposits and a T. distichum canopy. The disturbed cypress domes are partially surrounded by retention ponds that fill with rainfall and surface runoff during the wet season.

2.2 Methane sampling

The most common way to sample methane fluxes from soils is by using non-steady-state chambers (Livingston and Hutchinson 1995). However, chamber design and operation can alter the natural development of CH4 concentration within the chamber resulting in under or overestimation of CH4 fluxes (Hutchinson and Livingston 2001; Christiansen et al. 2011).

Potential sources of error are pressure perturbations within the chamber, gas leakage and lack of air mixing in the headspace chamber (Davidson et al. 2002; Christiansen et al. 2011; Pihlatie et al. 2013).

The use of a vent tube is recommended to reduce pressure perturbations because it balances chamber air with ambient air pressure (Livingston and Hutchinson 1995; Xu et al.

2006). Bekku et al. (1995) found that CO2 sampling from a non-vented chamber resulted in flux overestimation. The study stated that sampling depressurizes the chamber headspace, thus, leading to a mass flow from the soil to the chamber. As an additional way to manage pressure perturbations, Pihlatie et al. (2013) found helpful waiting 3 to 5 minutes to sample, after closing the chamber. This time - break allowed recovery of the headspace air from small perturbations originated by chamber deployment (Davidson et al. 2002; Christiansen et al. 2011).

8

Leakage through the seal between the chamber parts is unavoidable but it can be minimized by implementing a seal made with a closed-cell foam and clamps to hold tight the base and deployable top of the chamber (Hutchinson and Livingston 2001).

Mixing the headspace air homogenizes gas concentration inside the chamber.

Christiansen et al. (2011) showed that CH4 concentration development from non-mixed chambers was uneven over time and overall fluxes were underestimated. Mixing can be achieved by using a fan located inside the chamber or by flushing a syringe with headspace air before collecting the sample (Livingston and Hutchinson 1995; Pihlatie et al. 2013). Nonetheless, there is still debate on what option is better and on how they should be implemented (Pihlatie et al.

2013).

Three non-steady-state chambers, following the design used by Sha et al. (2011), were used for gas sampling in each of the six sampling sites. Chambers were made with 53 L plastic storage containers and consisted of a bottomless base and a deployable top. The base was permanently installed by burying it 10 cm into the soil to serve as an air-soil interface. The top was a complete inverted 53 L container fitted with a 1 °C increment thermometer, a butyl rubber port for gas sampling and a tygon vent tube (1.6 mm inside diameter, 60 cm length) for pressure relief. During sampling, the deployable tops were sealed around the permanent bases using a vinyl foam weather seal bond with duct tape and petroleum jelly, and they were held together with large binder clips. According to Hutchinson and Livingston (2001) this type of seal is an efficient seal that can reduce gas leakage. When water level exceeded 10 cm in height, slightly modified tops with foam around the bottoms were used for buoyancy. These floating chambers were deployed on the water surface, over the bases and held still with four small diameter-poles placed around the chambers to avoid drifting.

9

Gas samples were taken at two times of the day (morning, i.e., 8:00-12:00 and afternoon, i.e., 14:00-18:00) with initial samples taken within 10 min of site arrival to minimize soil disturbance effects. Initial samples were ambient air samples, after which chambers were closed to take five remaining samples over 25 min. First sample during the enclosure period was taken at minute 5 to reduce the initial disturbance due to chamber deployment (Pihlatie et al. 2013;

Davidson 2002). The collection of six samples represented one chamber run. Twenty milliliters of gas were collected through the butyl port using a 30 mL syringe, fitted with a stopcock, and injected into pre-evacuated 10 mL glass vials sealed with butyl septa. The use of small syringes

(≈20 mL) and pre-evacuated glass vials is recommended to minimize pressure disturbance during gas sampling (Pihlatie et al. 2013). The vials were stored and transported to the lab for analysis.

Ten samplings (i.e., 10 visits to each site) were performed from January to December

2014. Disturbed cypress dome 2 was added to the sampling starting June 2014. Chamber air temperature and time of day were registered every 5 min, when a sample was extracted. On each research site except the newly added disturbed cypress dome 2, hourly water levels and soil temperature at 2 cm depth were recorded using HOBO U20 water level and water temp pro v2 data loggers, respectively. Daily precipitation data from the Corkscrew Swamp Sanctuary rain gauge (26° 2’ N, 81° 3’ W) and the Southwest Florida international airport (26° 5’ N, 81° 7’ W) weather station were used to define rainy season.

2.3 Laboratory analysis

Samples were analyzed by flame ionization detection on a gas chromatograph (Shimadzu

GC-2014 Kyoto, Japan) equipped with a 64 - position AOC-5000 headspace autosampler. A 1.8 m Porapack Q column detector with Helium as the carrier gas was used for sample preparation.

Six-point calibration curves were performed with Matheson CH4 standards and one check

10

standard (blanks) with known concentration of CH4 was run every 32 samples for quality control. The GC detection limit was 0.1 mg/L. The chromatograph results of CH4

-1 volume/volume concentrations (Cv, µL CH4 L ) were transformed into mass/volume

-3 concentration (Cm, mg CH4-C m ) using the ideal gas law as follows:

(Eq. 1) �� = !"×!×! !×!

-1 where M is the molar mass of carbon in methane (M = 12.01 µg CH4-C µmole CH4 ); P is barometric pressure assumed as 1 atm; R is the universal gas constant (R = 0.0820575 L atm K-1 mole-1) and T is temperature of the chamber at the time of sampling. Linear regression analysis between resulting Cm’s and sampling time for each chamber was developed in order to determine

-3 -1 the rate of emission (Crate, mg CH4-C m min ). Crate was the slope of the linear regression and

-2 -1 was used to calculate hourly CH4 flux (fh, mg CH4-C m h ) as:

(Eq. 2) �ℎ = !×!!"#$ ×60 ��� ! where V is the internal volume of the chamber (m3) and A is the surface area covered by the chamber (m2). The chamber volume varied according the water levels during sampling as follows: 1) 73 L under dry conditions, 2) 53 L when water level was greater than 10 cm, and 3) variable, calculated subtracting the volume occupied by water, when water level was lower than

10 cm.

2.4 Data analysis

Linear regressions used to determine CH4 emission rates were conducted at a significance level of α =0.05. Regressions that are not statistically significant have a slope of zero, thus, rates calculated from not significant regressions were considered as zero net emissions and their fluxes were counted in the sampling estimates. Infrequent high rates that resulted from significant

11

regressions were not removed as outliers because natural spikes in CH4 emissions can occur. Cv’s that were lower than the chromatograph detection limit (0.1 mg/L) were given a value of zero and were included in the regressions. Discontinuous increases in Cm’s during each chamber sampling were assumed to be due to ebullition (Baulch et al. 2011; Villa and Mitsch 2014) or to the un-mixed headspace air (Christiansen et al. 2011). Regardless, regressions excluded the data after this episode to account only for the diffusive fluxes (Happell and Chanton 1993; Baulch et al. 2011; Villa and Mitsch 2014) or to reduce estimation errors.

