Ecological and Biogeochemical Trajectories of Newly Constructed Salt Marshes at Poplar Island
A foundational conceptual model describing the successional trajectories of constructed salt marshes was proposed by Craft et al. [49], based on extensive examinations of 8 pairs of reference and constructed salt marshes ranging in age from 1 to 28 years old. According to this model, properties of salt marsh function directly tied to hydrology develop “almost instantaneously” with the establishment of appropriate hydrology and salt marsh vegetation. These properties include rates of sedimentation and organic carbon and nitrogen accumulation [49]. Platform elevations played a key factor in driving this immediate recovery of salt marsh functions, allowing these newly constructed marshes to rapidly gain elevation from sediment accumulation [49, 103]. Although marshes described in Craft et al. [49] were constructed to elevations lower than equilibrium to allow for inorganic sediment deposition during tidal flooding, more recently due to the accelerating sea level rise, marshes are usually constructed with elevations above equilibrium to aid in elevation gain through increased production. Properties associated with biological and biogeochemical processes like primary production, heterotrophic microbial activity, and marsh vegetation and invertebrate densities reach reference conditions on a scale of 5–15 years [49]. This time frame is theoretically set by the amount of time required to build up minimum inventories (i.e., thresholds) of soil nitrogen to support emergent vegetation (est. ~ 100 g N m− 2) and soil carbon to support heterotrophic activity (est. ~ 1000 g C m− 2; [49]). Finally, according to this model, properties associated with the development of mature salt marsh soils, such as depth-integrated organic carbon and nitrogen inventories, require time scales on the order of 3 or more decades to develop [49]. Comparable studies have found that timeframes for created and restored marshes to attain carbon and nitrogen inventories equivalent to reference conditions may be as fast as 32 years (for organic matter in created marshes on the Gulf Coast of LA; [104]), but frequently take much longer, e.g., 70 to 90 years for carbon and nitrogen pools in created marshes in North Carolina [105], 124 and 54 years for carbon and nitrogen pools, respectively, in created marshes in North Carolina [106], and 100 years for carbon pools in created marshes in Louisiana [107] and restored marshes in the UK [108, 109].
Marshes at Poplar Island share some characteristics with those examined in Craft et al. [49] in terms of geography (i.e., North American mid-Atlantic coast) and dominant vegetation type at low marsh elevations (i.e, S. alterniflora). Yet, the marshes at Poplar Island also differ in several important ways which demonstrably accelerated many of the biological trajectories compared with those examined in Craft et al. [49]. Seven of the eight constructed marshes in Craft et al. [49], and elsewhere [51, 105, 110–114], were constructed using coarse-grained (sandy) material, which is initially low in organic nitrogen and carbon. By contrast, the parent material used to construct marshes at Poplar Island was primarily fine-grained dredged material from the upper Chesapeake Bay, which was initially rich in nitrogen and organic carbon (Soil nitrogen (2.17 mg g− 1), carbon (23.5 mg g− 1), and LOI (6.9%) at 1-year-old marsh; [115]). The parent material, therefore, exceeds the minimum threshold requirements for marsh vegetation growth from the onset [47], rather than requiring 5–15 years to accrue (sensu [49]). There is also no apparent inhibition associated with other initially high constituents such as sulfate and iron concentration [47, 48, 78]. Additionally, the rates of marsh accretion among the fine-grained dredge material cells at Poplar Island match or exceed rates observed in reference marshes, by the second year of vegetation growth [47]. Secondly, the marshes at Poplar Island are constructed within containment dikes (with tidal exchange via channels and inlets), which allows more organic matter from aboveground biomass to be retained within the marsh cells compared to open systems [47]. Thirdly, the marshes at Poplar Island range in size from ~ 12–20 ha, while marshes reported by Craft et al. [49] were 0.2–1.2 ha. According to a meta-analysis of restored and created wetlands, the size of wetlands affects recovery rates of biological structure (i.e. related to the assemblage of vertebrates, macroinvertebrates, and plants) and biogeochemical functions, where larger wetlands recover faster than smaller ones [42]. As a result of these features, aboveground vegetation biomass reached a peak within 2 years after planting [47, 48]. Within approximately this time frame, organic matter accumulation, denitrification, and carbon sequestration rates also reached equivalency with reference marshes, despite supporting lower belowground biomass [47, 48].
