Identifying Sources and Oxidation of Methane in Standing Dead Trees in Freshwater Forested Wetlands

Wetlands are large sources of methane (CH4), therefore it is vital to understand the pathways, mechanisms, and sources to anticipate future positive feedbacks to climate change. Plant mediated transport of CH4 from sediment-borne gases is thought to be a major contributor in wetland ecosystems, though few studies have measured standing dead trees (snags). Snags are expected to become more common across the southeastern coast as marshes migrate into freshwater forested wetlands. In this study, our goal was to distinguish the main sources of CH4 being emitted from snags, that is, from soil or in situ origin. The δ2H and δ13C stable isotopic composition from various sources was sampled for source determination. We measured CH4 in various components: emissions from snag stem sides and the soil-atmosphere interface; and concentrations from snag trunk airspace at various heights from ground level (30, 60, and 120 cm), and soil porewater. Potential CH4 production and oxidation in tree cores from two heights (60 and 120 cm) was also measured to examine the potential for CH4 generation or oxidation in stems. We found that CH4 concentrations inside snags (∼10–200 ppm) were 2–50 times higher than atmospheric levels, and generally decreased with increasing stem height. The stable isotopes δ13C and δ2H showed an enrichment from porewater to soils and snag stems. δ13C enrichment of CH4 in snag stems suggests that CH4 is being oxidized as it moves through snags. The tree core vial incubations showed that very few cores produced small amounts of CH4 under anaerobic conditions (n = 5 out of 50), and very few cores oxidized CH4 under more aerobic conditions (n = 5 out of 50). It is possible that a small amount of CH4 is produced in-situ within the heartwood, but it is likely this depends on the density, porosity, and aeration of snags (degree of decay). Our results highlight that high concentrations of CH4 can persist within the heartwood of snags long after initial decay, and that CH4 emitted from snags is largely derived from deep wetland soils and oxidized during transport (via diffusion) throughout the stem of snags.


