Practical Guide to Measuring Wetland Carbon Pools and Fluxes.

Wetlands cover a small portion of the world, but have disproportionate influence on global carbon (C) sequestration, carbon dioxide and methane emissions, and aquatic C fluxes. However, the underlying biogeochemical processes that affect wetland C pools and fluxes are complex and dynamic, making measurements of wetland C challenging. Over decades of research, many observational, experimental, and analytical approaches have been developed to understand and quantify pools and fluxes of wetland C. Sampling approaches range in their representation of wetland C from short to long timeframes and local to landscape spatial scales. This review summarizes common and cutting-edge methodological approaches for quantifying wetland C pools and fluxes. We first define each of the major C pools and fluxes and provide rationale for their importance to wetland C dynamics. For each approach, we clarify what component of wetland C is measured and its spatial and temporal representativeness and constraints. We describe practical considerations for each approach, such as where and when an approach is typically used, who can conduct the measurements (expertise, training requirements), and how approaches are conducted, including considerations on equipment complexity and costs. Finally, we review key covariates and ancillary measurements that enhance the interpretation of findings and facilitate model development. The protocols that we describe to measure soil, water, vegetation, and gases are also relevant for related disciplines such as ecology. Improved quality and consistency of data collection and reporting across studies will help reduce global uncertainties and develop management strategies to use wetlands as nature-based climate solutions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13157-023-01722-2.

Dirt cheap: an experimental test of controls on resource exchange in an ectomycorrhizal symbiosis.

To distinguish among hypotheses on the importance of resource-exchange ratios in outcomes of mutualisms, we measured resource (carbon (C), nitrogen (N), and phosphorus (P)) transfers and their ratios, between Pinus taeda seedlings and two ectomycorrhizal (EM) fungal species, Rhizopogon roseolus and Pisolithus arhizus in a laboratory experiment. We evaluated how ambient light affected those resource fluxes and ratios over three time periods (10, 20, and 30 wk) and the consequences for plant and fungal biomass accrual, in environmental chambers. Our results suggest that light availability is an important factor driving absolute fluxes of N, P, and C, but not exchange ratios, although its effects vary among EM fungal species. Declines in N : C and P : C exchange ratios over time, as soil nutrient availability likely declined, were consistent with predictions of biological market models. Absolute transfer of P was an important predictor of both plant and fungal biomass, consistent with the excess resource-exchange hypothesis, and N transfer to plants was positively associated with fungal biomass. Altogether, light effects on resource fluxes indicated mixed support for various theoretical frameworks, while results on biomass accrual better supported the excess resource-exchange hypothesis, although among-species variability is in need of further characterization.

Brown meets green: light and nutrients alter detritivore assimilation of microbial nutrients from leaf litter.

In aquatic detrital-based food webs, research suggests that autotroph-heterotroph microbial interactions exert bottom-up controls on energy and nutrient transfer. To address this emerging topic, we investigated microbial responses to nutrient and light treatments during Liriodendron tulipifera litter decomposition and fed litter to the caddisfly larvae Pycnopsyche sp. We measured litter-associated algal, fungal, and bacterial biomass and production. Microbes were also labeled with 14 C and 33 P to trace distinct microbial carbon (C) and phosphorus (P) supporting Pycnopsyche assimilation and incorporation (growth). Litter-associated algal and fungal production rates additively increased with higher nutrient and light availability. Incorporation of microbial P did not differ across diets, except for higher incorporation efficiency of slower-turnover P on low-nutrient, shaded litter. On average, Pycnopsyche assimilated fungal C more efficiently than bacterial or algal C, and Pycnopsyche incorporated bacterial C more efficiently than algal or fungal C. Due to high litter fungal biomass, fungi supported 89.6-93.1% of Pycnopsyche C growth, compared to 0.2% to 3.6% supported by bacteria or algae. Overall, Pycnopsyche incorporated the most C in high nutrient and shaded litter. Our findings affirm others' regarding autotroph-heterotroph microbial interactions and extend into the trophic transfer of microbial energy and nutrients through detrital food webs.

Functional importance and diversity of fungi during standing grass litter decomposition.