Here we reported trajectories of several summer porewater constituents across the chronosequence, which all achieved reference conditions, though at variable rates. Dissolved inorganic carbon (DIC) concentrations declined somewhat more slowly than the other porewater constituents measured, reaching reference conditions within approximately 10 years. There was also one marsh (Cell 3D) that exhibited elevated values that did not match the temporal trend and is further discussed below. The decrease in DIC concentrations could be a result of declining respiration, but we suggest this is unlikely given the accumulation of organic matter content over time. Instead, we suggest this trend is likely driven by an increase in hydraulic conductivity over time, which would allow for greater porewater flushing and therefore lower DIC concentrations across the chronosequence. During salt marsh development, as organic matter increases and bulk density decreases, hydraulic conductivity and porosity typically increase [104, 116, 117].
As reported by Cornwell et al. [78], porewater NH4+ concentrations were initially very high (0.22–1.44 mM) but declined to reference conditions in all marshes (0.02–0.05 mM) within the 3 year of plant establishment, consistent with NH4+ uptake by emergent marsh vegetation at depths of < 15 cm during their growing season [48]. This contrasts with porewater NH4+ concentrations deeper in the parent material below the rooting depth which tend to remain high for longer periods of time (e.g., 0.36 mM and 0.16 mM at 4 years and 8 years, respectively, at > 20 cm ). This prolonged source of reactive nitrogen is likely accessible to the vegetation by upward diffusion [118].
The molar ratio of sulfate to chloride declined to reference levels within 3 years. Initially high concentrations of sulfate likely reflected the oxidation of iron sulfide minerals during the drying/dewatering of the dredged material, and the rapid decline could be accounted for by diffusion of sulfate to tidal flood water as well as sulfate reduction occurring at anoxic depths in the soils [78, 119].
Trajectories of Microbial Community Structure
Characterizing the temporal shifts in microbial diversity and community composition as constructed salt marshes mature may provide insight into successional changes in their biogeochemical function. Based on our space-for-time substitution approach using newly constructed S. alterniflora-dominated salt marshes, we found that alpha diversity (reported as Faith’s PD and Shannon diversity) increased rapidly in the transitional and parent material depth horizons following the planting of marsh vegetation and establishment of tidal inundation, with both indices achieving reference equivalency within the first 3 years. By contrast, in the surface soils of fine-grained dredge material marshes, alpha diversity was initially relatively high and remained unchanged across the chronosequence. These observations likely reflect less specialization of microbial communities associated with greater resource availability (i.e, oxygen and labile organic carbon; [120, 121]), and negligible dispersal limitation in the surface soils, which can affect microbial communities at depth [122]. We also observed that the microbial community structure was on a trajectory towards reference condition over time, rather than heading towards a new and/or alternative state. These time scales were 24 years in the surface soils, and 30–67 years in the subsurface soils, which are near or within the range reported for the development of carbon and nitrogen soil inventories (i.e., 32–124 years, as described above). We caution that based on the records available (chronosequence of 16 years) we assumed that the rate of change was linear, but longer timescales could reveal a curvilinear relationship not captured in our estimates. Nevertheless, there is a general trend of increasing community similarity toward reference marshes with age (except for the one marsh discussed), which we suggest likely reflects the development of complex organic matter soils that are becoming increasingly similar to natural reference marshes.
Several of the identified important microbial predictor taxa have putative involvement in sulfur cycling, suggesting the development of complex soil sulfur cycling over time in these marshes. Although rapid decreases in sulfate were observed in marsh soils across the chronosequence, concentrations remained sufficiently high to support sulfate reduction. At all depth horizons, there were multiple predictor taxa affiliated with Desulfobacterota, which are likely sulfate reducers [123, 124]. In the surface soil, the genus-level Sva0081 sediment group increased across the chronosequence, which have been identified as fast-growing micro-aerophilic sulfate reducers that can oxidize both acetate and hydrogen [125, 126]. In the transitional horizon, there were several predictor taxa affiliated with Desulfobacterota, of which most increased over time, including members of the genus Desulfatiglans, Desulfobacca, SEEP-SRB1, SEEP-SRB2, and unidentified members of the family Dissulfuribacteraceae. Desulfobacca is described as an acetate-degrading sulfate reducer [127], and SEEP-SRB groups are described as sulfate reducers, some of which can form consortia with Archaea to enable anaerobic oxidation of methane [128, 129]. In the parent material horizon, increases were observed among sulfate-reducing members of the genus Desulfatiglans, SEEP-SRB2, and unidentified members of the family Dissulfuribacteraceae, while decreases were observed in unidentified members of the family Desulfocapsaceae and genus MSBL7 (family Desulfurivibrionaceae), which have been described as specializing in sulfur-disproportionating metabolisms [130, 131]. Several additional predictor taxa affiliated with Chloroflexi and Firmicutes increased across the chronosequence which furthermore suggested an increasing role for fermentation and possibly organic sulfur cycling. Chloroflexi are putatively involved in carbon and sulfur cycles [132, 133]. While several predictor taxa among Chloroflexi decreased in surface soils (Caldilineaceae and JG30-KF-AS9), numerous taxa affiliated to this phylum increased in the subsurface horizons, including unclassified members of the classes Anaerolineae and Dehalococcoidia. Recent work has revealed that members of these classes have genetic repertoires indicative of high metabolic plasticity with putative roles in deep sea and benthic sulfur cycling, including genes involved in organosulfur compound degradation and sulfite oxidation [132], as well as possible sulfate reduction [134, 135]. Similarly, in the transitional and parent material horizons, members of the class Clostridia (Firmicutes) increased across the chronosequence, which are broadly saprophytic fermenters and have diverse metabolic abilities including potentially iron oxide reduction [68, 136].