INTRODUCTION
Wetlands play a major role in the global carbon cycle, especially in methane (CH 4 ) emissions due to the anaerobic conditions created from standing water and saturated soils (Reddy and DeLaune, 2008). CH 4 is an important greenhouse gas because it has a sustained-flux global warming potential 45 times that of carbon dioxide (CO 2 ), averaged over 100 years (Neubauer and Megonigal, 2015).
CH 4 accounts for about 30% of all long-lived greenhouse gases (GHGs) globally, with a mean residence time of 9 years (Whalen, 2005). Wetlands are the largest natural source of CH 4 , contributing as much as 20-30% of the global emissions (Reddy and DeLaune, 2008;Saunois et al., 2020). Freshwater wetlands tend to produce more CH 4 than saline wetlands, because of the abundant sulfate (SO 4 2− ) found in seawater, which is a preferred electron acceptor and reduces methanogenesis (Reddy and DeLaune, 2008). In freshwater wetlands CH 4 is produced mostly through the acetoclastic pathway, using acetate as the substrate (Conrad, 2005). Once CH 4 is produced there are two relevant fates in wetland ecosystems: consumption by methanotrophs or flux to the atmosphere. The amount emitted to the atmosphere is ultimately a balance between CH 4 production and oxidation as it is transported through the soil-water-plant continuum (Reddy and DeLaune, 2008).
CH 4 pathways to the atmosphere include the soil-atmosphere interface, water-atmosphere interface, or plant-mediated transport to the atmosphere (Carmichael et al., 2014). Many studies have shown that both live and dead herbaceous vegetation contribute a significant amount of CH 4 to the atmosphere (Covey and Megonigal, 2019), and more recent studies have focused on the role of woody vegetation as a CH 4 pathway to the atmosphere (Carmichael et al., 2014;Barba et al., 2018;Martinez and Ardón, 2021). Studies from both upland and wetland ecosystems have shown tree stems to be sources of CH 4 to the atmosphere (Covey and Megonigal, 2019;Vargas and Barba, 2019). While upland ecosystems are generally thought of as CH 4 sinks, studies have shown CH 4 emitted through tree stems can be sourced from within the tree due to wood decomposition, or from deep soil layers and transported through the plant continuum (Megonigal and Guenther, 2008;Covey et al., 2012). Tree stem emissions have been found to be about 42.7 Tg yr −1 from the wetland Amazon basin alone, demonstrating the importance of tree stem surfaces (Pangala et al., 2017). While we know more about the source and sink dynamics of soil CH 4 production at broader scales, the biogeochemical pathways of production, oxidation, and pathways of tree stem CH 4 emissions remains poorly understood (Barba et al., 2018).
Fewer studies have focused on CH 4 emissions/pathways from standing dead trees (i.e., snags), which are becoming more common in freshwater forested wetlands across the southeastern United States (White and Kaplan, 2021). These areas with abundant snags are often called ghost forests and are striking features of climate change (Kirwan and Gedan, 2019). White and Kaplan (2021) estimate that 19,480 km 2 of coastal forested wetlands have transitioned to other habitat types from 1986 to 2016, some of which were ghost forests at some point during the transition. Snags have the potential to transport soil produced CH 4 through the root and plant tissue cells to the atmosphere, but CH 4 emitted could also be produced internally due to decomposition (Carmichael and Smith, 2016;Carmichael et al., 2017). As the climate changes, ghost forests are projected to expand in area (Kirwan and Gedan, 2019), and therefore could emit a substantial amount of greenhouse gases to the local and regional carbon cycle (Martinez and Ardón, 2021). It is important to understand the emissions of CH 4 from different pathways for better management in response to a changing climate (Barba et al., 2018).
Stable isotopes ratios (δ 2 H-CH 4 and δ 13 C-CH 4 ) are useful tools to attribute carbon from different sources and differentiate between gas transport mechanisms (Sanci and Panarello, 2015). The isotopic composition of CH 4 will depend on the metabolic pathway (CO 2 reduction vs. acetate fermentation), and potential oxidation by CH 4 oxidizing bacteria (Barba et al., 2018). Microbially mediated oxidation of CH 4 leaves residual CH 4 enriched (δ 13 C-CH 4 is more positive). Acetoclastic CH 4 production has δ 13 C ranging −65 to −50‰, and δ 2 H from −400 to −250%, while hydrogenotrophic pathways result in more depleted δ 13 C values ranging from −110 to −60‰, and δ 2 H ranging from −250 to −170‰ (Hornibrook et al., 1997). δ 13 C and δ 2 H values of CH 4 can be altered by secondary processes in anaerobic environments (Hornibrook et al., 2000). Evaporation will lead to observable fractionation, yielding relatively light vapor and δ 2 H-CH 4 rich water left behind (more positive) (Michener and Lajtha, 2007). A recent study showed an enrichment of δ 13 C-CH 4 with increasing tree stem height, estimating that 33% of the CH 4 was oxidized from the lower stem towards the upper stem (Jeffrey et al., 2021). Stable isotopes have not been used to examine sources and potential transformations of CH 4 in snags.
Previously, we showed that snag stem CH 4 fluxes were not as predictable as soil CH 4 fluxes using environmental parameters such as water levels, salinity, and soil sulfate concentrations (Martinez and Ardón, 2021). The disconnect between soil and snag CH 4 drivers was thought to be driven by oxidation taking place within snags. Although soil CH 4 fluxes (5.9 ± 1.9 mg CH 4 m −2 hr −1 ) were 20 times greater than snag CH 4 fluxes (0.3 mg CH 4 m −2 hr −1 ), snag fluxes should still be taken into account given the cumulative GHGs ghost forests stands can potentially contribute.
Our goal in this study was to identify the main sources and pathways of CH 4 emitted from snags in ghost forests by quantifying CH 4 fluxes and concentrations. Stable isotopes, and lab incubation experiments were combined to examine if the CH 4 emitted through the snag stems was soil-derived or produced internally due to heartwood rot. We hypothesized that CH 4 emitted from snag stems was produced in soils, and snags served as conduits to the atmosphere due to the intricate network of open cells within decaying stems. We expected that most of the CH 4 in snags is formed by acetate cleavage (acetoclastic pathway as opposed to CO 2 reduction), therefore we used stable isotope data to examine the degree of oxidation. Lastly, we hypothesized that little CH 4 would be produced from snag tree cores under anaerobic conditions, and CH 4 could be oxidized under higher CH 4 concentrations and aerobic conditions.