Descriptive statistics (mean, median, standard error) were used to characterize water level, soil temperature and methane flux data. Daily fluxes for the study period were used to assess the seasonal variability of the six sites. Water level, soil temperature and CH4 data failed to meet criteria for normal distribution as indicated by the Kolmogorov-Smirnov one-sample test

(p < 0.001 for all variables). Therefore, a non-parametric Kruskal-Wallis chi-squared test was performed to test for temporal variation in water level and soil temperature within sites and for differences in CH4 daily fluxes among sites. Following pair-wise comparisons tests, within each variable, were conducted using a Mann-Whitney U test. Spearman's rank-order correlation of the average fh from each sampling and its correspondent water level and soil temperature was used to test for associations between the fluxes and the environmental variables. All difference tests and correlations were conducted at a significance level of � = 0.05. Statistical analyses were performed using JMP 12 for Mac (SAS Institute, Cary, NC) and SPSS Statistics 23 for PC (IBM

Corporation, Armonk, NY).

12

Chapter 3

Results

3.1 Precipitation, water levels and soil temperature

Monthly precipitation followed the characteristic pattern of precipitation in South Florida

(Duever et al. 1994) at the reference and disturbed swamp with a rainy June through September

(when more than 65% of the rain fell) and a dry remainder of the year. Total rainfall in the region ranged from 1,108 to 1,672 mm at the two weather stations.

Water levels in the reference and disturbed swamp showed seasonal hydroperiods (Fig. 2,

Table 1) lagging behind the precipitation events. The reference swamp’s flooding phase started between late June and early July 2014, and all three sites there stayed inundated through the end of the study period (December, 2014). Conversely, the flooding phase in the disturbed swamp was much shorter—from late July to early October—with some days at the end of July, August and September having no surface water. The disturbed sites were inundated for a 6-month duration (mid June to mid December, 2014). Water levels from January to mid-June were lower than the piezometer depth (70 cm), so there were no water level readings then. Mean monthly water levels within each one of the sites was significantly different (p < 0.05) from one another with a few exceptions during transitional months, from wet to dry season, or vice versa, (i.e.,

May and June) and during the same season (i.e., August and September, October and December).

Overall, the reference swamps had deeper surface water than the disturbed swamps. The maximum water levels were observed in the reference bald cypress slough, while the shortest flood duration and lowest water levels were found in the disturbed cypress dome 1 (Table 1).

13

Soil temperatures followed expected seasonal patterns (Table 1; See also Appendix A).

The reference swamp had highest soil temperatures during early spring (bald cypress slough) and summer (pond cypress slough and cypress dome) and the lowest during early winter. In the disturbed swamp, the highest soil temperatures occurred in mid-spring and summer, while the lowest occurred during early spring and winter. Mean monthly soil temperatures within each research site were significantly different (p < 0.05) from one another; few exceptions were found in months that belonged to the same, wet or dry, season (i.e., Feb. and Mar., May and Oct., Nov. and Dec.). Soil temperatures were least variable in the wettest sites and during the wettest months, and most variable in the driest swamps and driest months; this trend was more evident at the reference swamps.

3.2 Methane fluxes

A total of 2052 gas samples were collected during the study, from which 7 resulted in methane concentrations (Cv) below the GC detection limit of 0.1 mg/L. The slopes of the methane curves from these samples led to the estimation of 342 methane fluxes (fh), with 29 (or

8.5%) of the flux estimations proving to be significant. In addition, of the 342 methane flux estimates, noticeable discontinuous increases in the CH4 concentrations were identified on 10 occasions, all of which were at the reference swamps.

Daily CH4 fluxes at the six research sites are summarized in Figs. 3 and 4 and Table 2.

During the non-flooded phase, all the reference sites except the reference pond cypress slough showed CH4 fluxes equal to zero. Conversely, the disturbed sites showed CH4 fluxes different to zero only in the non-flooded phase. Regardless, this apparent difference in CH4 fluxes within the sites between phases, no statistical differences between fluxes during non-flooded and flooded phase were found.

14

The disturbed swamp had relatively more sampling periods with zero net emissions than the reference swamp, with 78% (21 of 27) daily fluxes estimated to be equal to zero for the former compared to 40% (12 of 30) for the latter (Figs. 3 and 4). Over the entire study period,

CH4 fluxes were more variable in the reference than in the disturbed sites (Fig. 4), ranging from -

-2 -1 -2 -1 7.0 to 218 CH4-C m d versus -6.6 to 38.6 mg CH4-C m d , respectively. The maximum values of the CH4 fluxes range (Table 2), especially in the reference sites, are an indication of the infrequent high spikes in emissions from wetlands. Conversely, the minimum values in the range indicate that methane oxidation is occurring in both reference and disturbed swamp. The

-2 disturbed cypress dome 1 acted as a CH4 net sink with an uptake rate of 1.4 ± 0.8 mg CH4-C m

-1 d (mean ± SE); additionally, three sites out of six were temporary sinks (Table 2). Daily CH4 fluxes from the reference swamp sites were significantly higher than fluxes from the disturbed

2 -2 -1 swamp (X = 5.61, df = 1, p < 0.05). Mean CH4 emissions ± SE (in mg CH4-C m d ) were 32.4

± 11.9 and 1.1 ± 1.5 for the reference and disturbed swamp, respectively. Methane emission medians for both swamps were zero. Within the reference swamp, emissions were not significantly different between sites (X2 = 3.5, df = 2, p = 0.18). However, significant differences were found between the disturbed sites (X2 = 6.2, df = 2, p < 0.05). Pairwise comparisons showed that fluxes from the disturbed pond cypress slough were significantly higher than fluxes from the disturbed cypress dome 1 (U = 28, df = 18, p < 0.05), but fluxes from both of these sites were similar to fluxes from the disturbed cypress dome 2 (Fig. 4).

3.3 Relationship of CH4 fluxes to physiochemistry

Considering only fluxes that were different than zero, CH4 emissions from the reference sites were usually higher when water was deeper. Overall for all sites, the correlation between methane emissions and water level was poor (Fig. 5). CH4 fluxes did not show any relationship

15

with soil temperature. Spearman’s correlations, between CH4 fluxes and water level or soil temperature, were not statistically significant for any of the six sampling sites or the 2 swamps

(i.e., reference and disturbed) (Table 3).

16

Chapter 4

Discussion

4.1 Methane fluxes

Mean CH4 fluxes from the reference and disturbed sites were within and towards the low range of fluxes reported from other subtropical and tropical freshwater forested wetlands (Table

4) and were lower than or within ranges reported for other studies of freshwater swamps located in Florida (Table 4). Previous studies at the reference swamp (Corkscrew Swamp Sanctuary)

- reported wider ranges of mean fluxes than the current study: 4.5 - 55 and 8.2 - 265 mg CH4-C m

2 d-1 by Villa and Mitsch (2014) and Harriss and Sebacher (1981) respectively.

