DOI: 10.5061/dryad.3ffbg79rn
title: Does European-introduced Phragmites australis experience below-ground microbial enemy release in North America?

description: Escape from native range enemies can give invasive species a competitive
 edge according to the enemy-release hypothesis. While more commonly
 associated with predators and herbivores, release from belowground
 microbial antagonists has been recently demonstrated to benefit invasive
 plants. Biogeographic variation in dominance and comparisons of soil
 communities suggest that invasive European Phragmites australis may have
 also benefitted from belowground enemy release in North America. Here we
 examine the effects of native range (Europe) versus introduced range
 (North America) soil communities on European native and North American
 introduced P. australis using a reciprocal inoculation seedling growth
 experiment. Contrary to the enemy-release hypothesis, we found that North
 American-introduced P. australis was sensitive to soil community origin in
 that the seedlings grown in European soil communities (native) had higher
 total biomass than seedlings grown in North American soil communities
 (introduced). This pattern was not observed in the European native P.
 australis seedlings which had similar biomass when grown with North
 American or European soil communities. Notably, introduced P. australis
 had higher biomass than native P. australis regardless of which soil
 community it was grown in, suggesting a growth-defense tradeoff. Though
 the relative abundance of mutualists and pathogens composition did not
 differ between the two ranges, an indicator analysis revealed that
 mutualistic fungi and bacteria were key components of European soil
 communities but not in North American communities. Interestingly, North
 American soil communities had lower β-diversity than European communities
 suggesting higher levels of community conservation amongst North American
 populations. This research represents the first evidence of growth-defense
 trade-offs in introduced P. australis and offers a novel
 mechanism for understanding the invasion of P. australis in North America.
description: <strong>Field sampling</strong>
 North American and European <em>Phragmites </em>seed
 and soil were collected from three different habitats, tidal brackish
 wetland, freshwater marsh, and inundated urban areas. All plant material
 was confirmed to be haplotype “M” using visual identification methods
 (Swearigan & Saltonstall 2010). This stratification allowed us to
 detect the effects of range-level differences in soil community across our
 different response variables and account for environmental filtering
 through sampling. North American sites were located along the mid-Atlantic
 coast of the United States, the urban (NA-U) and freshwater (NA-F) sites
 were located around the greater Philadelphia, PA (USA) area, while the
 tidal site (NA-T) was located 150 km south on the eastern shore of
 Maryland along King’s Creek, MD (USA) (Table 1). This site is a
 well-studied and extensively sampled site previously used in a multitude
 of <em>Phragmites </em>studies and ranges in salinity between
 0.1-8 ppt (Meadows & Saltonstall 2007; Mozdzer & Zieman
 2010; Yarwood et al. 2016). European seeds and soil were collected along a
 23-km transect running along the Bay of Aarhus in Denmark (Table 1). The
 freshwater (EU-F) and urban (EU-U) sites were located inland from the bay
 while the tidal site (EU-T) was on the water’s edge and experienced
 salinities ranging from 14-23 ppt. A total of 6-8 inflorescences were
 collected at each site in the autumn of 2017 and were subsequently
 stripped to bare seed, pooled, and stored at room temperature until
 germination. Soil inocula were obtained through bulking and homogenizing 6
 soil cores (20.3 cm diameter x 30.5 cm depth) collected at the base of
 randomly selected <em>Phragmites </em>shoots to ensure a
 site-level representative sampling of the rhizosphere-associated soil
 community. During homogenization, the inocula was processed to remove live
 plant material such as rhizomes and roots. The soil was stored at 4°C
 until the time of use. A soil import permit was acquired from the Animal
 and Plant Health Inspection Service (APHIS) a subdivision of the United
 States Department of Agriculture (USDA). All soil was handled and disposed
 of in accordance with the issued permit.
