Primary Producers and Nutrient Concentrations: Marsh platform primary producer biomass and diversity as well as benthic microalgal biomass are quantified along with phytoplankton biomass in the channels adjacent to the marsh. Additionally, marsh soil and adjacent channel water are characterized in terms of salinity, temperature, pH, and carbon and nutrient concentrations.

Microbiology: Soil cores, sediment cores, and water samples are collected for analysis of relative and quantitative abundances for bacterial, archaea, and fungi (referred to as total microbial diversity), total environmental DNA, and microbial biomass. Data describe changes in microbial diversity, relative abundances, and community structure that may be similar or different among the sampled natural and created marshes.

Infaunal benthos: Infaunal benthos (> 0.5 mm) will be collected using core samples, preserved, and identified to lowest possible taxon. From these fundamental data, there will be a determination of mean species richness, abundance, diversity, and estimates of biomass for food web structure.

Macroinvertebrates: Epibenthic invertebrates (crabs, bivalves, and gastropods) will be collected, near the core samples and at the marsh edge. Specifically, density analysis will focus on three representative intertidal macroinvertebrates, Fiddler crabs, marsh periwinkles, and Gulf ribbed mussels. Crab burrow density is used as a proxy for crab abundance with burrow diameter as a proxy for crab size.

Aquatic and terrestrial insects and spiders: Terrestrial arthropods (insects and spiders) are sampled on plots using sweep nets. Emergence traps and aquatic net sampling are also incorporated to capture aquatic insects at the marsh edge and platform. Samples are frozen in the field and identified in the laboratory to the species level.

Fish: Marsh fish and nekton composition and abundance (catch per unit effort) are quantified using fyke nets and wire mesh traps. Off-marsh fish and nekton communities will be quantitatively sampled using replicated tows of a 5 m otter trawl behind a small research vessel. All captured specimens are identified to the lowest taxonomic level possible, measured, and subsampled for isotopic content as needed. Otoliths will be extracted from some samples.

Otolith chemistry is used to reconstruct the salinity regime experienced by marsh-resident fish and off-marsh juvenile fish over their lifetimes. An extracted left- or right-side otolith is used for laser ablation and  mass spectrometery to generate a geochemical time series experienced by the fish through its lifetime. The other otolith is used to compare fish growth between natural vs. restored marshes and is estimated using an analysis of daily growth rings, analysis of annual otolith width increments, or use of magnesium (Mg) as a proxy for fish growth.

A range of marsh primary producers, and invertebrate and vertebrate consumers are sampled from the marshes (see Species Abundances and Community Structure, above). Bulk tissue carbon (δ¹³C), nitrogen (δ¹⁵N), and sulfur (δ³⁴S) isotopes, and amino acid-specific stable isotopes (δ¹³C) help identify differences in food-web structure and carbon flow and variation among seasons and relative distance from the WPH siphon.

Primary producers and infauna will be analyzed whole, only muscle tissue will be analyzed for all other consumers. Bulk isotopic samples are analyzed through an interfaced Thermo Delta V Advantage CFIRMS, and δ¹³C CSIA-AA values are obtained via Gas Chromatography-IRMS following acid hydrolysis and derivatization.

Bayesian stable isotope mixing models are applied to estimate the percent contribution of organic matter sources by species, and IsoWeb is used to compare and contrast food web topologies based on bulk isotope data. Consumer trophic levels are calculated using static and scaled trophic enrichment factors and estimates of basal food web sourcesThis approach generates isotopic niche areas and volumes for each species in each food web. Changes in the size, overlap, and position of species and community level niche areas in isotopic space and changes in overall food chain length and carbon source usage are used to infer marsh food web structure and function along salinity gradients and among different-aged created marshes under the influences of the WPH siphon. Amino acid-specific (δ¹³C) values and Bayesian stable isotope mixing models are used to estimate the percent contribution of basal carbon sources that  support a subset of marsh consumers (e.g., penaeid shrimp, periwinkle snails, gulf killifish, and blue crabs). These data identify changes in carbon sources along a salinity gradient and over time in created marshes.

Interactions among species make it difficult to predict the results of restoration efforts from habitat models alone. The strength of species interactions plays an important role in conferring ecosystem stability, with weak trophic interactions tending to increase ecosystem stability and resilience. Quantifying differences in predator-prey interactions between natural and restored marshes provides insight on ecosystem resilience.

Data from the field sampling and biomarker studies are used to parameterize two base Ecopath models (one for natural and one for restored marshes) by modifying the diet matrix of an existing model for nearby Breton Sound trophic diversity/redundancy, a critical component of food web resilience.

Base Ecopath models  are used to identify emergent characteristics for created vs. natural marshes. Proposed metrics are net primary production, system total respiration, total biomass flows and total system throughput. Ecosystem properties of interest are ecosystem maturity, Kempton’s Q metric of biodiversity modified for Ecopath, pelagic:demersal ratio and the evenness of functional group biomass by Shannon’s entropy index. [de Mutsert, K. et al. 2012, Marine and Coastal Fisheries 4: 104–116, https://doi.org/10.1080/19425120.2012.672366]

A dynamic food web model (Ecosim) is used to understand the comparative resilience of natural and restored marsh food webs to different simulated perturbations, including: loss of individual top predator species, loss of species identified as most sensitive to salinity shifts and loss of species identified as critical network nodes (i.e., keystone species) from the Ecopath analysis. Resilience is assessed through percent change in each of the unperturbed components of the model at equilibrium and return time (time to reach 95% of new equilibrium biomass).