Overall, fluxes from the soil of the six sites described here are highly variable as shown by the standard errors that ranged from 50 to 99% and 53 to 100% of the mean for the reference and disturbed swamp, respectively. A two-year study, also at Corkscrew Swamp Sanctuary, found daily fluxes with high standard errors (Villa and Mitsch 2014). A three-year study in cypress swamps in Louisiana measured fluxes with standard errors higher than the mean (146 ±

-2 -1 199 in mg CH4-C m d ) (Alford et al. 1997). This high variability is common with the measurement of CH4 emissions from wetlands; emissions can vary greatly from short to long periods of time (i.e., hours to years) and from spatial scales lower than 1 m, as a response to changes in the factors that control CH4 fluxes (Windsor et al. 1992; Bartlett and Harriss 1993;

Whalen 2005). The higher variability of CH4 fluxes from the reference compared to the disturbed swamp could be related to the level of disturbance of the swamp. Variability in biogeochemical processes such as methanogenesis tends to be higher in natural wetlands because the drivers of

CH4 production (e.g. substrate availability, hydrology, soil properties, vegetation, etc.) are

17 usually more dynamic than in restored or disturbed wetlands (Altor and Mitsch 2008).

Fluxes from the reference swamp area were significantly higher than fluxes from the disturbed swamp area. Previous research on cypress swamps in the southeast United States compared CH4 fluxes from disturbed and undisturbed swamps and found that Corkscrew Swamp

Sanctuary had the highest CH4 fluxes of the 3 undisturbed swamps, (Harriss and Sebacher 1981).

This study attributed its findings to the differences in soil properties, specifically, organic content and soil-thickness. Observations of the soil cores taken from the research sites coincide with

Harriss and Sebacher's (1981) report of Corkscrew’s thick and organic-rich soils. Soils from the reference sites accumulated more peat than soils in the disturbed sites, with thickness of the peat deposits ranging from 8 to 25 cm, and 5 to 17 cm, respectively.

Within the reference swamp, CH4 fluxes among the sites were not significantly different.

These sites have similar: 1) hydroperiods (with long flooding phases and wide ranges of water level) and 2) soils (medium or high accumulation of peat, from 8 to 25 cm deep), which could explain the lack of significant difference among them. Another plausible explanation could be the high variability of the fluxes (Bartlett and Harriss 1993). All sites have a wide range of fluxes, with CH4 uptakes and emissions of one to three orders of magnitude, respectively.

Contrary to the findings in the reference sites, methane fluxes from the disturbed cypress dome 1 were significantly lower than the fluxes from the disturbed pond cypress slough. Both sites have similar hydroperiods and water levels but not similar hydrogeomorphology, as the dome is more isolated than the slough. Vegetation (e.g., macrophytes) can promote CH4 emissions by contributing to the net primary productivity and by being sources of methanogenic substrates (Bartlett and Harriss 1993; Whalen 2005). The forest floor of the disturbed pond

18 cypress slough had more understory vegetation coverage than the disturbed cypress dome 1, and the presence of this vegetation could explain the higher fluxes in the disturbed pond cypress slough.

4.2 Methane oxidation

Methane oxidation rates, measured especially in the disturbed sites, were similar to those seen previously in these and other freshwater wetlands. CH4 uptake fluctuated from -7 to -1 mg

-2 -1 -2 -1 CH4-C m d compared with -4 to -2 mg CH4-C m d measured by Villa and Mitsch (2014) in

-2 -1 Corkscrew Swamp Sanctuary and -4 mg CH4-C m d measured by Nahlik and Mitsch (2011) in tropical wetlands in Costa Rica. Methane fluxes from the disturbed sites were usually negative or

-2 -1 very low (i.e., <4 mg CH4-C m d ), suggesting these sites could be net sinks of CH4.

4.3 Discontinuities in methane emissions and ebullition

Discontinuous increases in CH4 concentration were found in 10 occasions, or 3% of the

342 chamber runs. Methane concentration in chambers with no headspace mixing is uneven over time, usually showing a systematic variation with high values followed by low values or vice versa (Christiansen et al. 2011). However, a substantial spike on CH4 concentration over time represented most of the identified increases. The variation in the slope of the linear regression between CH4 and time, after and before this spike, was constant. This is a pattern more related to ebullition (Villa and Mitsch 2014). The current research was not designed to identify ebullitive fluxes or potential errors due to chamber operation but it is important to note that, since these episodes were not included in the linear regressions used to calculate emission rates, it is possible that reported fluxes are slightly over or underestimated.

19

4.4 Methane fluxes and hydrology

Water depth is expected to be the major controlling variable of methanogenesis in tropical and subtropical wetlands with marked wet and dry seasons (Bartlett and Harriss 1993;

Whalen 2005). The reference swamp had significantly higher CH4 fluxes than the disturbed swamp and also had deeper and longer periods of standing water than the disturbed swamp. This supports the first hypothesis that cypress swamps with longer flooded phases and higher water levels will result in higher CH4 emissions than those with shorter flooded phases and lower water levels. The drier settings and intermittent drawdowns of the water table at the disturbed swamp caused a severe decline in methanogenesis over historical conditions due to oxidation of labile carbon and/or inhibition of methanogens after exposing them to oxygen (Basiliko et al. 2007;

Altor and Mitsch 2008). Methanogens are highly sensitive to oxygen and stop the production of

CH4 soon after soils are drained (Whalen 2005).

Methane fluxes from the disturbed sites occurred only during the non-flooded phase, suggesting that factors other than inundation could be controlling these fluxes. These sites showed a saturated water regime, meaning the substrate is saturated during the wet season but standing water is infrequent. This saturated condition probably allowed the soils to remain chemically reduced when the water table went below ground. A study in temperate zone wetlands with different levels of disturbance (natural, actively mined, mined-abandoned and restored) found significant correlation between moisture content and CH4 production, and concluded that methane dynamics at the mined-abandoned and restored sites were mainly controlled by soil moisture (Basiliko et al. 2007). This explanation does not apply for the fluxes from the disturbed cypress dome 1, however, which acted as a net sink. During the samplings that resulted in CH4 uptake soils were not saturated, rather, the water table was >69 cm below

20 ground, preventing anaerobic conditions in the surface soils. Usually, when water levels decrease

CH4 oxidation increases (Kayranli et al. 2010).

4.5. Comparison within swamps

In spite of the clear differences between the two researched swamps (i.e., reference and disturbed) concerning the fluxes during flooded and non – flooded phase, no statistical differences were found among sites in each swamp. The high variability and the uneven number of samplings per flood phase could be one reason for the lack of significant differences (Bartlett and Harriss 1993). In addition to water table depth, the type of flow through a wetland is influential in methane dynamics. Previous studies found that freshwater wetlands with steady inflows had higher fluxes than those exposed to pulsed inflows (Whalen and Reeburgh 2000;

Mitsch et al. 2005; Altor and Mitsch 2008; Koh et al. 2009). Research results support the second hypothesis that fluxes from the swamp with more consistent flooding conditions (i.e., reference swamp) were significantly higher than those of the swamp with pulsed hydrologic regimes (i.e., disturbed swamp).

