Salt Marsh Elevation Drives Root Microbial Composition of the Native Invasive Grass Elytrigia atherica

Elytrigia atherica is a native invasive plant species whose expansion on salt marshes is attributed to genotypic and phenotypic adaptations to non-ideal environmental conditions, forming two ecotypes. It is unknown how E. atherica–microbiome interactions are contributing to its adaptation. Here we investigated the effect of sea-water flooding frequency and associated soil (a)biotic conditions on plant traits and root-associated microbial community composition and potential functions of two E. atherica ecotypes. We observed higher endomycorrhizal colonization in high-elevation ecotypes (HE, low inundation frequency), whereas low-elevation ecotypes (LE, high inundation frequency) had higher specific leaf area. Similarly, rhizosphere and endosphere bacterial communities grouped according to ecotypes. Soil ammonium content and elevation explained rhizosphere bacterial composition. Around 60% the endosphere amplicon sequence variants (ASVs) were also found in soil and around 30% of the ASVs were ecotype-specific. The endosphere of HE-ecotype harbored more unique sequences than the LE-ecotype, the latter being abundant in halophylic bacterial species. The composition of the endosphere may explain salinity and drought tolerance in relation to the local environmental needs of each ecotype. Overall, these results suggest that E. atherica is flexible in its association with soil bacteria and ecotype-specific dissimilar, which may enhance its competitive strength in salt marshes.

S2. Data of the plant traits, intensity of mycorrhizal colonization and plant litter biomass from each of the sampling sites. Sites H1-H3 are located at salt marsh high elevation and L1-L3 at low elevation.

S3. Soil physicochemical parameters methods
The soil physicochemical parameters tested were texture, pH, soil water content (SWC), organic matter (OM), sodium (Na), nitrates (N-NO3 -) and ammonium (N-NH4 + ), total carbon and nitrogen (TC/TN). The soil parameters were carried out in collaboration with the Department of Community and Conservation Ecology in the University of Groningen, except for texture that was performed in the Netherlands Institute of Ecology (NIOO-KNAW). Soil texture determined the grain size distribution by laser diffraction on a particle sizer (Malvern, Worchester, UK). The pH was measured weighting 15 g and adding 20 ml of distilled water. The tubes were shaken and left stand overnight, then the pH was measured using a potentiometer. Soil moisture was measured by oven-drying 10 g of soil at 105°C for ~16 h. Moisture percentage was calculated as fresh weight minus dry weight, divided by fresh weight multiplied by 100. After that, the dried samples were placed in a muffle furnace (Naberthermn, Germany) at 550 °C for 4 h. The soil organic matter content was calculated as dry soil weight -dry weigh after ignition, divided by dry soil weight x 100 [1]. To measure N content in nitrate and ammonium, 12.5 g soil was mixed with 30 ml KCl (1M), shaken for ~16 h using a custom-made overhead shaker (1 turn/s). Afterwards, the suspension was filtered with a paper filter by gravity and the extract was analyzed for N-NO 3and N-NH 4+ on a continuous flow auto analyzer (Type 5100; Skalar-40 BV, Breda, the Netherlands) using a colorimetric method [2]. For TC, TN and Na content, 10 g soil was first dried at 40 °C in a stove for 16 h and then ground to a fine powder in a Cyclotec 1093 mill. Sodium exchangeable ion content was measured by extraction of 5 g soil with ammonium acetate (1M, pH 7), mixed in the overhead shaker for 1 h and then filtered with a paper filter by

S5.
Bacterial Amplicon Sequence Variants (ASVs) richness comparing type of communities (A) and in each community separately: bulk soil (B), rhizosphere (C) and endosphere (D) in each sampling site. Sites H1-H3 are located at salt marsh high elevation and L1-L3 at low elevation. Letters denotes significant differences after a pairwise comparison of the least square means (p<0.001, Tukey adjustment).

S6. Shannon diversity index of the Amplicon Sequence Variants (ASVs) comparing communities (A) and in each community separately: bulk soil (B)
, rhizosphere (C) and endosphere (D) in each sampling site. Sites H1-H3 are located at salt marsh high elevation and L1-L3 at low elevation. Letters denotes significant differences after a pairwise comparison of the least square means (p<0.001, Tukey adjustment).

S7. Phylogenetic diversity index of the Amplicon Sequence Variants (ASVs) comparing communities (A) and in each community separately: bulk soil (B)
, rhizosphere (C) and endosphere (D) in each sampling site. Sites H1-H3 are located at salt marsh high elevation and L1-L3 at low elevation. In panel A and B, letters denote significant differences after a pairwise comparison of the least square means (p<0.001, Tukey adjustment), degrees of freedom method Kenward-Roger.
S8. Effect of elevation on soil, rhizosphere and endosphere bacterial community composition. Principal coordinate analysis based on unweighted Unifrac and Bray-Curtis dissimilarity distances of the bacterial community inhabit all type of communities i.e. endosphere, rhizosphere and soil as indicated at the top of each plot. Percentage of community variance explained by each axis is indicated in parentheses and summary of the permutational multivariate analysis of variance (PERMANOVA, 999 permutations) testing the effect of elevation, type of sample and stage of succession are reported in the table below the plots.