Study Site
Our study took place during the growing season in June and July 2019 on the Albemarle Pamlico Peninsula (APP), in eastern North Carolina. We selected five areas: Palmetto-Peartree  (Martinez and Ardón, 2021). The coastal wetlands along the APP include non-riverine swamp forests, pocosins, and freshwater and brackish marshes. The APP has generally low salinity (one to seven parts per thousand) and is subject to frequent flooding from wind tides (Manda et al., 2014). More than 50% of the peninsula is within 1.5 m of mean sea level, making it highly vulnerable to sea level rise and saltwater intrusion (Moorhead and Brinson, 1995). The APP has been drained via extensive artificial canals and ditches to improve the agricultural value of the land (Manda et al., 2014), but these canals also allow saltwater to propagate further inland especially during storms, imperiling nearby vegetation communities (Bhattachan et al., 2019). Saltwater intrusion and increasing flooding events have increased the amount of forest die back in the region, creating extensive ghost forests (Smart et al., 2020).

Snag-Stem and Soil CH 4 Emissions
Five snags were selected at each site (n 25) based on stability (i.e., intact and not at risk of falling). There are different stages of decay for standing dead trees, which can be classified from 1 to 5, with stage 1 being intact and recently deceased while stage 5 being close to falling over (Odion et al., 2011). The snags selected for all sites, except PC, were all pine trees with most of the bark removed classified as stages ranging from 1 to 3. The snags selected at PC were a mixture of pine and cypress trees with most of the bark removed, also ranging from 1 to 3 stages of decay. Snag and soil CH 4 concentrations were measured using clear semi-rigid chambers at various heights ranging from 60-70 cm from ground, depending on optimal sealing conditions ( Figure 1). The snag-stem chambers were designed after Siegenthaler et al. (2016), which is a flexible type of chamber made from a clear, polycarbonate sheet. Soil chambers (30 cm diameter) were installed 1-2 m a way from snags. The soil chamber tops included a vent tube, internal fan (0.003 m 3 /s; Jameco Electronics, Belmont, California, United States), and three sampling ports, two hose barbed connectors for portable gas analyzer tubing (Input/Output air sample) and air-tight tubing (12 inches) with a three-way stopcock for extra gas samples. The soil chambers were inserted 10 cm into the soil and left to stabilize for at least 1 week before taking measurements. The CH 4 in snag and soils chambers was measured using two different methods: Gasmet portable gas analyzer (Gasmet Technologies, Finland) and the syringe method ( Figure 1). The Gasmet is capable of analyzing a variety of greenhouse gases, but this study is focused only on CH 4 . Chambers were attached for 10-20 min (10 min for snags and 20 for soils), and measuring gas concentrations every 20 s using the Gasmet, or 30-40 min measuring every 5 min using the syringe method. The syringe method was used at times when the Gasmet was not optimal due to either weather or battery failures. Gas samples using syringe method were stored in Exetainer vials (Labco-Lampeter, Wales, United Kingdom). Prior to field use, Exetainer vials were evacuated using a vacuum pump connected to a manifold and flushed repeatedly with ultra-pure N 2 gas. Gas samples were stored in vials and run within a 6-week period, which has been shown to maintain sample concentrations (Sturm et al., 2015). CH 4 gases were analyzed using an Agilent gas analyzer (7890 A GC system -Santa Clara, CA) equipped with a methanizer and Flame ionization Detector for CH 4 analysis. Detailed descriptions of the region, site, and methods can be found in Martinez and Ardón (2021). FIGURE 1 | Conceptual diagram of monitoring setup for each site. Gas samples were taken from snag and soil greenhouse gas chambers, and water samples from porewater for dissolved CH 4 concentrations. Red dot on tree chamber is rubber septum which is used when extracting samples with syringe. Snags were drilled at three heights to measure CH 4 concentrations within heartwood of snag. Tree cores were also taken from two heights (60 and 120 cm) for lab incubation experiments to measure potential CH 4 production and oxidation.
Frontiers in Environmental Science | www.frontiersin.org February 2022 | Volume 9 | Article 737379 CH 4 concentration inside snag heartwood were assessed following a similar protocol to Covey et al. (2012). A 1.27 cm diameter drill bit was used to drill to the center of the tree at three heights (30, 60, and 120 cm). After drilling, each hole was plugged using a rubber septum and pierced with a sampling wand connected to the Gasmet to sample for ∼3 min. The air immediately outside the drilled hole was also sampled for comparison and to allow for the higher gas concentration to flow out of the tubing.
Gas samples for stable isotope analysis were sampled once at each site in June 2019 for GR and PP and July 2019 for PC, SQ, and PPP from inside the snag (n 25) and soil (n 25) chambers after chamber incubation using a gas tight syringe. Both snag and soil chambers were modified to sample with a syringe either through a rubber septum (snag chambers) or tubing using a three-way stopcock (soil chambers). Heartwood gas samples were also collected for stable isotope analysis with a syringe through a rubber septum 60 cm from ground (n 25).