Although microbial participation in litter decomposition is widely known within terrestrial soils, the role and significance of microorganisms during the aerial standing litter phase of decomposition remains poorly investigated. We examined the fungi inhabiting standing leaf litter of Schizachyrium scoparium and Schizachyrium tenerum in a Longleaf Pine savanna ecosystem and estimated their contribution to litter decomposition. We identified fungal phylotypes associated with leaf litter and quantified leaf C mass loss, fungal biomass production, and microbial respiration during decomposition. These data were used to construct budgets estimating C flow into and through fungi. Significant losses in S. scoparium (55%) and S. tenerum (67%) leaf C mass were observed during standing decomposition along with concomitant increases in fungal biomass, which reached a maximum of 36 and 33 mgC/g detrital C, respectively. Cumulative fungal production during decomposition totaled 99 ± 6 mgC/g initial detrital C in S. scoparium and 73 ± 5 mgC/g initial detrital C in S. tenerum, indicating that 18 and 11% of the litter C was converted into fungal biomass, respectively. Corresponding estimates of cumulative fungal respiration totaled 106 ± 7 and 174 ± 11 mgC/g initial detrital C in S. scoparium and S. tenerum, respectively. Next generation sequencing identified several fungal phylotypes, with the majority of sequences belonging to the Ascomycota (Dothideomycetes) and Basidiomycota (Agaricomycetes). Fungal phylotypes were similar between litter species and changed over time, showing a successional pattern. These findings extend our understanding of fungal processes to standing litter in terrestrial ecosystems, and highlight the quantitative importance of fungi in C cycling processes.

Filter-feeders have differential bottom-up impacts on green and brown food webs.

Nutrient recycling by consumers can strongly impact nutrient availability for autotrophic and heterotrophic microbes, thus impacting functions such as primary production and decomposition. Filter-feeding freshwater mussels form dense, multispecies assemblages in aquatic ecosystems and have been shown to play a critical role in nutrient cycling. Mussel excretion can enhance benthic primary production and influence algal species composition. However, the role of mussels in brown or detritus-based food webs and species-specific differences has received considerably less attention. Here, using mesocosm experiments, we assessed how three species of freshwater mussels that occupy three different phylogenetic tribes influenced benthic algal accrual, ecosystem metabolism, cotton strip decomposition, leaf litter (Acer saccharum) decomposition, and litter-associated fungal biomass measured as ergosterol. Additionally, we measured mussel excretion and biodeposition rates and assessed the stoichiometry (C:N, C:P, and N:P) of the benthic algae, cotton strips, and leaf litter. In comparison to controls without mussels, generally, mussel treatments had higher benthic algal biomass composed of more diatoms, higher gross primary productivity and net ecosystem production rates, and higher cotton strip tensile strength loss, but there was not a difference in ecosystem respiration rates, leaf litter decomposition rates, or fungal biomass. Benthic algae had lower C:N and higher N:P in mussel treatment tanks and cotton strip C:N was lower in mesocosms with mussels. Our results suggest that nutrient regeneration by mussels most strongly regulates green food webs, with some impacts to brown food webs, suggesting that consumers have interactive effects on microbial functioning in freshwaters.

Periphytic algae decouple fungal activity from leaf litter decomposition via negative priming.

1. Well-documented in terrestrial settings, priming effects describe stimulated heterotrophic microbial activity and decomposition of recalcitrant carbon by additions of labile carbon. In aquatic settings, algae produce labile exudates which may elicit priming during organic matter decomposition, yet the directions and mechanisms of aquatic priming effects remain poorly tested. 2. We tested algal-induced priming during decomposition of two leaf species of contrasting recalcitrance, Liriodendron tulipifera and Quercus nigra, in experimental streams under light or dark conditions. We measured litter-associated algal, bacterial, and fungal biomass and activity, stoichiometry, and litter decomposition rates over 43 days. 3. Light increased algal biomass and production rates and increased bacterial abundance 141-733% and fungal production rates 20-157%. Incubations with a photosynthesis inhibitor established that algal activity directly stimulated fungal production rates in the short-term. 4. Algal-stimulated fungal production rates on both leaf species were not coupled to long-term increases in fungal biomass accrual or litter decomposition rates, which were 154-157% and 164-455% greater in the dark, respectively. The similar patterns on fast- vs. slow-decomposing L. tulipifera and Q. nigra, respectively, indicated that substrate recalcitrance may not mediate priming strength or direction. 5. In this example of negative priming, periphytic algae decoupled fungal activity from decomposition, likely by providing labile carbon invested toward greater fungal growth and reproduction instead of recalcitrant carbon degradation. If common, algal-induced negative priming could stimulate heterotrophy reliant on labile carbon yet suppress decomposition of recalcitrant carbon, modifying energy and nutrients available to upper trophic levels and enhancing organic carbon storage or export in well-lit aquatic habitats.