Taken together, the observed community shifts in the subsurface horizons suggest a proliferation of microbes involved in primary degradation pathways including fermentation, as well as increased sulfate reduction, with sufficient niche space for multiple sulfate reducers to concurrently proliferate and potentially support complex sulfur cycling (e.g., sulfurization of organic matter). Increases in the relative importance of fermentation and sulfate reduction could be expected based on the stimulation of anaerobic respiration associated with DOC supplied through roots during the growing season, and decaying root biomass following senescence [119, 137]. Sulfate reduction in brackish salt marshes may account for 50 to 95% of the total anaerobic respiration (e.g., Jack Bay, reported in [138]). Another significant proportion of respiration in salt marshes may proceed via iron respiration [138], which would be best quantified with metagenomic and metatranscriptomic sequencing to examine functional gene abundance and activity.
Nevertheless, despite microbial community evidence suggesting a possible increase in sulfate reduction, previous reports found free sulfide to be non-detectable in the porewaters of these marshes across the chronosequence, with a single exception at Cell 3D, which is further described below [139]. Any sulfide produced is therefore likely rapidly removed via a combination of iron sulfide mineral formation and/or oxidation. We also observed an increase in the predictor taxon Thiogranum (family Ectothiorhodospiraceae) in surface soils, which are described as obligately chemolithoautotrophic sulfur oxidizer [140], suggesting that at least some of the sulfide may be recycled by microbial oxidation.
In transitional and parent material horizons, large monotonic increases were also observed among Archaea. Small increases were observed within the family Geothermarchaeaceae (Crenarchaeota), which are affiliated with Nitrososphaeria, a class of putative ammonia-oxidizing archaea [141], suggesting potential increases in nitrogen cycling in the subsurface horizons. The largest increases across the chronosequence among all Archaea, however, were observed among unidentified Bathyarchaea and Crenarchaeota. At both the transitional and parent material horizons, unidentified Bathyarchaea increased from ~ 0.16% in the 1-year-old marsh (Cell 5AB) up to ~ 7% in the 14-year-old marsh (Cell 3D) and 16-year-old marsh (Cell 4D) and ~ 9% at the reference marsh. Although their functional role in salt marsh soils is uncertain, these archaea are potentially acetogenic and/or methanogenic [142]. Concurrently across the chronosequence, in the parent material horizon, increases were observed among Methanosaeta (order Methanosarcinales), which are obligate acetoclastic methanogens [134, 143]. Methanosaeta commonly co-occurs with sulfate reducers and fermenters [134, 143] and/or in syntrophic association with acetate-producing Bathyarchaea [144]. Concurrent with the development of subsurface anaerobic metabolisms, there was evidence for the development of methane oxidizers in surface soils. In the surface horizon, none of the predictor taxa driving the community shifts were affiliated with Archaea, which includes all known methanogens. Instead, there were increases across the chronosequence among aerobic methanotrophic or methylotrophic taxa affiliated with Gammaproteobacteria. Specifically, there were increases in Methylocaldum, a type Ib aerobic methane oxidizer [145, 146]; Methyloparacoccus, an obligate utilizer of methane or methanol as sole carbon and energy sources [147]; genus-level IheB2-23 clade and unidentified members of the family Methylomonadaceae, a type Ia methanotroph [148]; and an unidentified member of the family B1-7BS, a probable methylotroph based on a newly described Candidatus Methylophosphatis from this family [149]. In salt marshes, increases in methane production have been reported as a consequence of slowly accumulating inventories of organic matter [27, 49, 150]. These observations suggest that as methane production is potentially increasing at subsurface depths across the chronosequence, a biological filter comprised of aerobic methane- and methyl oxidizers were concurrently developing in the surface soils.