Regardless of the undeniable influence of water levels on CH4 fluxes, these two variables failed to correlate in the current study. A thorough review of several studies concluded that correlations with environmental variables (e.g., water table) could explain only 34% of variability in methane production and 10% of the variability in oxidation (Segers 1998). Similar results have been reported by Harriss and Sebacher (1981), Wilson et al. (1989), Alford et al.

(1997) and Bloom et al. (2010). Records from the Amazon wetlands showed a negligible correlation between hydrology and CH4 emissions, with changes in the CH4 emissions lagged behind changes in water table depth by 1 to 3 month (Bloom et al. 2010). Another plausible explanation for the lack of correlation could be the high number of CH4 fluxes equal to zero,

21 indicating that factors other than water level (e.g., organic soil thickness or peat accumulation, soil moisture, and presence of macrophytes) may be controlling the methane biogeochemical processes in the researched swamps.

4. 6 Methane fluxes and soil temperature

Soil temperature affects the metabolism of both methanogens and methanotrophs. High temperatures increase the activity of most microbes, resulting in higher decomposition that leads to fewer methanogenic substrates (Alford et al. 1997). Methanogens isolated in pure cultures are usually mesophilic, with an optimal temperature range for growth between 30 and 40 °C (Zinder

1993). Similar optima values (35 to 42 °C) were found for methanogens from a temperate lake in

Wisconsin (Zeikus and Winfrey 1976). Overall, this research’s recorded soil temperatures lie outside the optimal temperature range of methanogens; mean soil temperature from the reference and disturbed swamp were 23 °C and 24 °C, respectively. Previous studies at Corkscrew Swamp

Sanctuary found CH4 fluxes and mean soil temperature similar to what I recorded, and also failed to find a correlation between these variables (Harris and Sebacher 1981; Villa and Mitsch 2014).

Despite the noted importance of soil temperature on CH4 fluxes, correlations between these variables are usually weak or non-existent (Windsor et al. 1992; Bartlett and Harriss 1993).

Variation in CH4 in the subtropics and tropics is poorly explained by changes in soil temperature

(Bloom et al. 2010), in comparison with fluxes at higher latitudes, where temperature is a more important control of methanogenesis (Whalen 2005).

4. 7 Study significance

In wetlands, methane emission is intimately related to carbon sequestration and storage.

Thus, an analysis of carbon sequestration as well as methane emissions should be factored in to fully comprehend the impact of land use modifications in wetland areas. The balance between

22

CH4 and CO2 cycling through a wetland are essential for understanding a wetland’s contribution to climate regulation (Whitting and Chanton 2001, Mitsch et al. 2013). Bondavalli et al. (2000) developed a carbon budget for southwest Florida cypress swamps and found these ecosystems act as carbon sinks. Similarly, Villa and Mitsch (2015) measured carbon sequestration and provided an analysis of CH4 emission and C sequestration rates from Corkscrew Swamp

Sanctuary to assess the sequestering potential of this ecosystem. Results suggested that

Corkscrew acts as a net greenhouse gas (GHG) source when analyzed for a short-term (i.e., 20 yrs) but as a net sink over a long-term (i.e., 500 yrs). Of course the wetlands there are well older than 500 years.

23

Chapter 5

Conclusions

This study presents methane emission rates for six cypress (Taxodium) swamps with different hydroperiods and degrees of disturbance located in southwest Florida. Methane emissions among and within swamps were compared to determine spatial and temporal variation.

Also, emissions were examined in relation to water levels and soil temperatures to determine the influence of these environmental variables on methane fluxes. Conclusions are as follows:

1. Methane fluxes from the reference and disturbed swamp were on the low range

of fluxes reported from other subtropical and tropical freshwater wetlands.

2. Methane fluxes from sites with longer flooded phases and higher water levels

(reference swamp) were significantly higher than fluxes from the sites with

shorter flooded phases and lower water levels (disturbed swamp).

3. For the reference sites, the hydroperiods, by themselves, could not explain the

lack of significant differences in methane fluxes.

4. At the disturbed swamp, methane fluxes from the site that showed the longest

flood duration and highest water levels (disturbed pond cypress slough) were

significantly higher than the other disturbed sites.

Future studies of CH4 dynamics from subtropical and tropical cypress swamps should include a characterization and analysis of soil properties such as organic matter and soil moisture, microbial communities, and vegetation communities, to determine and understand the interaction of other known factors that affect methane emissions. To better comprehend the

24 impact of land use modifications in wetland areas, an analysis of carbon sequestration should be factored in. The balance between carbon emission and sequestration are essential for understanding a wetland’s contribution to climate regulation. The results of this research, especially those related to the disturbed wetlands, can provide a better understanding on CH4 emissions from cypress swamps located within an altered wetland landscape like that of southwest Florida.

25

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33

Figures and Tables

34

Fig 1. Locations of two research swamp areas in southwest Florida: Florida Gulf Coast

University (FGCU) campus in Lee County and Corkscrew Swamp Sanctuary (CSS) in

Collier County.

35

Fig. 2 Water levels for a) three reference cypress sites at Corkscrew Swamp Sanctuary (CSS) and

b) two of the three disturbed cypress sites on the campus of Florida Gulf Coast University

(FGCU). Data were collected from December 2013 to December 2014. Water levels

were more than 40 cm below the surface for the sites at FGCU for the first 6 months of

the study. Ground level corresponds to 0 cm; grey shade shows below-ground elevations.

36

Fig. 3 Mean (triangles), standard error (vertical lines), and median (diamonds) methane

-2 -1 emissions (mg C-CH4 m d ) for the reference (CSS) and disturbed (FGCU) cypress

swamp for 2013-14: a) Reference bald cypress slough; b) Reference pond cypress slough;

c) Reference cypress dome; d) Disturbed pond cypress slough; e) Disturbed cypress dome

1; and f) Disturbed cypress dome 2. Solid lines illustrate the water depth for each site.

37

Fig. 3. continued

38

Fig. 4. Boxplot with methane (CH4) average and media fluxes for the 6 sampling sites for the

entire study period. Dashed line and horizontal line within the boxes respectively

represent the mean and median values for the study period. Whiskers represent the full

range of the values and the edges of the boxes represent the 25th and 75th percentiles.

Outliers are represented by hollow circles. Pairwise differences are represented with

letters.

39

Fig. 5 Linear regression for methane (CH4) flux vs. water level for all swamps combined.

40

Table 1 Ranges, mean ± standard error (SE) of water level and soil temperature at 2 cm depth and flood duration by site during the study period. Ground level is 0 cm; negative values represent water below ground surface and positive numbers indicate water levels above the ground surface.