 <strong>Greenhouse Experimental Set-up &
 Design</strong> All seeds were surface sterilized
 in two batches in the first week of January 2018 in 70% ethanol and then
 3% bleach solution and plated on 2% water-agarose (no added nutrients) for
 germination in a Conviron growth chamber set to 30°C (16 hr)-15°C (8 hr)
 day/night cycle (Conviron, Pembina, North Dakota). Two weeks post-plating,
 seedlings (~1 cm tall) were potted in 6.35 cm diameter x 35.6 cm 1L pots
 filled with 700 g (dry) of autoclave-sterilized
 <em>Sphagnum</em> peat moss as well as 100 g (wet) of the
 corresponding soil inocula (sterilized treatments received 100g of
 sterilized inocula). Earth Juice Microblast-liquid micronutrients were
 added to each pot during planting to help seedlings overcome transplant
 shock at a concentration of 660uL/L of water (Earth Juice, Chico, CA,
 USA). Seedlings were planted in the center of the pot with enough media to
 cover all visible root tissue. Each pot was placed in its own 13.9-cm tall
 water reservoir to ensure minimal cross-contamination between different
 soil communities. The seeds and soil from each of the
 six populations (NA-U, NA-F, NA-T, EU-U, EU-F, and EU-T + sterilized soil)
 were planted in a fully factorial manner, giving a total of 42 different
 soil x seed combinations, and replicated four times (6 seed populations x
 (6 soil populations) x 4 replicates = 144 pots) (Fig. 1). Pots were
 arranged in racks corresponding to each inoculum source to minimize
 chances of accidental cross contamination. Pots were randomly rotated
 every two weeks between the two benches and pots were randomly moved
 within each rack as to reduce environmental effects. All pots were top
 watered every other day until the water reservoir was filled, and average
 daily light intensity (0.504 ± 0.019 mmol m<sup>-2</sup>
 s<sup>-1</sup>) and temperature (25.31°C ± 0.10) were recorded
 using the Onset HOBO Pendant<sup>®</sup> Temperature/Light 8K
 Data Logger (Onset, Bourne, MA). Dead seedlings were replaced with the
 corresponding seedlings from maintained populations up until day 120;
 thereafter pots with dead plants were omitted from the study. Plants were
 harvested on day 230 after planting. If seedlings accumulated less than
 0.5g of biomass (dry weight) at the time of harvest, they were omitted
 from the study. Harvest was performed 230 days
 post-planting. Aboveground biomass was clipped at the soil line and
 oven-dried at 65°C to constant mass. Belowground biomass (roots and
 rhizomes) was washed from the associated soil using a hose and a series of
 3mm and 1mm sieves and dried at 65°C to constant mass (Roots
 Lab<sup>1</sup>). The dried aboveground biomass and the dried
 roots and rhizomes were sorted and weighed separately and summed to obtain
 total biomass. <strong>Soil community
 sequencing</strong> Ten grams of rhizosphere soil
 was collected from each pot planted with NA-U plants during harvest. After
 collection, samples were immediately flash-frozen in liquid nitrogen and
 stored at -80ºC. In total, 21 soil samples were collected, 3 samples from
 each of the soil sources (NA-U, NA-T, NA-F, EU-U, EU-T, EU-F, Sterilized).
 Total DNA was extracted using the Qiagen DNeasy Powersoil Kit, extractions
 were performed following the provided kit protocol, with a small
 modification of using 200 ml of phenol:chloroform:isoamyl alcohol instead
 of the provided bead solution to maximize DNA yields (Sigma-Aldrich, St.
 Louis, MO, USA; Qiagen, Hilden, Germany). DNA was
 quantified using QuantiFlour dsDNA System (Promega, Madison, WI). 12.5 ng
 of DNA was used as input into Illumina’s 16S / ITS metagenomics Library
 preparation workflow. The V4 region of the bacterial 16S rRNA gene
 sequences and ITS2 region of fungal rRNA were amplified using the primer
 pair containing the gene‐specific sequences and Illumina adapter overhang
 nucleotide sequences. 16S rRNA was amplified using forward primer 515f
 (5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA) and reverse
 primer 806r (5'
 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAC-TACHVGGGTWTCTAAT). ITS rRNA
 amplification was amplified using forward primer ITS3
 (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCATCGATGAAGAA-CGCAGC) and reverse
 primer ITS4
 (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA-GTCCTCCGCTTATTGATATGC).