Porewater CH 4
Belowground porewater sippers were installed at 30 cm near three snags at each site. Porewater was analyzed to help provide insights into whether CH 4 emitted by stems was produced in soils or groundwater (Barba et al., 2018). Porewater was drawn after snag and soil gas sample collections and measured for δ 13 C-CH 4 , δ 2 H-CH 4 , and CH 4 concentration. CH 4 concentrations were measured using ultra-high purity N 2 using headspace equilibration methods (Sturm et al., 2015). Approximately 6 ml of porewater was injected into a pre-evacuated 12 ml exetainer. Liquid exetainer samples were stored upside down to prevent gas leakage and placed immediately in a cooler in the field. At the end of each field day collection, 6-7 ml of ultra-pure N 2 gas was added to provide over-pressure preventing access of atmospheric air during transport. Samples were wrapped in aluminum foil, stored upside down, and kept in cool refrigerator (4°C) until lab analysis. Samples were taken out of fridge 24 h prior to GC analysis to reach room temperature to allow equilibration of gases in headspace and water column. The volume of the porewater sample and headspace was determined by weighing the exetainer vial. The concentration of gas in the original water sample was determined by calculating the aqueous concentration in water and headspace after equilibration (Helton et al., 2014).
Porewater samples collected for isotopes δ 2 H-CH 4 and δ 13 C-CH 4 were acidified with HCl acid in the field to a pH < 2 and capped with no headspace bubbles. Two sets of porewater samples were collected per sipper for isotopes δ 2 H-CH 4 and δ 13 C-CH 4 , which are analyzed separately. All gas and porewater samples for stable isotope measurements were shipped to UC Davis Stable Isotope Facility. There the stable isotope ratios of carbon (δ 13 C) and hydrogen (δ 2 H) in CH 4 were measured using a ThermoScientific Precon concentration unit interfaced to a ThermoScientific Delta V Plus isotope ratio mass spectrometer (ThermoScientific, Bremen, Germany).

