Salt marshes of the Atlantic and northern Gulf of Mexico coasts of temperate North America are highly productive [1
] and commonly dominated by the smooth cordgrass, Spartina alterniflora
. In these systems, Spartina
grows in extensive, often monotypic stands [1
]. Several other plant species are also present, growing in distinct zones based on edaphic conditions, minor differences in elevation, and interspecific competition [2
]. Semi-diurnal tides maintain moderate levels of interstitial solutes in the sediments of these systems [5
], contributing to their high plant biomass and productivity.
Nitrogen frequently limits primary productivity in salt marshes [9
] and plant-associated diazotrophs provide much of the ‘new’ nitrogen in marsh systems that lack riverine freshwater inputs [12
]. Diazotrophic Bacteria
are very active in the rhizospheres of salt marsh plants [14
] and this activity is tightly coupled to the photosynthetic activity of these macrophytes and to the decomposition of dead plant biomass [14
]. The assemblages of diazotrophs associated with the rhizospheres of salt marsh plants are diverse, mostly novel and mainly belong to the classes α-, β-, γ-, δ-, and ε-Proteobacteria
]. These assemblages consist of mixtures of ubiquitous (non-responsive to season or plant host type), seasonally responsive, and plant host specific organisms [27
]. The assemblages are relatively stable to at least some types of perturbations as long and short-term fertilization and above ground plant biomass removal experiments resulted in only minor changes in the Spartina
rhizosphere diazotroph assemblage [23
]. DNA sequences specific to numerous rhizosphere diazotrophs have been recovered in multiple studies spanning more than a decade and including various marsh conditions [6
]. This indicates that these organisms consistently maintained detectable populations in the rhizospheres.
Hydrology is a major driver of species composition in salt marshes and drought can have a significant effect on the structure and function of these coastal ecosystems [32
]. Increased average temperatures and reduced precipitation lead to prolonged dry conditions and frequent droughts. These conditions have resulted in mass diebacks in Atlantic coast and Gulf coast marshes [33
]. Macro and meiofauna could also be negatively impacted by drought and inadequate tidal flooding [32
], but the effects of these conditions on microbial communities in salt marsh sediments are not known. The impact of increasingly severe conditions might be magnified if the plant associated diazotrophic bacteria are strongly affected due to the importance of these organisms to marsh productivity.
It will become increasingly important to understand how and why diazotroph assemblages change in response to environmental conditions. Global change is predicted to produce rising mean sea levels, increases in average global temperatures, and reduced frequency of precipitation events in some areas [35
]. These changes have negatively affected coastal salt marshes worldwide and are especially important to consider due to the large contributions of these ecosystems to coastal productivity. Interannual variability in mean sea level results in fluctuations in tidal heights, which also negatively affect coastal marshes [37
]. Lowered local sea levels result in insufficient tidal flooding, with concomitant increases in sediment porewater salinities and strong negative impacts on above ground plant productivity. Tidal deficiency displaces marsh plants and changes species diversity when freshwater zones become mesohaline [34
A 4-year study undertaken to investigate how diazotroph assemblages associated with highly stable plant zones varied, and to determine if changes in environmental conditions in the marsh had any effect on assemblage composition captured a period of unusually low tides and two drought events. This provided a rare opportunity to determine the effects of drought on diazotroph assemblages in situ and on the drivers of coastal ecosystem structure and function. The results of this study indicate that portions of the diazotroph assemblage persisted during and after a drought event and significant changes in the plant zone associated assemblages were correlated with abiotic and soil chemistry parameters. These data provide much needed information on the effects of frequent drought events on salt marsh diazotrophy, the foundation of marsh productivity. Increasing frequency of drought has immediate effects on acute marsh dieback which is expected to limit the ability of intertidal marshes to accommodate expected rising sea levels [39
From September 2003 to May 2007 the salt marsh system in Crab Haul Creek Basin, North Inlet, SC went through several dramatic changes. The moderate drought event in combination with the lack of tidal inundation during the growing season of 2004 had a significant effect on the marsh ecosystem that was easily observable. In addition, during the years 2004 and 2005 the mean high water relative to mean sea level was at the lowest level it had been in approximately 10 years [37
]. Droughts lead to substantial changes in saltmarshes, and in the period of this study, this marsh landscape experienced two such events (June–July 2004, and May 2007). The lowered sea level had a great effect on flooding frequency of the marshes, consequently resulting in higher porewater salinity especially during warmer summer months. Increased salinities in turn results in lower plant productivity [38
]. Lack of tidal inundation and drought conditions in the system had lasting effects that were obvious in the porewater chemistry, and the shifts in the composition of the diazotroph assemblages.