 Amplicon PCR was performed to amplify the template out of input
 DNA samples. Briefly, each 25 μL of polymerase chain reaction (PCR)
 reaction contains 12.5 ng of sample DNA as input, 12.5 μL 2x KAPA HiFi
 HotStart ReadyMix (Kapa Biosystems, Wilmington, MA), and 5 μL of 1 μM of
 each primer. PCR reactions were carried out using the following protocol:
 an initial denaturation step performed at 95°C for 3 min followed by 25
 cycles of denaturation (95°C, 30 s), annealing (55°C, 30 s), and extension
 (72°C, 30 sec), and a final elongation of 5 min at 72°C. The PCR product
 was cleaned up from the reaction mix with Mag-Bind RxnPure Plus magnetic
 beads (Omega Bio-tek, Norcross, GA).  A second index PCR amplification,
 used to incorporate barcodes and sequencing adapters into the final PCR
 product, was performed in 25 μL reactions, using the same master mix
 conditions as described above. Cycling conditions were as follows: 95°C
 for 3 minutes, followed by 8 cycles of 95°C for 30”, 55°C for 30” and 72°C
 for 30”. A final, 5 minutes elongation step was performed at 72°C.  The
 libraries were normalized with Mag-Bind® EquiPure Library Normalization
 Kit (Omega Bio-tek, Norcross, GA) and then pooled. The pooled library ~600
 bases in size was checked using an Agilent 2200 TapeStation and and
 sequenced (2 x 300 bp paired-end read setting) on the MiSeq (Illumina, San
 Diego, CA). Results were analyzed via Illumina BaseSpace 16S metagenomics
 application using module version Isis v2.5.35.6 and Illumina-curated
 version of the May 2013 Greengenes taxonomic database.
 <strong>Data analyses</strong> All
 data were analyzed in RStudio version 4.0.3. We assessed the effect of
 soil range, seed range, and their interaction on total biomass, shoot
 growth, shoot emergence, and percent guild composition (“endophyte”,
 “plant pathogen/plant parasite”, “mycorrhizae”, and “plant saprotroph”)
 using either linear mixed-effects models (LME) or generalized least
 squares models (GLS) using the “nlme” R package (Pinheiro et al. 2023).
 The model selection process starts with a base GLS model for backward
 selection using soil range * seed range as fixed effects. Then, we tested
 whether the random effect of soil and seed habitat creates a better model
 than a model without the incorporation of random effects. We then selected
 the model with the lowest AIC value. We determined whether there are
 heterogeneous variances across seed and soil habitats and fit an
 alternative variance structure to the model. Again, AIC values are
 assessed between models, and the best model is selected. We then run a
 type III ANOVA on the best model to assess the significance of soil and
 seed range in explaining the variance of the response variable and perform
 model validation by plotting fitted values against residuals, each of the
 fixed effects against residuals, and a histogram of the residuals alone.
 Tukey’s HSD values were also obtained for the soil range*seed range
 comparisons using the function “glht” in the R package “multcomp” (Hothorn
 et al. 2008). Indicator analysis was used to assess the
 level of conservation within North American and European 16s/ITS soil
 communities. We performed indicator analysis on16s/ITS genus-level OTU
 reads of the sampled soil communities at a probability threshold of
 p&lt;0.01 using the “indval” function from the R package “labdsv”
 (version 2.1-0) (Roberts 2023). Permutational analysis
 of variance (PERMANOVA) and principal coordinate analysis (PCoA) was
 performed to assess whether 16s and ITS soil communities on the genus
 level from North America and Europe differed significantly from one
 another. PERMANOVA was performed using Bray-Curtis dissimilarity values
 for the 16s/ITS soil communities using the “adonis” function in the R
 package “vegan”, Tukey’s HSD values were also attained for the group
 comparisons using the function “TukeyHSD” in R package “vegan” (Oksanen et
 al. 2022). Principal coordinate analysis (PCoA) was performed for the
 16s/ITS soil communities on the genus level in R using the function
 “metaMDS” in R package “vegan”, on Bray-Curtis distance matrices (Oksanen
 et al. 2022). To assess multivariate homogeneity of dispersion between
 North American and European 16s/ITS soil communities was calculated using
 the mean distance of each sample to the centroid for both in R using the
 “betadisper” function in R package “vegan” (Oksanen et al.
 2022). FUNGuild was used to assign ecological roles to
 fungal OTUs at the species level (Nguyen et al. 2016). FUNGuild was run
 using a Python 3 environment (Van Rossum & Drake 2009). The
 FUNGuild output assigned each fungal OTU a guild classification that lists
 known ecological roles/functions. This output was parsed to extract OTUs
 that fall into the guild classifications of “endophyte”, “plant
 pathogen/plant parasite”, “mycorrhizae”, and “plant saprotroph”. The
 counts of each of the extracted OTUs were acquired for each sample. All
 OTUs belonging to the same classification were then summed and divided by
 total OTUs in each respective sample to derive the percent composition of
 each guild.
description: # Does European-introduced *Phragmites australis* experience below-ground
 microbial enemy release in North America?