Potential CH 4 Incubations
Snag tree cores were taken (June and July 2019) to provide estimates of CH 4 emissions by incubation to test the potential of wood to produce CH 4 (n 100; PMP and PMO). Tree cores (5 mm diameter × 10-30 cm length) included the bark (if present), sapwood, and heartwood. Heartwood rot/wet wood conditions provide a conducive habitat for microbial CH 4 production, therefore potential CH 4 production (PMP) from incubations could suggest tree decomposition as an important source of CH 4 (Covey et al., 2012;Barba et al., 2018). Tree cores were taken at two heights (120 and 60 cm) from the five sites, to perform wood incubations to measure potential CH 4 production (n 50, 25 per each height). Cores were placed in 120 ml serum vials and flushed with N 2 for 8-10 min. Gas samples were taken from the vial at 0, 12, 24, 48, and 72 h after incubating. A separate empty vial was flushed with N 2 as a field blank to measure potential background concentrations. Snag tree cores from GR and PP were run in June 2019 measuring at time points 0, 12, 24, and 48 h, while snag tree cores from PPP, SQ, and PC sites were run in July 2019 measuring at time points 0, 24, 48, and 72 h. The 12 h time point for July incubation was deemed unnecessary, and we extended the incubation to 72 h to determine if a longer incubation was needed to measure change in CH 4 concentration during treatments.
In addition to measuring potential CH 4 produced under anaerobic conditions, snag cores from similar heights (120 and 60 cm from ground) were incubated under higher concentrations of CH 4 to measure potential CH 4 oxidation (PMO) under aerobic conditions (n 50). Vials were flushed with a gas standard mixture of CO 2 , CH 4 , and N 2 O near ambient levels with a much higher CH 4 concentration. Snag cores from GR and PP were incubated at a CH 4 concentration of ∼45 ppm, while snag cores from PPP, SQ, and PC were incubated at a CH 4 concentration of ∼120 ppm. The difference in CH 4 concentrations was due to difficulties diluting Pure CH 4 (99.999%). CH 4 can potentially be oxidized by methanotrophs and/or nitrifying bacteria which have an affinity for CH 4 (Chan and Parkin, 2001). Gas samples were taken at similar intervals as the potential CH 4 production incubations. An empty vial flushed with the same gas standard mixture was measured as a field blank to assess potential gas leaks or air seepage inside the vial.
We calculated snag tree core CH 4 fluxes, represented as ng g −1 hr −1 , using the volume of the vial (minus the volume of the snag tree core) and the weight of the snag tree core as a substitute for area. Criteria were used to filter data using a minimum detectable concentration difference (MDCD) between the initial and final measurements. The MDCD in this study is based on the detection limits of the GC, which is 20% (or 0.2 ppm) for CH 4 , so |Final CH4 Concentration -Initial CH4 Concentration | > 0.2 (meets MDCD criteria). Observations that did not meet the criteria were not used (marked as NA). The incubation fluxes were calculated using the HMR approach (HMR R package), which uses both linear and nonlinear approaches (Pedersen et al., 2010). Only data with significant correlations (p < 0.1) were kept.

Statistics
Due to the lack of normality in the data, Kruskal-Wallis was used to test differences in stable isotopes (δ 13 C-CH 4 and δ 2 H-CH 4 ) among source types, and heartwood CH 4 concentrations among Frontiers in Environmental Science | www.frontiersin.org February 2022 | Volume 9 | Article 737379 sites, and between heights (30, 60, and 120 cm). Dunn's multiple pairwise comparisons were then used for post-hoc analysis when Kruskal-Wallis showed significant differences, with a Bonferroni p-value adjustment (p < 0.05). Linear regression models were used to assess the relationships between stable isotopes and CH 4 concentrations with all source types (snag chambers, soil chambers, heartwood, and porewater), and also between CH 4 heartwood concentrations and stem height. CH 4 concentration data was log transformed due to lack of normality. All statistical analyses were performed in R program (R Core Team, 2020).

RESULTS
Salinity levels were the lowest at PPP (∼1 ppt) and highest at SQ (∼7 ppt), with PC, PP, and GR in between, increasing in salinity respectively (Table 1). Sulfate concentrations from porewater sippers followed similar patterns as salinity with the lowest concentration in PPP (2.39 mg/L) and highest in SQ (245 mg/L). Air temperature during the sampling period ranged from 27 to 34°C, while soil temperature ranged from 21 to 27°C (Table 1). Water levels during the sampling period were highest at PC (∼6 cm above ground) and lowest at PP (∼20 cm below ground).

Heartwood CH 4
Heartwood CH 4 concentrations differed among sites and between heights (p < 0.01, Figure 2). Concentrations from PC (mean ± SE: 904 ± 415 ppm) were an order of magnitude higher than all other sites, which were on average 13 ppm (p < 0.01, Figure 2). For all sites, CH 4 concentrations decreased with increasing height, with measurements from 30 cm being 2-4 times higher than
Overall, there was enrichment in isotopic signature in both δ 13 C and δ 2 H for all source types with decreasing CH 4 concentrations across all sites (Figures 3, 4), except for δ 13 C from SQ.