Site Water level (cm) Soil T (°C) Range Mean ± SE Flood Range Mean ± SE duration

(days) Reference bald cypress slough -71.9 - 98 43.3 ± 2.14 292 5.95 - 34.20 23.07 ± 0.04 Reference pond cypress slough -114.3 - 53.8 -0.4 ± 2.12 209 5.77 - 38.67 22.89 ± 0.04 Reference cypress dome -74.1 - 39.7 12.0 ± 1.14 280 7.17 - 25.35 22.76 ± 0.03 Disturbed pond cypress slough -62.6 - 12.4 -11.0 ± 1.28 67 9.51 - 54.64 23.67 ± 0.05 Disturbed cypress dome 1 -65.2 - 11.1 -17.1 ± 1.53 58 10.76 - 42 24.02 ± 0.05

41

Table 2. Mean ± standard error (SE), number of measured fluxes (n) and median of methane (CH4) fluxes by site and by locality for the study period and inundation condition.

-2 -1 Site Daily fluxes (mg C-CH4 m d ) Non-flooded Study Flooded phase phase

Range Mean ± SE (n) Median Mean ± SE (n) Median Mean ± SE (n) Median Ref. bald cypress slough 0 - 139.13 25.86 ± 15.62 (10) 0.00 28.73 ± 17.16 (9) 0.00 0 ± -- (1) 0 Ref. pond cypress slough -4.57 - 218.03 22.02 ± 21.8 (10) 0.00 30.03 ± 31.34 (7) 0.00 3.36 ± 1.68 (3) 4.86 Ref. cypress dome -6.99 - 201.65 49.47 ± 24.71 (10) 12.88 54.97 ± 26.93 (9) 14.07 0 ± -- (1) 0 Ref. sites -6.99 - 218.03 32.45 ± 11.94 (30) 0.00 38.54 ± 14.05 (25) 0.00 2.02 ± 1.24 (5) 0 Dist. pond cypress slough 0 - 38.65 4.0 ± 3.85 (10) 0.00 0 ± 0 (2) 0 5.00 ± 4.81 (8) 0 Dist. cypress dome 1 -6.59 - 0 -1.44 ± 0.77 (10) 0.00 0 ± 0 (1) 0 -1.60 ± 0.85 (9) 0 Dist. cypress dome 2 0 - 3.84 0.55 ± 0.55 (7) 0.00 -- -- 0.55 ± 0.55 (7) 0 Dist. sites -6.59 - 38.65 1.09 ± 1.49 (27) 0 0 ± 0 (4) 0 1.28 ± 1.75 (23) 0

42

Table 3. Spearman's rank order correlation between methane (CH4) fluxes, water level and soil temperature at 2 cm depth.

Site Water level Soil temperature Reference bald cypress slough 0.23 (10), 0.82 0.18 (10), 0.75 Reference pond cypress slough 0.002 (10), 0.14 0.073 (10), 0.35 Reference cypress dome 0.23 (10), 0.3 0.07 (9), 0.72 Reference sites 0.1 (30), 0.67 0.01 (29), 0.6 Disturbed pond cypress slough 0.39 (6), 0.56 0.07 (10), 0.53 Disturbed cypress dome 1 -- (6), -- 0.24 (10), 0.7 Disturbed sites 0.12 (12), 0.47 0.01 (20), 0.84 Data is presented as correlation coefficient (n), p-value.

There were no significant correlations at a level of α =0.05.

43

Table 4. Methane emissions (mean ± standard error) from tropical and subtropical freshwater forested swamps.

Methane emissions -2 -1 (mg CH4 - C m d ) Study location Source 25.9 ± 15.6 Corkscrew Swamp, Florida, U.S. This study 22 ± 21.9 Corkscrew Swamp, Florida, U.S. This study 49.5 ± 24.7 Corkscrew Swamp, Florida, U.S. This study 55.3 ± 21.5 Corkscrew Swamp, Florida, U.S. Villa and Mitsch 2014 4.5 ± 3.6 Corkscrew Swamp, Florida, U.S. Villa and Mitsch 2014 5.5 ± 2.5 Corkscrew Swamp, Florida, U.S. Villa and Mitsch 2014 67 Corkscrew Swamp, Florida, U.S. Harris and Sebacher 1981 4.0 ± 3.8 Florida Gulf Coast University, Florida, U.S. This study -1.4 ± 0.8 Florida Gulf Coast University, Florida, U.S. This study 0.5 ± 0.5 Florida Gulf Coast University, Florida, U.S. This study 146 ± 199 Mississippi Delta, Louisiana, U.S. Alford et al. 1997 92.3 Okefenokee Swamp, Georgia, U.S. Harris and Sebacher 1981 10 Four Holes Swamp, South Carolina, U.S. Harris and Sebacher 1981 68.9 ± 22.5 Everglades, Florida, U.S. Bartlett et al. 1989 601 La Selva Biological Station, Costa Rica Nahlik and Mitsch 2011 117 Newport News Swamp, Virginia, U.S. Wilson et al. 1989 140.4 Clear Springs, Mississippi, U.S. Koh et al. 2009 106 Congo River, Congo Tathy et al. 1992 71 ± 74 St. Marks National Wildlife Refuge, Florida, U.S. Happell and Chanton 1993 31 ± 13 Ogeeche River Floodplain, Georgia, U.S. Pulliam 1993 72 ± 27 Ogeeche River Floodplain, Georgia, U.S. Pulliam 1993 122.4 ± 28.8 Earth University, Costa Rica Mitsch et al. 2008 81 Orinoco River Floodplain, Venezuela Smith et al. 2000

44

Appendix A

Soil temperature at 2 cm depth at the reference sites

45

Soil temperature at 2 cm depth at the disturbed sites

46

Appendix B

Original data

Methane fluxes for the different samplings in the Reference bald cypress slough.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 1/24/14 Reference bald cypress slough M 1 0.00 1/24/14 Reference bald cypress slough M 2 0.00 1/24/14 Reference bald cypress slough M 3 0.00 1/24/14 Reference bald cypress slough A 1 0.00 1/24/14 Reference bald cypress slough A 2 0.00 1/24/14 Reference bald cypress slough A 3 0.92 2/20/14 Reference bald cypress slough M 1 0.00 2/20/14 Reference bald cypress slough M 2 0.00 2/20/14 Reference bald cypress slough M 3 0.00 2/20/14 Reference bald cypress slough A 1 3.90 2/20/14 Reference bald cypress slough A 2 19.53 2/20/14 Reference bald cypress slough A 3 0.00 4/12/14 Reference bald cypress slough M 1 0.00 4/12/14 Reference bald cypress slough M 2 0.00 4/12/14 Reference bald cypress slough M 3 0.00 4/12/14 Reference bald cypress slough A 1 0.00 4/12/14 Reference bald cypress slough A 2 0.00 4/12/14 Reference bald cypress slough A 3 0.00 6/4/14 Reference bald cypress slough M 1 0.00 6/4/14 Reference bald cypress slough M 2 0.00 6/4/14 Reference bald cypress slough M 3 0.00 6/4/14 Reference bald cypress slough A 1 0.00 6/4/14 Reference bald cypress slough A 2 0.00 6/4/14 Reference bald cypress slough A 3 0.00 7/10/14 Reference bald cypress slough M 1 1.68 7/10/14 Reference bald cypress slough M 2 0.00 7/10/14 Reference bald cypress slough M 3 0.73