The use of amplicon sequencing is a valuable first step in assessing the trajectories of microbial community structure over time and allowed us to infer the potential development of some microbe-mediated processes. However, to accurately understand how functional genes are shifting over time towards reference conditions, additional tools such as metagenomic sequencing will be necessary. Geochemical measurements to characterize rates of methane and sulfur cycling are also worthy of further investigation.
Although differences in soil texture, initial nutrient content, and root depth distribution were observed between areas with different parent material types (i.e., fine-grained versus sandy material), counter to expectation, there were no detectable differences in the microbial community composition between the parent materials. This was somewhat surprising since it is well-established that soil texture influences water content, nutrient retention, and oxygen penetration of soils, which in turn all play an important role in structuring microbial communities [43, 151, 152]. The lack of a detectable difference may have been due to the fact that the sandy areas were primarily limited in distribution. In Cell 3C, the sandy parent material was applied as a lens over fine-grained dredge material, and in Cell 5AB the sandy areas were very limited in size. Within these areas of Cells 3C and 5AB, the sandy parent material receives upwardly and possibly laterally diffusing ammonium from the underlying fine-grained dredge material [78], which may be minimizing the differences in the microbial communities between the two parent material types. Additionally, our sample size for the sandy areas was smaller than the fully fine-grained dredge material areas, limiting our statistical power.
Microbes as Bioindicators of Excessive Inundation
Interestingly, the surface soil microbial composition and DIC concentrations of the 14-year-old marsh (Cell 3D) did not follow the same trajectory as the other marshes. Moreover, past surveys found detectable sulfide concentrations in the porewater only at this marsh [139]. These results suggest that marsh conditions were more anaerobic and had less hydraulic conductivity compared to the reference and similarly aged marshes. This was most likely attributed to more prolonged periods of tidal inundation. Indeed, the low marshes within Cell 3D were initially graded to a lower elevation range (0.37–0.55 m above Mean Lower Low Water; MLLW) compared to the cells constructed subsequently (0.48–0.66 m above MLLW). Consequently, this marsh has not been gaining elevation at the same pace as the other marsh cells examined in this study. While limited areas with clumps of healthy vegetation are gaining elevation, bare areas that previously experienced diebacks have been losing elevation [153]. These bare areas within Cell 3D are exhibiting signs of inundation stress, including ponding, creek bank erosion, thinning of vegetation, hummock formation, and migration of low marsh plant, S. alterniflora, into the zones built as a high marsh platform [153]. Other marshes exhibiting these traits of marsh degradation and instability tend to also exhibit an accumulation of porewater sulfide and corresponding inhibition of nitrogen uptake and primary production of the emergent vegetation and therefore decline in vertical accretion [21, 103, 154, 155].
Our study therefore additionally identified that microbial community composition may be an effective bioindicator of marsh inundation period and by extension marsh health in the context of sea level rise. We observed that the surface soil samples collected from Cell 3D (14-year-old) exhibited an elevated relative abundance of the Bacterial phyla Desulfobacterota, Chlorofexi, and Latescibacterota, and the Archaeal phyla Asgardarchaeota, Thermoplasmatota, and Crenarchaeota, relative to the other marsh cells. These taxa are known to be predominantly or exclusively anaerobic. The surface soil samples also exhibited a lower relative abundance of Bacteroida, Planctomycetota, Alphaproteobacteria, and Gammaproteobacteria, which are typically aerobic. Other recent studies in salt marshes have similarly observed decreases in the relative abundance of aerobic taxa within Bacteroida and increases in the relative abundance of anaerobic sulfate-reducing bacteria in unvegetated sediments (as a result of sudden vegetation dieback) with significantly elevated water content when compared to healthy vegetated patches in a S. alterniflora dominant salt marsh [156]. Likewise, Gao et al. [157] tested the impacts of the inundation period on salt marsh soils in mesocosm experiments and found that greater soil water content, due to lower platform elevation, was associated with higher relative abundances of anaerobic taxa and lower relative abundances of aerobic taxa. Our analysis suggests that, with further testing and optimization, monitoring of microbial communities could be an efficient tool to assess excessive inundation in salt marshes, and potentially to assess stability following thin-layer application to supplement marsh elevation [32, 33, 158].