4.1. Sediment Chemistry is Somewhat Affected by Environmental Conditions
Porewater chemistry exhibited clear differences among ‘drought’ years and ‘normal’ years. Drought conditions led to significantly higher sediment temperatures, and salinity, and lower pH (Figure 6
), however over the four years of sampling there were also significant similarities in porewater chemistry parameters based on location in the marsh, season, and plant zone. Higher salinity levels in the mid-marsh zones were most likely due to being exposed for longer periods in between tides and evaporation. The high marsh and low marsh salinities were significantly different from that experienced in the mid-marsh, as the high marsh is inundated less frequently, and the low marsh is constantly in contact with the creek. Similar patterns were observed with sediment temperatures; however, pH was significantly lower in the mid-marsh than the high and low marsh environments. There were no significant patterns observed with porewater chemistry except for significant levels of sulfide in the short form Spartina alterniflora
(S) zone and during the 2006 growing season.
Near neutral pH was typical for this system, except during the drought year (2004) and the year immediately following (2005). Drought conditions and a lack of tidal inundation resulted in drained sediments which were more aerated and likely resulted in formation of sulfuric acid from abiotic and microbial oxidation of pyrite and sulfide [74
]. In 2005 pH values were close to neutral but lower than those recorded in all other years in the data set. Concentrations of soluble sulfide were lowest during the drought year, consistent with the increased oxidation of sulfide, however in September 2005 they were exceptionally high. This indicates that either rates of sulfate reduction were also exceptionally high during that growing season or a prolonged accumulation of sulfide occurred in the sediment. High concentrations of sulfide inhibit uptake of ammonium by plants [75
], possibly contributing to the high concentrations of soluble ammonium recorded in all plant zones on that sampling date. These concentrations could also be indicative of high rates of microbial decomposition of dead plant material. Considerable decomposition of dead Spartina
biomass occurs relatively quickly, resulting in losses of up to 61% of dry weight in 23 days [76
]. However, if conditions in the sediment were not conducive to microbial activity, the high concentrations of soluble ions could suggest a delayed response to an increase in dead plant material left over from a stress event. Acute stress events, such as drought, have resulted in mass dieback in southeastern US marshes [33
] and this drought event resulted in large amounts of dieback observed in all zones of this marsh system (Davis, personal observation). High concentrations of ammonium ion and increased aeration of drained sediments (during the drought years) may have increased rates of nitrification resulting in higher concentrations of nitrate and nitrite [78
Throughout this period nitrogen fixation was detected (through ARA activity) and the highest ARA rates recorded in this data set occurred in the growing season of 2005 which was after the drought event. During the drought acetylene reduction activity (a proxy for nitrogen fixation) was not detectable in the high marsh and was lower than normal in the mid-marsh mixed plant zone (co-occurring short form Spartina alterniflora
and Salicornia virginica,
SS), and low marsh S plant zone. However, in the low marsh tall form Spartina alterniflora
(T) plant zone, the only marsh zone that remained wet during the drought period, rates of acetylene reduction were typical for that zone when compared to other dates. Acetylene reduction rates increased to typical levels as conditions in the marsh improved with higher rates observed at the beginning of the growing season. The high rates of acetylene reduction observed in the SV and SS zones are typical for this system [6
4.2. Diazotroph Assemblages
The patterns of clustering in PCA analysis observed for the September 2003 and May 2007 samples were typical for this system. Previous studies observed clustering of samples from plant zones that contained the same dominant macrophyte or were located at a similar elevation in the marsh [6
]. The data obtained in this study indicate that the diazotroph assemblages associated with the rhizospheres of these salt marsh plants are similar to those previously documented. The diazotroph assemblages associated with salt marsh plant rhizospheres are somewhat stable. Only minor changes in the assemblages were observed when long and short-term fertilization and above ground biomass removal experiments were performed [29
]. The drastic difference in the diazotroph assemblages in 2005 and 2006 as observed in DGGE band determination and deemed significant by PCA cluster analysis, indicate that the moderate drought event that occurred in 2004 directly affected these assemblages. The mass mortalities of flora and fauna that were evident across the marsh landscape were immediate, yet the assemblages remained intact and changes were not immediately evident.