47

-2 -1 Date Site Time Chamber mg CH4 - C m h 7/9/14 Reference bald cypress slough A 1 3.11 7/9/14 Reference bald cypress slough A 2 0.00 7/9/14 Reference bald cypress slough A 3 0.00 8/5/14 Reference bald cypress slough M 1 0.00 8/5/14 Reference bald cypress slough M 2 0.00 8/5/14 Reference bald cypress slough M 3 0.00 8/5/14 Reference bald cypress slough A 1 0.00 8/5/14 Reference bald cypress slough A 2 0.00 8/5/14 Reference bald cypress slough A 3 0.00 9/21/14 Reference bald cypress slough M 1 0.00 9/21/14 Reference bald cypress slough M 2 0.00 9/21/14 Reference bald cypress slough M 3 0.00 9/21/14 Reference bald cypress slough A 1 0.00 9/21/14 Reference bald cypress slough A 2 0.00 9/21/14 Reference bald cypress slough A 3 0.00 10/18/14 Reference bald cypress slough M 1 18.60 10/18/14 Reference bald cypress slough M 2 0.00 10/18/14 Reference bald cypress slough M 3 0.00 10/18/14 Reference bald cypress slough A 1 14.34 10/18/14 Reference bald cypress slough A 2 0.00 10/18/14 Reference bald cypress slough A 3 1.85 11/16/14 Reference bald cypress slough M 1 0.00 11/16/14 Reference bald cypress slough M 2 0.00 11/16/14 Reference bald cypress slough M 3 0.00 11/16/14 Reference bald cypress slough A 1 0.00 11/16/14 Reference bald cypress slough A 2 0.00 11/16/14 Reference bald cypress slough A 3 0.00 12/14/14 Reference bald cypress slough M 1 0.00 12/14/14 Reference bald cypress slough M 2 0.00 12/14/14 Reference bald cypress slough M 3 0.00 12/14/14 Reference bald cypress slough A 1 0.00 12/14/14 Reference bald cypress slough A 2 0.00 12/14/14 Reference bald cypress slough A 3 0.00

48

Methane fluxes for the different samplings in the Reference pond cypress slough.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 1/24/14 Reference pond cypress slough M 1 0.00 1/24/14 Reference pond cypress slough M 2 0.00 1/24/14 Reference pond cypress slough M 3 0.00 1/24/14 Reference pond cypress slough A 1 0.00 1/24/14 Reference pond cypress slough A 2 0.00 1/24/14 Reference pond cypress slough A 3 1.21 2/20/14 Reference pond cypress slough M 1 26.10 2/20/14 Reference pond cypress slough M 2 0.00 2/20/14 Reference pond cypress slough M 3 3.72 2/20/14 Reference pond cypress slough A 1 22.50 2/20/14 Reference pond cypress slough A 2 2.18 2/20/14 Reference pond cypress slough A 3 0.00 4/12/14 Reference pond cypress slough M 1 0.00 4/12/14 Reference pond cypress slough M 2 0.00 4/12/14 Reference pond cypress slough M 3 0.00 4/12/14 Reference pond cypress slough A 1 0.00 4/12/14 Reference pond cypress slough A 2 0.00 4/12/14 Reference pond cypress slough A 3 1.31 6/4/14 Reference pond cypress slough M 1 0.00 6/4/14 Reference pond cypress slough M 2 0.00 6/4/14 Reference pond cypress slough M 3 0.00 6/4/14 Reference pond cypress slough A 1 0.00 6/4/14 Reference pond cypress slough A 2 0.00 6/4/14 Reference pond cypress slough A 3 0.00 7/9/14 Reference pond cypress slough M 1 0.00 7/9/14 Reference pond cypress slough M 2 0.00 7/9/14 Reference pond cypress slough M 3 0.00 7/9/14 Reference pond cypress slough A 1 -1.14 7/9/14 Reference pond cypress slough A 2 0.00 7/9/14 Reference pond cypress slough A 3 0.00

49

-2 -1 Date Site Time Chamber mg CH4 - C m h 8/5/14 Reference pond cypress slough M 1 0.00 8/5/14 Reference pond cypress slough M 2 0.00 8/5/14 Reference pond cypress slough M 3 0.00 8/5/14 Reference pond cypress slough A 1 0.00 8/5/14 Reference pond cypress slough A 2 0.00 8/5/14 Reference pond cypress slough A 3 0.00 9/21/14 Reference pond cypress slough M 1 0.00 9/21/14 Reference pond cypress slough M 2 0.00 9/21/14 Reference pond cypress slough M 3 0.00 9/21/14 Reference pond cypress slough A 1 0.00 9/21/14 Reference pond cypress slough A 2 0.00 9/21/14 Reference pond cypress slough A 3 0.00 10/18/14 Reference pond cypress slough M 1 0.00 10/18/14 Reference pond cypress slough M 2 -0.66 10/18/14 Reference pond cypress slough M 3 0.00 10/18/14 Reference pond cypress slough A 1 0.00 10/18/14 Reference pond cypress slough A 2 0.00 10/18/14 Reference pond cypress slough A 3 0.00 11/16/14 Reference pond cypress slough M 1 0.00 11/16/14 Reference pond cypress slough M 2 0.00 11/16/14 Reference pond cypress slough M 3 0.00 11/16/14 Reference pond cypress slough A 1 0.00 11/16/14 Reference pond cypress slough A 2 -0.17 11/16/14 Reference pond cypress slough A 3 0.00 12/14/14 Reference pond cypress slough M 1 0.00 12/14/14 Reference pond cypress slough M 2 0.00 12/14/14 Reference pond cypress slough M 3 0.00 12/14/14 Reference pond cypress slough A 1 0.00 12/14/14 Reference pond cypress slough A 2 0.00 12/14/14 Reference pond cypress slough A 3 0.00