Changes in nutrient stoichiometry, elemental homeostasis and growth rate of aquatic litter-associated fungi in response to inorganic nutrient supply.

Aquatic fungi mediate important energy and nutrient transfers in freshwater ecosystems, a role potentially altered by widespread eutrophication. We studied the effects of dissolved nitrogen (N) and phosphorus (P) concentrations and ratios on fungal stoichiometry, elemental homeostasis, nutrient uptake and growth rate in two experiments that used (1) liquid media and a relatively recalcitrant carbon (C) source and (2) fungi grown on leaf litter in microcosms. Two monospecific fungal cultures and a multi-species assemblage were assessed in each experiment. Combining a radioactive tracer to estimate fungal production (C accrual) with N and P uptake measurements provided an ecologically relevant estimate of mean fungal C:N:P of 107:9:1 in litter-associated fungi, similar to the 92:9:1 obtained from liquid cultures. Aquatic fungi were found to be relatively homeostatic with respect to their C:N ratio (~11:1), but non-homeostatic with respect to C:P and N:P. Dissolved N greatly affected fungal growth rate and production, with little effect on C:nutrient stoichiometry. Conversely, dissolved P did not affect fungal growth and production but controlled biomass C:P and N:P, probably via luxury P uptake and storage. The ability of fungi to immobilize and store excess P may alter nutrient flow through aquatic food webs and affect ecosystem functioning.

Discrimination in Degradability of Soil Pyrogenic Organic Matter Follows a Return-On-Energy-Investment Principle.

A fundamental understanding of biodegradability is central to elucidating the role(s) of pyrogenic organic matter (PyOM) in biogeochemical cycles. Since microbial community and ecosystem dynamics are driven by net energy flows, then a quantitative assessment of energy value versus energy requirement for oxidation of PyOM should yield important insights into their biodegradability. We used bomb calorimetry, stepwise isothermal thermogravimetric analysis (isoTGA), and 5-year in situ bidegradation data to develop energy-biodegradability relationships for a suite of plant- and manure-derived PyOM (n = 10). The net energy value (ΔE) for PyOM was between 4.0 and 175 kJ mol(-1); with manure-derived PyOM having the highest ΔE. Thermal-oxidation activation energy (Ea) requirements ranged from 51 to 125 kJ mol(-1), with wood-derived PyOM having the highest Ea requirements. We propose a return-on-investment (ROI) parameter (ΔE/Ea) for differentiating short-to-medium term biodegradability of PyOM and deciphering if biodegradation will most likely proceed via cometabolism (ROI < 1) or direct metabolism (ROI ≥ 1). The ROI-biodegradability relationship was sigmoidal with higher biodegradability associated with PyOM of higher ROI; indicating that microbes exhibit a higher preference for "high investment value" PyOM.

Ectomycorrhizal fungal mycelia turnover in a longleaf pine forest.

Elucidation of the patterns and controls of carbon (C) flow and nitrogen (N) cycling in forests has been hindered by a poor understanding of ectomycorrhizal fungal mycelia (EFM) dynamics. In this study, EFM standing biomass (based on soil ergosterol concentrations), production (based on ergosterol accrual in ingrowth cores), and turnover rate (the quotient of annual production and average standing biomass estimates) were assessed in a 25-yr-old longleaf pine (Pinus palustris) plantation where C flow was manipulated by foliar scorching and N fertilization for 5 yr before study initiation. In the controls, EFM standing biomass was 30 ± 7 g m(-2) , production was 279 ± 63 g m(-2)  yr(-1) , and turnover rate was 10 ± 3 times yr(-1) . The scorched × fertilized treatment had significantly higher EFM standing biomass (38 ± 8 g m(-2) ), significantly lower production (205 ± 28 g m(-2)  yr(-1) ), and a trend of decreased turnover rate (6 ± 1 times yr(-1) ). The EFM turnover estimates, which are among the first reported for natural systems, indicate that EFM are a dynamic component of ecosystems, and that conventional assessments have probably underestimated the role of EFM in C flow and nutrient cycling.

Priming in the microbial landscape: periphytic algal stimulation of litter-associated microbial decomposers.

