Bioinvasions trigger undeniable changes in ecosystem functioning of estuarine and coastal ecosystems(Carlton 1996; Chakraborty 2019). Salt marshes are no exception, with Spartina bioinvasions occurring all over the globe, leading to significant changes in the wetland systems, namely at the floristic biodiversity level (Daehler and Strong 1996; Zhong-Yi et al. 2004; Li et al. 2009; Baumel et al. 2016; Duarte et al. 2018b), and with consequent effects at the ecosystem services level (e.g., Human et al. 2020). In Mediterranean and Atlantic marshes, invasion by S. patens has been putting middle-upper marsh endemic species such as H. portulacoides under increased competitive pressure. Spartina species are characterized by high biomass production due to their highly efficient C4 photosynthetic mechanism, and strong rhizomatous radicular system that allows them to efficiently capture nutrients (Cheng et al. 2006). But in the Tejo estuary, H. portulacoides, a C3 shrub plant, is the most abundant species in the middle-upper marsh (Duarte et al. 2013c). But as S. patens generate fewer necromass (48-52% of the total biomass produced) (Vera et al. 2009) when compared to the endemic H. portulacoides (Caçador et al. 2009; Duarte et al. 2010), bioinvasions by S. patens decrease the necromass input to the system and inevitably affecting the decomposition and biogeochemical cycling process occurring in the salt marsh sediments. This biogeochemical cycling is known to be modulated by the colonizing species (Costa et al. 2007; Duarte et al. 2008, 2009) along the salt marsh floristic inventory. The introduction of a new and aggressive NIS is a game-changer for the marsh biogeochemical functioning. This is demonstrated when analyzing the relationship between the EEA profiles and the physic-chemical characteristics in the sediments colonized by both species. While in H. portulacoides there is an evident control of the EEA by the sediment physico-chemical traits (high number of significant correlations observed), these relationships are weaker and in fewer number when comparing the EEA and the sediment abiotic traits in the sediments colonized by S. patens. This indicates a prevalence of the plant effect over the abiotic traits in controlling the extracellular enzymatic activities. In fact, the highly active photosynthetic mechanism present in the C4 S. patens, allied to a dense rhizospheric system, promotes oxygen input to the sediments but also the exudation of low molecular weight organic acids (LMWOA), known to stimulate the rhizosphere activity, namely at the microbial level (Duarte et al. 2009, 2011).
Mediterranean salt marsh necromass production has its peak at the end of the growing season (late summer and autumn), concomitant with the peak extracellular enzymatic activity normally observed in the rhizosediments of the endemic halophyte species (Duarte et al. 2008, 2009, 2010; Caçador et al. 2009). This allows the system to recycle the organic input from the necromass plants, into inorganic forms bioavailable for the primary producers to incorporate into their biomass in the next growing season (Duarte et al. 2008; Freitas et al. 2014). When observing the enzymatic activity peaks in the sediments colonized by S. patens, the onset of biogeochemical functioning disruption is evident. In the sediments colonized by this NIS, the activity peaks occurred during spring and summer (observed e.g. for N-acetylglucosaminidase, urease, phenol oxidase and b-glucosidase activities), evidencing a temporal disruption compared to the seasonal pattern associated with the endemic species. Moreover, during the cold seasons (autumn and winter) several extracellular enzymatic activities showed significantly higher values in S. patens rhizosediments. If on one hand, there is a temporal disruption of the biogeochemical recycling processes, on the other, there is a very active decomposition system occurring during the cold seasons. This completely shifts the marsh biogeochemical functioning. Salt marshes are considered efficient organic matter sinks, retaining large amounts of the so-called blue carbon, nitrogen and phosphorous (Caçador et al. 2016; Geraldi et al. 2019; Duarte et al. 2021), not only due to their high productivity but also due to their low decomposition rates, especially when compared to other terrestrial ecosystems (Pereira et al. 2007). This allows marshes to retain, in the form of recalcitrant organic matter, large amounts of necromass, storing high amounts of carbon and reducing the eutrophication loading of the estuarine systems where they are present (Caçador et al. 2004; Duarte et al. 2021). This extracellular enzymatic activity observed in the sediments colonized by S. patens during the warmer seasons (spring and summer) alongside the comparatively higher activity assessed during the cold seasons, has as an ultimate consequence the acceleration of the necromass decomposition processes and thus a reduced marsh storage and remediation capacity (Duarte et al. 2008).