 [https://doi.org/10.5061/dryad.3ffbg79rn](https://doi.org/10.5061/dryad.3ffbg79rn) This repository contains all the datasets and R code needed to analyze the study, "Does European introduced *Phragmites australis* experience below-ground microbial enemy-release in North America?". Datasets are presented in .csv format and titles will correspond to the analysis performed. The detailed annotated code used to analyze each of the datasets is included in the Ecosphere Models.R file. The code used to reproduce the figures found in the study is also included in the Ecosphere Figures 2.R file.  For any additional information please contact the corresponding author Sean Lee at ([slee38@tulane.edu)](mailto:slee38@tulane.e) ## Description of the data and file structure ### *Biomass analysis* We explored the effects of soil inocula on *Phragmites* seedling biomass sourced from different habitats across both North America (invasive range) and Europe (native range). *Phragmites* seedlings were sourced from the same corresponding habitats across the two ranges as well and used to populate a fully factorially crossed seedling growth experiment. These datasets described below were used to create the stacked barplot representing the total, aboveground, and belowground biomass of *Phragmites* seedlings (Fig. 2) and also create the linear mixed effect models used to analyze the effects of seed and soil inocula range on explaining the variation in observed biomass. 1. ER_BIOMASS_FIGS_2.csv * Contains aboveground, belowground, and total biomass of *Phragmites* seedlings at harvest in grams as well as variables such as seed and soil range of the seeds used in the experiment and the soil inocula used to inoculate each pot. * These data were used to create the stacked barplot representing total, aboveground, and belowground biomass of *Phragmites* seedlings (Fig. 2). * "SEED_RANGE": Describes which continent the seed originated from (US_SDR: seed collected from North America, and DE_SDR: seed collected from Europe) * "SOIL_RANGE": Describes the continent the soil inoculum was sourced from (US_SOR: North American soil, DE_SOR: European soil, and STER_SOR: sterilized inoculum). * "AGB": Aboveground biomass of the seedling in grams. * "BGB": Belowground biomass of the seedling in grams. * "TOT_BM": Total biomass of the seedling in grams. 2. "ER_BIOMASS_new_2.csv" * Contains aboveground, belowground, and total biomass of *Phragmites* seedlings at harvest in grams as well as variables such as seed range and soil range which were used as fixed effects in our linear mixed effects models, as well as soil habitat which was used as a random effect in the total biomass model. * These data were used to create the linear mixed effects models to analyze the effects of seed and soil range on total, aboveground, and belowground biomass. * "SEED_RANGE": Describes the continent the seeds were sourced from (US_SER: North American seeds, and DE_SER: European seeds). * "SOIL_RANGE": Describes the continent the soil inoculum was sourced from (US_SOR: North American soil, DE_SOR: European soil, and STER_SOR: sterilized inoculum). * "SOIL_HAB_TYPE": Describes the habitat type the soil inoculum was sourced from (URB_SOH: soil taken from an urban environment, TID_SOH: soil taken from a tidal environment, FRESH_SOH: soil taken from a freshwater environment, and STER_SOH: sterilized soil inoculum). * "AGB": Aboveground biomass of the seedling in grams. * "BGB": Belowground biomass of the seedling in grams. * "TOT_BM": Total biomass of the seedling in grams. ### *Soil microbial community analysis* To explore the composition and structuring of soil bacteria and fungi communities in *Phragmites* associated soils we performed 16S and ITS rRNA sequencing. These datasets were generated from the sequencing data and used to create NMDS plots, perform indicator analyses, perform PERMANOVA, and for fungal communities, analyze the proportion of fungi belonging to different guilds according to FUNGuild. 1. SIXTEEN_GENUS_ORD_NO_STER.csv * Contains OTU counts for bacterial 16S rRNA on the genus level, without the incorporation of samples taken from sterilized treatments. * This dataset is used to create NMDS plots for Fig. 3, and code for this figure can be found in the *Ecosphere Figures.R* file. 2. ITS_GENUS_ORD_NO_STER.csv * Contains OTU counts for fungal ITS rRNA on the genus level, without the incorporation from samples taken from sterilized treatments. * This dataset is used to create NMDS plots for Fig. 3, and code for this figure can be found in the *Ecosphere Figures.R* file. 3. SIXTEEN_GENUS_AGGREGATE.csv * Contains OTU counts for bacterial 16S rRNA on the genus level, without the incorporation of samples taken from sterilized treatments. * This dataset is used to perform the bacterial indicator analysis based on soil range, hence the first three columns represent different geographic and environmental variables. * "SOURCE": Describes the soil source of the inoculum (USU: North American soil taken from an urban environment, UST: North American soil taken from a tidal environment, USF: North American soil taken