Potential CH 4 Incubations
CH 4 concentrations in snag tree core incubations changed very little over time ( Figure 5). The overall number of snag tree incubation cores that had a measurable flux was 10 out of 100 cores taken. From the measurable fluxes, 5 were from the potential CH 4 oxidation (PMO) treatment, while the other five were from the potential CH 4 production (PMP) treatment ( Figure 5). The highest CH 4 flux within the PMP treatment was 2.12 ng g −1 hr −1 for cores 60 cm from ground, while the smallest increase was 0.35 ng g −1 hr −1 for cores 120 cm from ground. One core from 120 cm height produced a small amount of CH 4 under PMO conditions, 0.5 ng g −1 hr −1 . The highest oxidized CH 4 within the PMO treatment was -2.5 ng g −1 hr −1 from 120 cm height core. The sample size was too low to perform any statistical analysis on the incubation flux calculations.

DISCUSSION
Our main goal was to determine the main source of CH 4 found and emitted from snags, and predicted that it was produced in the soil and transported up the stem. We found three lines of evidence to support this hypothesis: 1) there was a decrease in CH 4 concentrations with increasing stem height, 2) the isotopic composition of CH 4 within snags was more enriched in snags relative to porewater, and 3) very little CH 4 was produced or consumed when tree cores were incubated under anaerobic and aerobic conditions, respectively. As expected, most of the CH 4 from snag stems, soil, and porewater was produced through the acetoclastic pathway based on the isotopic signature (δ 13 C: −45 to  Frontiers in Environmental Science | www.frontiersin.org February 2022 | Volume 9 | Article 737379 −60‰). Our results show that high concentrations of CH 4 can persist within the heartwood of snags long after initial decay (stage 1) (Odion et al., 2011), and undergo oxidation before being emitted through the stem. The stable isotopes showed enrichment of both δ 13 C-CH 4 and δ 2 H-CH 4 from porewater, to heartwood, to snag chamber indicating that CH 4 was being oxidized throughout this pathway. Although it is important to note that we did not sample water from the various locations, so while the δ 2 H-CH 4 pattern is consistent with the δ 13 C-CH 4 suggesting oxidation, we cannot rule out other water sources. The tree core vial incubations indicated that small amounts of CH 4 can be produced under anaerobic conditions, about 2.12 ng g −1 hr −1 , but also oxidized under higher CH 4 concentrations (−2.5 ng g −1 hr −1 ). Based on these results, the CH 4 emitted from snags is largely derived from the soil, and progressively oxidized as it passes throughout the stem. It is possible that a small amount of CH 4 is produced in situ within the heartwood, but it is likely this depends on the density, porosity, and aeration status of snags (degree of decay and water content). A recent study in forested wetlands showed that gas diffusion barriers may be what ultimately determines the magnitude of stem CH 4 emissions, which in the case of snags is porosity from decay and wood moisture content (Norwood et al., 2021). Overall, our results are consistent with the hypothesis that CH 4 is primarily produced in soils, and is oxidized as it moves along the soil-stem-atmosphere pathway.
Heartwood CH 4 CH 4 concentrations in heartwood varied across the sites. The CH 4 concentrations at PC were higher (904 ± 415 ppm) because there was standing water year-round, unlike all other sites ( Figure 2; Table 1). When drilling into the heartwood at PC, some snags had a small stream of dripping water from within the tree (personal observation). The high moisture (and likely low oxygen levels) within the snags were conducive to CH 4 production, which would explain why their concentrations were so much higher than all other sites. The mean heartwood CH 4 concentration for the other sites (∼13 ppm) were below those found in snags from a similar region within the APP (100 ppm) (Carmichael et al., 2017). The difference between snags from the Carmichael et al. (2017) study and ours could be due to site differences similar to PC within our own study. The snags from Carmichael et al. (2017) were in standing water yearround.
There was a gradual decrease in CH 4 concentration with increasing stem height (Figure 2), which has been found in other studies when measuring heartwood CH 4 concentrations in live and dead trees (Covey et al., 2016;Carmichael et al., 2017), but this decrease also affects tree stem CH 4 fluxes (Pangala et al., 2017;Jeffrey et al., 2019). The decrease in heartwood CH 4 is likely due to distance from source (CH 4 originating from within soils) and increasing oxidation with increasing stem height, which is also why tree-stem CH 4 flux would be affected (Pangala et al., 2017;Jeffrey et al., 2019). Using a linear regression model, we calculated that CH 4 concentrations inside the heartwood would decrease to 0 near 3.34 m (Eq. 1). Snags across the study sites ranged in height from 3 to 15 m. Our results suggest that snags can be important areas for CH 4 oxidation. It is important to note that measurements were taken during a relatively warm time of year (Table 1), so it is possible that during time periods when the air is cooler, there might be less CH 4 oxidation in the snags (King and Adamsen, 1992).