Analyzing the extracellular enzymatic activity profiles, the abovementioned facts acquire added importance when associated with each of the biogeochemical cycles for which these ecosystems play a key role. Observing S. patens rhizosediments, phosphatase activity peaks during winter, with values far above the ones observed in the sediments colonized by the endemic H. portulacoides, points to an enhanced release of the inorganic phosphorous into the system, with potential consequences in the eutrophication degree of the estuary. Previous studies have shown there is also a clear relationship between the phosphatase activity and the inorganic phosphorous generation in the vegetated marsh rhizosediments, with vegetated sediments acting as phosphorus sinks, with the recycling of the organic forms of this element being modulated by the plant species present (Freitas et al. 2014). Thus, this change in colonization promoted by S. patens has potential consequences on the ecosystem inorganic phosphorous load. In line with this, it could also be observed a higher urease activity year-round in the sediments colonized by the NIS, as well as a peak of proteasic activity during autumn. Urease and protease act in the hydrolysis of organic to inorganic nitrogen, the former using urea-type substrates and the latter simple peptidic substrates (Caravaca et al. 2005). These enzymes produce inorganic nitrogen forms, namely ammonia, one of the key compounds, that when in elevated concentrations is responsible for eutrophication in estuarine systems (Domingues et al. 2011). Moreover, this is not the preferred form of inorganic nitrogen assimilable by phytoplankton (Domingues et al. 2011), nor by halophytic vegetation (Stewart et al. 1972), thus no phytoremediation process will filter this increase ammonia concentration.
Impacts on carbon biogeochemical cycling is also a matter of concern in sediments colonized by S. patens. The two oxidoreductases (POX and FOX) showed higher activities in the sediments colonized by the NIS, in more than one of the sampling seasons, along with high values of b-glucosamidase and N-acetylglucosaminidase. All these enzymes actively participate in the decomposition of carbon-based substrates, namely, phenolic compounds (e.g., originated from plant lignin decomposition), carbohydrates and chitin exoskeletons (Freeman et al. 2004; Duarte et al. 2008). The increase in these enzymes activities increases the availability of carbon-based respiratory substrates, that end in carbon release from the sediments in the form of CO2 from bacterial respiration (Alonso-Sáez et al. 2008). Moreover, these carbon decomposition activity peaks in S. patens rhizosediments were concomitant with an increase in the dehydrogenase activity. Dehydrogenase activity is considered to be a proxy of microbial biomass (Kelley et al. 2011). The higher activity of carbon-based substrates decomposition activity allied to this increase in dehydrogenase activity reinforces the need for carbon use for microbial heterotrophic growth and thus a correspondent increase in respiration, with an inevitable decrease in the carbon sink capacity of the sediments and reduction of the blue carbon storage ecosystem service (Duarte et al. 2021). Although sulfur storage in salt marshes is not among the most recognized ecosystem services provided by these habitats, changes in its biogeochemistry have tremendous implications in other key services such as metal remediation (Duarte et al. 2008). Previous studies ( e.g., van Hullebusch et al. 2005) indicate that high sulfatase activity promotes the conversion of the sulfate produced by this enzyme into sulfides by sulfate-reducing bacteria (SRB). The sulfides establish chemically stable bonds with heavy metals, maintaining these elements in a reduced form (residual fraction) for extended periods of time (Tabak et al. 2005), thus reducing the heavy metal bioavailable fraction. Previous studies focusing on the metal speciation in the rhizosediments of the NIS S. patens showed that these metals are present in higher proportions in the more bioavailable chemical fractions (lower residual fraction) (Human et al. 2020). This is aligns with our present findings. In Autumn, the period of typically higher organic matter recycling activity (Duarte et al. 2008, 2009), the sulfatase activity in the S. patens rhizosediments showed a significant decrease. This would lead to reduced production of sulfate, and consequently lower production in sulfides to reduce the metals present in these sediments into more stable forms (residual fraction) (Tabak et al. 2005; van Hullebusch et al. 2005; Duarte et al. 2008).