DOI: 10.5061/dryad.3j9kd51h6
title: Plant and microbial impacts of an invasive species vary across an environmental gradient

description: Invasive plants often successfully occupy large areas encompassing broad
 environmental gradients in their invaded range, yet how invader dominance
 and effects on ecological communities vary across the landscape has rarely
 been explored. Furthermore, while the impacts of invasion on plant
 communities are well studied, it is not well understood whether responses
 of aboveground (plant) and belowground (microbial) communities are
 coupled. Here we test patterns in Phragmites australis (common
 reed) invasion in a field survey of eight sites situated across a salinity
 gradient, ranging from freshwater to saline marsh, in Southeast Louisiana.
 At each site, we surveyed plant composition and used metabarcoding methods
 to assess soil fungal and bacterial composition in plots within the dense
 Phragmites stand, in a transition zone of ~50:50 Phragmites:native plants,
 and in native-only areas. We hypothesized that Phragmites’ abundance and
 impact on above and belowground communities would vary across the salinity
 gradient and that the responses of above and belowground communities to
 invasion would be coupled. We found weak evidence that invasion varied
 across the gradient: Phragmites stem densities increased slightly
 with salinity, and Phragmites increased aboveground litter accumulation
 more in fresh and saline areas compared to brackish. We found stronger
 evidence that plant and microbial responses to invasion varied with
 salinity. Phragmites strongly reduced native plant density across the
 gradient, with slightly greater reductions in fresh and saline areas.
 Plant species richness displayed consistent decreases with invasion across
 the salinity gradient; however, fungal and bacterial richness increased
 sharply with invasion only in brackish sites. Furthermore, the effect of
 Phragmites on plant and microbial community composition became stronger as
 salinity increased. Plants and microbes exhibited coupled responses to
 invasion in the magnitude of compositional shifts brought on by
 Phragmites, but Phragmites’ effects on richness were not coupled.
 Synthesis. Overall, the variability in Phragmites impacts across the
 gradient, particularly soil microbial impacts, suggests that it may be
 difficult to generalize invader effects from single-site or
 single-ecosystem studies. However, above and belowground communities show
 some coupled responses to Phragmites; thus understanding plant community
 responses to invasion gives insight into impacts occurring belowground.
description: Eight field sites across SE Louisiana were surveyed, ranging from
 freshwater to saline marsh. We classified sites based on dominant
 vegetation. Freshwater marshes were Barataria Preserve and Turtle Cove
 Research Station. Intermediate/Brackish mashes were Pearl River WMA,
 Fontainebleau State Park, Big Branch NWR, and Bayou Sauvage NWR. Saline
 marshes were two sites at the Louisiana Universities Marine Consortium,
 LUMCON. In 2017 at each site, 21 permanent 1
 m<sup>2</sup> plots were established in three transects based
 on vegetation type: <em>Phragmites </em>stand, transition, and
 native, each with 7 plots. The <em>Phragmites </em>stand
 transect plots were located entirely within an area where
 <em>Phragmites</em> was highly dominant. The native plots ran
 parallel to, but outside of, the <em>Phragmites</em> stand and
 contained only native plants representing the native marsh community. The
 transition plots ran along the edge of the <em>Phragmites</em>
 stand, capturing the interface of the native community and the
 invading<em> Phragmites</em> front. Plots were spaced
 approximately 10 m apart.  Plant species composition
 was sampled from September to November 2017 by performing stem counts of
 all plant species rooted in each 1 m<sup>2</sup> plot. For
 bunch grasses, each ramet (culm) was counted separately. Live biomass and
 litter mass per m<sup>2</sup> were estimated for each plot by
 harvesting from a 20 × 20 cm area, drying at
 60<strong>°</strong>C for 48 hrs, and weighing.
 Soil samples for microbial analysis were collected in October and
 November 2017. Soils were collected with a sterilized soil corer (5 cm
 diameter, approximately 10 cm depth), homogenized in a plastic bag, and a
 subsample collected in a sample tube. Samples were placed directly into a
 liquid nitrogen container in the field and transferred to a
 -80<strong>°</strong>C freezer upon returning to the
 lab. DNA was extracted from the soil samples with a
 Qiagen DNeasy PowerSoil Kit. We used a dual-index, two step PCR approach.
 We first amplified the fungal ITS region (primers ITS1f/ITS2) and the
 bacterial 16S region (primers 515F/806R). PCR was done in duplicate and
 duplicates were pooled. We then performed a second PCR to attach barcodes
 and Illumina adaptors. Samples were purified and standardized with a
 SequalPrep kit (Invitrogen Inc.), and pooled into ITS and 16S libraries.
 Libraries were sequenced on two lanes of an Illumina Miseq v3 (300bp PE)
 by Duke Sequencing Core, Duke University, NC. Sequence
 data were processed using the amplicon sequence variants (ASV) method in
 QIIME2 (Bolyen et al., 2019) and DADA2 (Callahan et al., 2016). We first
 trimmed reads where the median quality score fell below ~30, then
 quality-filtered the reads (no N’s, max expected errors 2, truncated at
 quality score 2, minimum read length 50) and denoised the data and joined
 paired reads using DADA2. We assigned taxonomy using a pre-trained Naïve
 Bayes classifier. The classifier was trained on the UNITE 8.2 database
 (Abarenkov et al., 2020) for ITS and Greengenes 13.8 (DeSantis et al.,
 2006) for 16S. Prior to analyses, both data sets were rarefied to an even
 sampling depth (fungi to 5334 reads and bacteria rarefied to 9316 reads
 per sample). We used FUNGuild (Nguyen et al., 2016) to
 classify fungal taxa into functional guilds. We focused on taxa classified
 as “plant pathogen” and “arbuscular mycorrhizae” as there were sufficient
 taxa classified as such for these two guilds. For each of these two
 guilds, we summed the relative abundance of taxa classified as plant
 pathogens and AMF to yield total pathogen and AMF relative abundance per
 sample.
description: Phragmites haplotyping was performed at each site, and we found
 that the Phragmites at our freshwater and brackish sites was haplotype I,
 and the Phragmites at our saline sites was haplotype M1.
 Some ITS and 16S samples did not sequence well and thus are NAs
 in this dataset. One 16S sample had an extraordinarily
 high number of reads and a very high richness and Chao1. Thus we added a
 separate column omitting this sample from the column (the value is
 NA). The Plant Pathogen relative abundance also
 contained two outliers, thus we added a separate column omitting these
 samples from the column (the value is NA).