50

Methane fluxes for the different samplings in the Reference cypress dome.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 1/24/14 Reference cypress dome M 1 0.00 1/24/14 Reference cypress dome M 2 0.00 1/24/14 Reference cypress dome M 3 0.00 1/24/14 Reference cypress dome A 1 0.00 1/24/14 Reference cypress dome A 2 0.00 1/24/14 Reference cypress dome A 3 1.23 2/20/14 Reference cypress dome M 1 50.41 2/20/14 Reference cypress dome M 2 0.00 2/20/14 Reference cypress dome M 3 0.00 2/20/14 Reference cypress dome A 1 0.00 2/20/14 Reference cypress dome A 2 0.00 2/20/14 Reference cypress dome A 3 0.00 4/12/14 Reference cypress dome M 1 0.00 4/12/14 Reference cypress dome M 2 0.00 4/12/14 Reference cypress dome M 3 0.00 4/12/14 Reference cypress dome A 1 -1.75 4/12/14 Reference cypress dome A 2 0.00 4/12/14 Reference cypress dome A 3 0.00 6/4/14 Reference cypress dome M 1 0.00 6/4/14 Reference cypress dome M 2 0.00 6/4/14 Reference cypress dome M 3 0.00 6/4/14 Reference cypress dome A 1 0.00 6/4/14 Reference cypress dome A 2 0.00 6/4/14 Reference cypress dome A 3 0.00 7/9/14 Reference cypress dome M 1 0.00 7/9/14 Reference cypress dome M 2 0.00 7/9/14 Reference cypress dome M 3 -0.77 7/9/14 Reference cypress dome A 1 4.29 7/9/14 Reference cypress dome A 2 0.00 7/9/14 Reference cypress dome A 3 0.00

51

-2 -1 Date Site Time Chamber mg CH4 - C m h 8/5/14 Reference cypress dome M 1 0.00 8/5/14 Reference cypress dome M 2 0.00 8/5/14 Reference cypress dome M 3 0.00 8/5/14 Reference cypress dome A 1 0.00 8/5/14 Reference cypress dome A 2 0.00 8/5/14 Reference cypress dome A 3 0.00 9/21/14 Reference cypress dome M 1 0.00 9/21/14 Reference cypress dome M 2 0.00 9/21/14 Reference cypress dome M 3 0.00 9/21/14 Reference cypress dome A 1 0.00 9/21/14 Reference cypress dome A 2 0.00 9/21/14 Reference cypress dome A 3 14.41 10/18/14 Reference cypress dome M 1 11.17 10/18/14 Reference cypress dome M 2 6.09 10/18/14 Reference cypress dome M 3 26.52 10/18/14 Reference cypress dome A 1 2.53 10/18/14 Reference cypress dome A 2 0.00 10/18/14 Reference cypress dome A 3 0.00 11/16/14 Reference cypress dome M 1 0.00 11/16/14 Reference cypress dome M 2 0.00 11/16/14 Reference cypress dome M 3 0.00 11/16/14 Reference cypress dome A 1 0.39 11/16/14 Reference cypress dome A 2 0.00 11/16/14 Reference cypress dome A 3 2.53 12/14/14 Reference cypress dome M 1 0.00 12/14/14 Reference cypress dome M 2 0.00 12/14/14 Reference cypress dome M 3 0.00 12/14/14 Reference cypress dome A 1 0.00 12/14/14 Reference cypress dome A 2 0.00 12/14/14 Reference cypress dome A 3 6.62

52

Methane fluxes for the different samplings in the Disturbed pond cypress slough.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 1/20/14 Disturbed pond cypress slough M 1 0.00 1/20/14 Disturbed pond cypress slough M 2 0.00 1/20/14 Disturbed pond cypress slough M 3 0.00 1/20/14 Disturbed pond cypress slough A 1 0.00 1/20/14 Disturbed pond cypress slough A 2 0.00 1/20/14 Disturbed pond cypress slough A 3 0.00 2/25/14 Disturbed pond cypress slough M 1 0.00 2/25/14 Disturbed pond cypress slough M 2 0.00 2/25/14 Disturbed pond cypress slough M 3 0.00 2/25/14 Disturbed pond cypress slough A 1 0.00 2/25/14 Disturbed pond cypress slough A 2 0.00 2/25/14 Disturbed pond cypress slough A 3 0.00 4/14/14 Disturbed pond cypress slough M 1 0.00 4/14/14 Disturbed pond cypress slough M 2 0.00 4/14/14 Disturbed pond cypress slough M 3 0.00 4/14/14 Disturbed pond cypress slough A 1 0.00 4/14/14 Disturbed pond cypress slough A 2 0.00 4/14/14 Disturbed pond cypress slough A 3 0.00 6/9/14 Disturbed pond cypress slough M 1 0.00 6/9/14 Disturbed pond cypress slough M 2 0.00 6/9/14 Disturbed pond cypress slough M 3 0.00 6/9/14 Disturbed pond cypress slough A 1 0.00 6/9/14 Disturbed pond cypress slough A 2 0.00 6/9/14 Disturbed pond cypress slough A 3 0.00 7/8/14 Disturbed pond cypress slough M 1 0.00 7/8/14 Disturbed pond cypress slough M 2 0.00 7/8/14 Disturbed pond cypress slough M 3 0.34 7/8/14 Disturbed pond cypress slough A 1 0.00 7/8/14 Disturbed pond cypress slough A 2 0.00 7/8/14 Disturbed pond cypress slough A 3 0.00

53

-2 -1 Date Site Time Chamber mg CH4 - C m h 8/3/14 Disturbed pond cypress slough M 1 0.00 8/3/14 Disturbed pond cypress slough M 2 0.00 8/3/14 Disturbed pond cypress slough M 3 0.00 8/3/14 Disturbed pond cypress slough A 1 0.00 8/3/14 Disturbed pond cypress slough A 2 0.00 8/3/14 Disturbed pond cypress slough A 3 0.00 9/17/14 Disturbed pond cypress slough M 1 0.00 9/17/14 Disturbed pond cypress slough M 2 0.00 9/17/14 Disturbed pond cypress slough M 3 0.00 9/17/14 Disturbed pond cypress slough A 1 0.00 9/17/14 Disturbed pond cypress slough A 2 0.00 9/17/14 Disturbed pond cypress slough A 3 0.00 10/16/14 Disturbed pond cypress slough M 1 0.00 10/16/14 Disturbed pond cypress slough M 2 9.66 10/16/14 Disturbed pond cypress slough M 3 0.00 10/16/14 Disturbed pond cypress slough A 1 0.00 10/16/14 Disturbed pond cypress slough A 2 0.00 10/16/14 Disturbed pond cypress slough A 3 0.00 11/12/14 Disturbed pond cypress slough M 1 0.00 11/12/14 Disturbed pond cypress slough M 2 0.00 11/12/14 Disturbed pond cypress slough M 3 0.00 11/12/14 Disturbed pond cypress slough A 1 0.00 11/12/14 Disturbed pond cypress slough A 2 0.00 11/12/14 Disturbed pond cypress slough A 3 0.00 12/11/14 Disturbed pond cypress slough M 1 0.00 12/11/14 Disturbed pond cypress slough M 2 0.00 12/11/14 Disturbed pond cypress slough M 3 0.00 12/11/14 Disturbed pond cypress slough A 1 0.00 12/11/14 Disturbed pond cypress slough A 2 0.00 12/11/14 Disturbed pond cypress slough A 3 0.00