Microbial communities associated with submerged detritus in aquatic ecosystems often comprise a diverse mixture of autotrophic and heterotrophic microbes, including algae, bacteria, protozoa, and fungi. Recent studies have documented increased rates of plant litter mass loss when periphytic algae are present. We conducted laboratory and field experiments to assess potential metabolic interactions between natural autotrophic and heterotrophic microbial communities inhabiting submerged decaying plant litter of Typha angustifolia and Schoenoplectus acutus. In the field, submerged plant litter was either exposed to natural sunlight or placed under experimental canopies that manipulated light availability and growth of periphytic algae. Litter was collected and returned to the laboratory, where algal photosynthesis was manipulated (light/dark incubation), while rates of bacterial and fungal growth and productivity were simultaneously quantified. Bacteria and fungi were rapidly stimulated by exposure to light, thus establishing the potential for algal priming of microbial heterotrophic decay activities. Experimental incubations of decaying litter with 14C- and 13C-bicarbonate established that inorganic C fixed by algal photosynthesis was rapidly transferred to and assimilated by heterotrophic microbial decomposers. Periphytic algal stimulation of microbial heterotrophs, especially fungal decomposers, is an important and largely unrecognized interaction within the detrital microbial landscape, which may transform our current conceptual understanding of microbial secondary production and organic matter decomposition in aquatic ecosystems.

Alteration of microbial communities colonizing leaf litter in a temperate woodland stream by growth of trees under conditions of elevated atmospheric CO2.

Elevated atmospheric CO(2) can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO(2) would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO(2), and their leaves were incubated in a woodland stream. Elevated-CO(2) treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO(2) treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall gram-positive bacterial sequences.

Nitrogen decreases and precipitation increases ectomycorrhizal extramatrical mycelia production in a longleaf pine forest.

The rates and controls of ectomycorrhizal fungal production were assessed in a 22-year-old longleaf pine (Pinus palustris Mill.) plantation using a complete factorial design that included two foliar scorching (control and 95% plus needle scorch) and two nitrogen (N) fertilization (control and 5 g N m(-2) year(-1)) treatments during an annual assessment. Ectomycorrhizal fungi production comprised of extramatrical mycelia, Hartig nets and mantles on fine root tips, and sporocarps was estimated to be 49 g m(-2) year(-1) in the control treatment plots. Extramatrical mycelia accounted for approximately 95% of the total mycorrhizal production estimate. Mycorrhizal production rates did not vary significantly among sample periods throughout the annual assessment (p = 0.1366). In addition, reduction in foliar leaf area via experimental scorching treatments did not influence mycorrhizal production (p = 0.9374), suggesting that stored carbon (C) may decouple the linkage between current photosynthate production and ectomycorrhizal fungi dynamics in this forest type. Nitrogen fertilization had a negative effect, whereas precipitation had a positive effect on mycorrhizal fungi production (p = 0.0292; r (2) = 0.42). These results support the widely speculated but poorly documented supposition that mycorrhizal fungi are a large and dynamic component of C flow and nutrient cycling dynamics in forest ecosystems.

Periphytic photosynthetic stimulation of extracellular enzyme activity in aquatic microbial communities associated with decaying typha litter.

We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular beta-glucosidase activity of litter-associated microbial communities. Activities of beta-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization).

Application of the [3H]leucine incorporation technique for quantification of bacterial secondary production associated with decaying wetland plant litter.

The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 microM) required to achieve saturation-level uptake. We assessed the application of the [(3)H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 microM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [(3)H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [(3)H]leucine into protein exhibited a biphasic saturation curve, with lower apparent K(m) values ranging from 400 nM to 4.2 microM depending on the plant species studied. Upper apparent K(m) values ranged from 1.3 to 59 microM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent K(m) values possibly representing bacterial uptake and higher apparent K(m) values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

Measuring external mycelia production of ectomycorrhizal fungi in the field: the soil matrix matters.

Assessing mycorrhizal fungi production in field settings has been hindered by the inability to measure external mycelia. Recently, external mycelia production was measured in the field using a novel in-growth core technique with acid-washed sand as the in-growth matrix. Here, we tested the assumption that external mycelia production in acid-washed sand is representative of that in native soil. External mycelia production was estimated as the difference in fungal growth between closed (allowing only saprotrophic fungal production) and open (allowing mycorrhizal and saprotrophic fungal production) cores using a factorial design of soil matrices (acid-washed sand vs native) and fertilization treatments (control vs nitrogen (N)) in a longleaf pine (Pinus palustris) plantation. In native soils, the ectomycorrhizal to saprotrophic fungal biomass signal was strong and consistent facilitating the assessment of external mycelia production, which was 300% higher than corresponding rates in acid-washed sand and inversely correlated with soil N. These results demonstrate the efficacy and importance of using native soil as the in-growth matrix to measure ectomycorrhizal fungi external mycelia production in field settings.