Stable Isotopes
Stable isotope data from all sites had similar patterns (enrichment from porewater < heartwood < soil chamber < snag chamber) with the exception of δ 13 C from SQ ( Figure 3). As we predicted most of the CH 4 produced from each source: snag chamber, heartwood, soil chamber, and porewater, was generated through the acetoclastic pathway which ranges from δ 13 C ∼ −65 to −50‰, and δ 2 H ∼ −400 to −250%, although a few samples (n 4) were less than δ 13 C −65‰ from porewater, soil, and heartwood samples. One sample from heartwood in PC had really high CH 4 concentrations (∼40,000 ppm) and a very depleted δ 13 C signature (−73‰), suggesting that hydrogenotrophic pathway might happen in stems standing in water. The δ 13 C-CH 4 from various freshwater wetlands have been determined to be within the range of −86‰ to −31‰ (Bréas et al., 2001). A previous study conducted in freshwater wetlands concluded that dissolved CH 4 in porewater at ca. 25−cm depth had δ 2 H values of ∼ -340‰ or less (Hornibrook et al., 2000), which is also within the range of our study. Overall, the δ 13 C and δ 2 H values indicate that most of the CH 4 measured was produced through the acetate fermentation pathway which is common in most freshwater soils (Bréas et al., 2001).
Both δ 2 H and δ 13 C of porewater CH 4 were significantly depleted compared to all other sources (Figures 3, 4). This would indicate that the CH 4 concentrations from porewater samples were closer in composition to that of the anoxic zone, or anoxic ''microsites'', leading to lower δ 2 H and δ 13 C (Sugimoto and Fujita, 2006). Porewater CH 4 concentrations are thought to be the best representation of CH 4 production, before any processes (i.e., diffusion and/oxidation) changes the stable isotope signature (Bréas et al., 2001), and therefore those values were used in this study to assess degree of oxidation in heartwood, snag chamber, and soil chamber. The end product of complete CH 4 oxidation is water vapor, which is also why δ 2 H becomes enriched (heavier), although rates of hydrogen transfer due to organic matter degradation can also affect δ 2 H (Bréas et al., 2001).
Stable isotopes from heartwood and soil chambers were more similar to each other and had wider ranges than snag chambers (Figures 3, 4). Heartwood δ 13 C and δ 2 H were on average similar to the mean porewater δ 13 C and δ 2 H, indicating that the degree of oxidation was less than the pathway from porewater to soil chamber, although there was a wide range for both δ 13 C and δ 2 H (Figures 3, 4). CH 4 oxidation occurred more in samples from snag chambers and were closest to ambient air stable isotopes (−46‰ and −114‰, δ 13 C and δ 2 H respectively), providing further evidence that is consistent with CH 4 continuing to be oxidized as it moves through the snags (Figures 3, 4). A previous study also showed δ 13 C-CH 4 enrichment with increasing stem height in a flooded forested ecosystem, suggesting oxidation during tree stem transport (Jeffrey et al., 2021). This same study also found methane Frontiers in Environmental Science | www.frontiersin.org February 2022 | Volume 9 | Article 737379 oxidizing bacteria (primarily Methylomonas) to be abundant within bark representing an important sink (Jeffrey et al., 2021). We have collected evidence of methanotrophs within snags in GR at ∼60 cm from ground, although the abundance is not yet known (unpublished data; Carmichael et al. In Prep).
The degree of oxidation in our study likely depends on the stage of decay of the snags. Later stages of decay (stages 4-5) likely having more porous stems (lower density) therefore increasing oxidation, whereas earlier stages of decay (stages 1-3) may continue emitting CH 4 due to moist tree stems which are more advantageous to methanogens, similar to the snags measured in this study. The overall positive correlation between δ 2 H and δ 13 C of CH 4 in freshwater wetlands has also been shown in other studies (Sugimoto and Wada, 1995;Sugimoto and Fujita, 2006). Isotopic fractionation, in both δ 13 C and δ 2 H, has been shown to decrease with decreasing CH 4 concentrations, which is what we observed in all sites, except SQ (Figures 3, 4) (Riveros-Iregui & King, 2008). It is possible that the higher salinities and thus higher SO 4 2content in the sediments in SQ inhibited methanogenesis because SO 4 2is a preferential electron acceptor in saturated sediments (Weston et al., 2006). If there is less CH 4 (substrate) in the sediment, then there will be less methanotrophy and less enrichment in δ 13 C signal (Edmonds et al., 2009).