54

Methane fluxes for the different samplings in the Disturbed cypress dome 1.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 1/20/14 Disturbed cypress dome 1 M 1 0.00 1/20/14 Disturbed cypress dome 1 M 2 0.00 1/20/14 Disturbed cypress dome 1 M 3 0.00 1/20/14 Disturbed cypress dome 1 A 1 0.00 1/20/14 Disturbed cypress dome 1 A 2 0.00 1/20/14 Disturbed cypress dome 1 A 3 0.00 2/25/14 Disturbed cypress dome 1 M 1 0.00 2/25/14 Disturbed cypress dome 1 M 2 0.00 2/25/14 Disturbed cypress dome 1 M 3 -1.14 2/25/14 Disturbed cypress dome 1 A 1 0.00 2/25/14 Disturbed cypress dome 1 A 2 0.00 2/25/14 Disturbed cypress dome 1 A 3 0.00 4/14/14 Disturbed cypress dome 1 M 1 0.00 4/14/14 Disturbed cypress dome 1 M 2 0.00 4/14/14 Disturbed cypress dome 1 M 3 0.00 4/14/14 Disturbed cypress dome 1 A 1 0.00 4/14/14 Disturbed cypress dome 1 A 2 -0.80 4/14/14 Disturbed cypress dome 1 A 3 0.00 6/9/14 Disturbed cypress dome 1 M 1 0.00 6/9/14 Disturbed cypress dome 1 M 2 0.00 6/9/14 Disturbed cypress dome 1 M 3 0.00 6/9/14 Disturbed cypress dome 1 A 1 0.00 6/9/14 Disturbed cypress dome 1 A 2 -1.65 6/9/14 Disturbed cypress dome 1 A 3 0.00 7/8/14 Disturbed cypress dome 1 M 1 0.00 7/8/14 Disturbed cypress dome 1 M 2 0.00 7/8/14 Disturbed cypress dome 1 M 3 0.00 7/8/14 Disturbed cypress dome 1 A 1 0.00 7/8/14 Disturbed cypress dome 1 A 2 0.00 7/8/14 Disturbed cypress dome 1 A 3 0.00

55

-2 -1 Date Site Time Chamber mg CH4 - C m h 8/3/14 Disturbed cypress dome 1 M 1 0.00 8/3/14 Disturbed cypress dome 1 M 2 0.00 8/3/14 Disturbed cypress dome 1 M 3 0.00 8/3/14 Disturbed cypress dome 1 A 1 0.00 8/3/14 Disturbed cypress dome 1 A 2 0.00 8/3/14 Disturbed cypress dome 1 A 3 0.00 9/17/14 Disturbed cypress dome 1 M 1 0.00 9/17/14 Disturbed cypress dome 1 M 2 0.00 9/17/14 Disturbed cypress dome 1 M 3 0.00 9/17/14 Disturbed cypress dome 1 A 1 0.00 9/17/14 Disturbed cypress dome 1 A 2 0.00 9/17/14 Disturbed cypress dome 1 A 3 0.00 10/16/14 Disturbed cypress dome 1 M 1 0.00 10/16/14 Disturbed cypress dome 1 M 2 0.00 10/16/14 Disturbed cypress dome 1 M 3 0.00 10/16/14 Disturbed cypress dome 1 A 1 0.00 10/16/14 Disturbed cypress dome 1 A 2 0.00 10/16/14 Disturbed cypress dome 1 A 3 0.00 11/12/14 Disturbed cypress dome 1 M 1 0.00 11/12/14 Disturbed cypress dome 1 M 2 0.00 11/12/14 Disturbed cypress dome 1 M 3 0.00 11/12/14 Disturbed cypress dome 1 A 1 0.00 11/12/14 Disturbed cypress dome 1 A 2 0.00 11/12/14 Disturbed cypress dome 1 A 3 0.00 12/11/14 Disturbed cypress dome 1 M 1 0.00 12/11/14 Disturbed cypress dome 1 M 2 0.00 12/11/14 Disturbed cypress dome 1 M 3 0.00 12/11/14 Disturbed cypress dome 1 A 1 0.00 12/11/14 Disturbed cypress dome 1 A 2 0.00 12/11/14 Disturbed cypress dome 1 A 3 0.00

56

Methane fluxes for the different samplings in the Disturbed cypress dome 2.

M=morning, A=afternoon

-2 -1 Date Site Time Chamber mg CH4 - C m h 6/9/14 Disturbed cypress dome 2 M 1 0.00 6/9/14 Disturbed cypress dome 2 M 2 0.00 6/9/14 Disturbed cypress dome 2 M 3 0.00 6/9/14 Disturbed cypress dome 2 A 1 0.00 6/9/14 Disturbed cypress dome 2 A 2 0.96 6/9/14 Disturbed cypress dome 2 A 3 0.00 7/8/14 Disturbed cypress dome 2 M 1 0.00 7/8/14 Disturbed cypress dome 2 M 2 0.00 7/8/14 Disturbed cypress dome 2 M 3 0.00 7/8/14 Disturbed cypress dome 2 A 1 0.00 7/8/14 Disturbed cypress dome 2 A 2 0.00 7/8/14 Disturbed cypress dome 2 A 3 0.00 8/3/14 Disturbed cypress dome 2 M 1 0.00 8/3/14 Disturbed cypress dome 2 M 2 0.00 8/3/14 Disturbed cypress dome 2 M 3 0.00 8/3/14 Disturbed cypress dome 2 A 1 0.00 8/3/14 Disturbed cypress dome 2 A 2 0.00 8/3/14 Disturbed cypress dome 2 A 3 0.00 9/17/14 Disturbed cypress dome 2 M 1 0.00 9/17/14 Disturbed cypress dome 2 M 2 0.00 9/17/14 Disturbed cypress dome 2 M 3 0.00 9/17/14 Disturbed cypress dome 2 A 1 0.00 9/17/14 Disturbed cypress dome 2 A 2 0.00 9/17/14 Disturbed cypress dome 2 A 3 0.00 10/16/14 Disturbed cypress dome 2 M 1 0.00 10/16/14 Disturbed cypress dome 2 M 2 0.00 10/16/14 Disturbed cypress dome 2 M 3 0.00 10/16/14 Disturbed cypress dome 2 A 1 0.00 10/16/14 Disturbed cypress dome 2 A 2 0.00 10/16/14 Disturbed cypress dome 2 A 3 0.00 11/12/14 Disturbed cypress dome 2 M 1 0.00 11/12/14 Disturbed cypress dome 2 M 2 0.00 11/12/14 Disturbed cypress dome 2 M 3 0.00

57

-2 -1 Date Site Time Chamber mg CH4 - C m h 11/12/14 Disturbed cypress dome 2 A 1 0.00 11/12/14 Disturbed cypress dome 2 A 2 0.00 11/12/14 Disturbed cypress dome 2 A 3 0.00 12/11/14 Disturbed cypress dome 2 M 1 0.00 12/11/14 Disturbed cypress dome 2 M 2 0.00 12/11/14 Disturbed cypress dome 2 M 3 0.00 12/11/14 Disturbed cypress dome 2 A 1 0.00 12/11/14 Disturbed cypress dome 2 A 2 0.00 12/11/14 Disturbed cypress dome 2 A 3 0.00