from a freshwater environment: DEU: European soil taken from an urban environment, DET: European soil taken from a tidal environment, and DEF: European soil taken from a freshwater environment). * "HAB_TYPE": Describes the habitat type the soil inoculum was sourced from (URBAN: soil taken from an urban environment, TIDAL: soil taken from a tidal environment, FRESHWATER: soil taken from a freshwater environment). * "RANGE": Describes the continent the soil inoculum was sourced from (US: North American soil, and EU: European soil). * The code for performing the bacterial indicator analysis is located in the *Ecosphere Models.R* file. 4. ITS_GENUS_AGGREGATE.csv * Contains OTU counts for fungal ITS rRNA on the genus level, without the incorporation from samples taken from sterilized treatments. * This dataset is used to perform the fungal indicator analysis and the PERMANOVA based on soil range, hence the first three columns representing different geographic and environmental variables. * "SOURCE": Describes the soil source of the inoculum (USU: North American soil taken from an urban environment, UST: North American soil taken from a tidal environment, USF: North American soil taken from a freshwater environment: DEU: European soil taken from an urban environment, DET: European soil taken from a tidal environment, and DEF: European soil taken from a freshwater environment). * "HAB_TYPE": Describes the habitat type the soil inoculum was sourced from (URBAN: soil taken from an urban environment, TIDAL: soil taken from a tidal environment, FRESHWATER: soil taken from a freshwater environment). * "RANGE": Describes the continent the soil inoculum was sourced from (US: North American soil, and EU: European soil). * The code for performing the fungal indicator analysis is located in the *Ecosphere Models.R* file. 5. PERMANOVA_16S_GENUS * Contains OTU counts for bacterial 16S rRNA on the genus level, with the incorporation of samples taken from sterilized treatments. * These sterilized samples were omitted from the PERMANOVA during the analysis (see the *Ecosphere Models.R* file). * This dataset is used to perform the PERMANOVA based on soil range, hence the first three columns represent sample ID, geographic, and environmental variables. * "SAMPLE_ID": Sample ID code, describes the soil source (see naming convention above) and the replicate number. * "SOIL_RANGE": Describes the continent the soil inoculum was sourced from (US: North American soil, DE: European soil, and STER: sterilized inoculum). * "HAB_TYPE": Describes the habitat type the soil inoculum was sourced from (URBAN: soil taken from an urban environment, TIDAL: soil taken from a tidal environment, FRESHWATER: soil taken from a freshwater environment). 6. ER_FUNGAL_GUILD_MODEL.csv * Contains proportion of fungal OTUs that are categorized into different guilds according to the FUNGuild database using OTUs collected from our ITS rRNA data, without the incorporation of samples taken from sterilized treatments. * These data were used to calculate the relative proportions of guilds based on soil range and whether or not North American soil fungal communities significantly differed in proportions of fungi belonging to different guilds compared to communities found in Europe (see the *Ecosphere Models.R* file). * "TRT": Sample ID code, describes the soil source (see naming convention above). * "HAB_TYPE": Describes the habitat type the soil inoculum was sourced from (URBAN: soil taken from an urban environment, TIDAL: soil taken from a tidal environment, FRESHWATER: soil taken from a freshwater environment). * "SOIL_RANGE": Describes the continent the soil inoculum was sourced from (US: North American soil, and EU: European soil). * "ENDOPHYTE": Proportion of fungal OTU counts classified as potential endophytic fungi. * "PATHOGEN_PARASITE": Proportion of fungal OTU counts classified as potential plant pathogens and parasites. * "MYCORRHIZAE": Proportion of fungal OTU counts classified as potential mycorrhizae. * "SAPROTROPH": Proportion of fungal OTU counts classified as potential plant saprotrophs. ### **.R Files** 1.  Ecosphere Figures 2.R * Contains R code to reproduce the figures in the manuscript. * Each section of code will correspond to the figure within the manuscript (i.e., Fig. 2, and Fig. 3) 2. Ecosphere Models.R * Contains R code for models used to analyze the data for this study including the models for biomass, FUNGuild guild classification proportions, distance-to-centroid of NMDS for 16S and ITS communities by soil range, bacterial and fungal indicators by soil range, and PERMANOVA of bacterial and fungal communities. 3. Ecosphere supplemental.R * Contains R code to reproduce the figures and models described in the supplemental materials. * Each section of code will correspond to the figure or analysis within the supplemental materials (i.e., Fig. S1, and Fig. S2) ## Sharing/Access information All data used in this study is contained here in this DRYAD page. A README associated with the supplemental materials can be found in the README (supplemental material) docx. file uploaded to Zenodo.