Potential CH 4 Incubations
Tree cores were incubated under two different simulated conditions to further investigate potential production and/or oxidation in snags internally (heartwood and sapwood). Very few (n 10) cores exhibited a measurable difference in CH 4 from the beginning to the end of the incubation ( Figure 5). This suggests that any amount of CH 4 that is present in the heartwood is largely soil derived. Under the PMP treatment, the highest measurable CH 4 flux occurred in cores taken from the base of the tree, while in the PMO treatment the highest measurable CH 4 oxidation flux occurred in cores from 120 cm from base of the tree. It is possible that there are more abundant methanogen communities closer to the base where conditions are more conductive for methanogenesis (i.e., higher moisture content, thus lower O 2 availability), but not as much as deep soil layers where majority of the CH 4 is derived. The fact that there was very little change in CH 4 concentrations throughout both treatments at both heights (PMP and PMO) further demonstrates that the majority of the CH 4 emitted from snag stems is largely soil derived ( Figure 5). A study looking at CH 4 fluxes from stems in the Amazon basin, similarly found that very few of their tree core incubations produced CH 4 , and interpreted those results as support for soil as the source of CH 4 (Pangala et al., 2017).

CONCLUSION
Analysis of greenhouse gas (GHG) fluxes from tree stems and snags is still an emerging field, but studies have shown that this pathway, previously unaccounted for in global budgets, is an important source to consider. Although fewer studies have measured the potential of standing dead trees (i.e. snags) to emit GHGs (Carmichael et al., 2017;Jeffrey et al., 2019;Martinez and Ardón, 2021), they have the potential to transport soil produced CH 4 through the plant tissue cells to the atmosphere, but CH 4 emitted could also be produced internally due to decomposition. Across the southeast coastline, many freshwater forested wetlands are rapidly transitioning to marshes due to increasing flooding and saltwater intrusion, leaving behind many snags, collectively referred to as ghost forests. CH 4 dynamics in ghost forests may be more complicated than previously thought due to the various sources and sinks. Our results show that: 1) the majority of CH 4 in soils of these ghost forests is formed through the acetoclastic pathway; 2) the majority of CH 4 being emitted from snags was likely produced deep in the soil, and 3) snags are important locations of CH 4 oxidation.

PERMISSION TO REUSE AND COPYRIGHT
We give permission for figures, tables, and images to be published under a Creative Commons CC-BY licence.

DATA AVAILABILITY STATEMENT
The datasets analyzed for this study can be found in Dryad Digital Repository https://datadryad.org/stash/dataset/doi:10.5061/ dryad.xksn02vhg.

AUTHOR CONTRIBUTIONS
MM and MA conceived the study and research methods. Material preparation, data collection and data analysis were performed by MM. The first draft of the manuscript was written by MM with feedback and comments from MA and MC. All authors reviewed, edited, and approved the manuscript to its final form.