Identification of sequences sampled from DGGE gels with DNA from May and September 2005, and May and July 2006, provided some insight into the types of diazotrophs that were present in the assemblages after the drought event. Two of the sequences grouped strongly with presumptive anaerobes, the fermentative δ-proteobacterium, P. carbinolicus
, and the anaerobic photolithoautotroph, C. tepidum
. Seven identical (sensu Venter [60
]) sequences also formed a group with the anaerobic photoautotroph γ-proteobacterium, H. halophila
. The other 12 sequences grouped strongly with presumptive oxygen utilizers, indicating that the assemblages shifted from having an equal representation of presumptive anaerobes and oxygen utilizers [6
]. One of these presumed oxygen utilizing taxa grouped strongly with G. diazotrophicus
, an α-proteobacterium that fixes nitrogen at low pO2
and low pH [80
]. These taxonomic groupings are representative of those previously observed in this salt-marsh system [6
4.3. Persistent Diazotrophs Maintain Marsh Dynamics Post Drought Event
Davis et al. [27
] and Gamble et al. [28
] have determined that the diazotroph assemblages associated with the rhizospheres of salt marsh plants respond to ordinary seasonal changes, however, such a radical shift in the composition of the assemblage has not been seen before. The long-term effects of water deficiency would change the oxic/anoxic transition in soils and could cause dramatic changes in the composition of the microbial community [81
]. Increasing the depth of the oxic layer of the sediment would reduce the strict anaerobe assemblage and decrease rates of anoxic and suboxic processes unique to microorganisms, such as nitrogen fixation. The recovery of sequences affiliated with photoautotrophic anaerobes and oxygen-utilizing diazotrophs that fix nitrogen under low concentrations of oxygen and acidic pH suggests that the diazotrophs present in the post-drought assemblages have maintained detectable populations based on their abilities to resist the drastic changes in edaphic conditions that occurred due to the lack of water. The presence of seasonally responsive diazotrophs suggests that these organisms can maintain low (undetectable by the methods used here) populations under severe environmental conditions, responding rapidly when conditions become conducive for growth. The presumably reduced levels of substrates in the sediment (due to lower plant productivity) could explain the recovery of nifH
sequences similar to those of photoautotrophic species belonging to the presumptively anaerobic portion of the assemblage.
Diazotroph assemblages were sorted into 5 distinct groups based on their dissimilarities, and ordination analysis indicates how the assemblage changed over seasons and differed between plant zones (Figure 10
b,c and Figure S3
). Assemblage group 1 was the most stable group as it was present in all zones, at all dates of sampling, and also accounted for the greatest proportion of the assemblages when grouped by plant zone or sampling date. Assemblage group 2 was also quite prevalent throughout the study period; it was present in all plant zones and most sampling dates. When the diazotroph assemblage groups are teased out per zone and assemblage, a clear pattern appears (Figure S3
) where group 1 and 2 dominate during ‘normal’ environmental conditions and during drought conditions, however during post-drought conditions an increase in groups 3, 4, and 5 can be observed throughout the various assemblages. This indicates that some diazotrophs persist and maintain detectable populations during and after the drought event, however diazotrophs that are not usually active and/or present in high numbers begin to significantly contribute within the diazotroph population. All the sequences obtained for phylogenetic analysis (Figure 5
) were from bands listed (in bold in group 1 only, Table S2
) in diazotroph assemblage groups. DNA from other bands was not successfully isolated, providing only a mere glimpse into the possible taxonomic groups to which these microorganisms belong.
Low but detectable rates of acetylene reduction also indicate that diazotrophs present in the assemblages were actively fixing nitrogen under the distressed conditions. In the post-drought sampling years, acetylene reduction rates were the highest recorded, indicating that the recovering assemblage could fix nitrogen very effectively. The physiological mechanisms by which salt marsh diazotrophs withstand acidic pHs, high salinities and low moisture conditions to maintain detectable populations and activity are unknown. Microorganisms acclimate to stressful conditions by reallocating their resources from growth to survival pathways, many ultimately entering a dormant status if the stressful conditions do not otherwise kill them [82
]. Diazotrophs capable of tolerating stressful conditions in salt marsh sediment and fixing nitrogen would be essential in maintaining nitrogen availability for plant uptake, thereby contributing significantly to plant productivity. Diazotrophs and rates of nitrogen fixation are tightly coupled to photosynthetic activity of the marsh grasses [14
]. It is not clear whether the plants require the microorganisms to be productive, but this additional source of ‘new’ nitrogen in a nitrogen limited system would certainly seem conducive to ecosystem function.
4.4. Implications and Future Studies
Due to the oscillation of mean high water relative to mean sea level, tidally-dominated salt marsh ecosystems will be subject to frequent perturbations of flooding or infrequent tidal inundation leading to drought conditions [37
]. These episodic perturbations have very obvious effects on flora and fauna in the marshes but the direct effects on microbial communities associated with these ecosystems would not have been predicted to be very significant [81
]. The data from this study shows that this expectation is not quite correct. The effects on microbial communities may not be immediate but are profound and lasting. The dramatic reduction in the detectable rhizosphere diazotrophs lasted for 2 years even though the marsh landscape seemed to have entered a recovery phase in the very next growing season as conditions improved. The recovery of previously defined ecologically significant sequences in the post-drought assemblages indicates their ability to maintain populations under severe conditions. It is reasonable to assume that these diazotrophs in this nitrogen-limited yet highly productive ecosystem are of fundamental importance to the vitality of this ecosystem. Further studies will be required to determine the physiologies of these organisms, their capacities for nitrogen fixation and their physiological responses to stressful conditions. Manipulated greenhouse experiments will also be necessary to gather informative data that can be used to determine a model of the effect these conditions have on diazotroph assemblages. Such information would provide much needed perspective on the role of microorganisms during severe weather-related events.