DOI: 10.5061/dryad.c866t1gfw
title: Data from: Arbuscular mycorrhizal communities respond to nutrient enrichment and plant invasion in phosphorus-limited eucalypt woodlands

description: Arbuscular mycorrhizal fungi (AMF) facilitate ecosystem functioning
 through provision of plant hosts with phosphorus (P), especially where
 soil P is limiting. Changes in soil nutrient regimes are expected to
 impact AMF, but the direction of the impact may depend on context. We
 predicted that nitrogen (N)-only enrichment promotes plant invasions and
 exacerbates their P limitation, increasing the utility of AMF and
 promoting AMF diversity. We expected that enrichment with N, P and other
 nutrients similarly promotes plant invasions, but decreases the benefit
 and diversity of AMF because P is readily available for both native and
 exotic plants. We tested these hypotheses in eucalypt woodlands of
 south-western Australia, that occur on soils naturally low in P. We
 evaluated AMF communities within three modified ground-layer states
 representing different types of nutrient enrichment and associated plant
 invasions. We compared these modified states to near-natural reference
 woodlands. AMF richness varied across ground-layer states. The moderately
 invaded/N-enriched state showed the highest AMF richness, while the highly
 invaded/NP-enriched state showed the lowest AMF richness. The reference
 state and the weakly invaded/enriched state were intermediate. AMF
 richness and colonisation were higher in roots of exotic than native plant
 species. AMF community composition differed among ground-layer states,
 with the highly invaded/NP-enriched state being most distinct.
 Distinctions among states were often driven by family-level patterns.
 Reference and moderately invaded/N-enriched states each supported distinct
 groups of zero-radius operational taxonomic units (zOTUs) in
 Acaulosporaceae, Gigasporaceae and Glomeraceae, whereas Gigasporaceae and
 Glomeraceae were nearly absent from the highly invaded/NP-enriched state.
 Further, Diversisporaceae and Glomeraceae were most diverse in the
 moderately invaded/N-enriched state.  Synthesis. Both the nature
 of soil nutrient enrichment and plant provenance matter for AMF. N-only
 enrichment of low-P soils increased AMF richness, likely due to
 introduction of AMF-dependent exotic plant species and exacerbation of
 their P-limitation. In contrast, multi-nutrient enrichment, decreased AMF
 richness potentially due to a decrease in host dependence on AMF,
 regardless of host provenance. The changes in AMF community composition
 with nutrient enrichment and plant invasion warrants further research into
 predicting the functional implications of these changes.
description: <em>Site selection</em> Sampling
 was centred on two nature reserves with areas of York gum
 (<em>Eucalyptus loxophleba</em> subsp.
 <em>loxophleba</em>) – Jam (<em>Acacia
 acuminata</em>) woodlands (hereafter York gum woodlands) in close to
 reference condition. The reserves are located in the central wheatbelt,
 Western Australia: Mount Caroline (31°45'25.3"S,
 117°38'38.3"E) and Namelkatchem Nature Reserves (31°10’47.9”S,
 117°11’18.1”E), and are ~ 70 km apart (Fig. 1). These reserves have a
 history of minimal livestock grazing and plant invasion, permitting the
 persistence of large areas of diverse understoreys dominated by native
 perennial and annual forbs and grasses (Prober and Wiehl 2012). The
 climate is Mediterranean type, with long-term (1990–2022) mean annual
 temperature and rainfall, respectively, of 17.8 °C and 321 mm at Mt
 Caroline and 17.6 °C and 333 mm at Namelkatchem (BoM, 2023).
 Our experimental design involved four ground-layer states
 representing different levels of nutrient enrichment and plant invasions
 as described above: a reference (control) state in near natural conditions
 with naturally low soil P and N, and three degraded states that we sought
 from different parts of the landscape: a weakly invaded/enriched state, a
 moderately invaded/N-enriched, and a highly invaded/NP-enriched state
 (Table 1; Fig. 1). We chose the term ‘ground-layer state’ to be consistent
 with previous research of this ecosystem (Prober et al. 2012).
 ‘Ground-layer’ is used rather than ‘understorey’ to distinguish the
 herbaceous ground-layer from a shrub layer that can occur in the woodland
 understorey. Two areas within each of the two nature
 reserves with few-to-no exotic plant species, were selected to sample the
 ‘reference’ state. Each reserve included localised patches invaded by
 exotic annuals, that likely arose historically due to localised
 disturbances (e.g. by introduced rabbits). These patches were used to
 represent the ‘moderately invaded/N-enriched state’, given that other
 studies have demonstrated that such exotic-invaded states of York gum
 woodlands are typically N-enriched (Prober and Wiehl, 2012).
 Because P-enriched areas rarely occur inside the nature reserves,
 to represent the ‘highly invaded/NP-enriched’ state we sampled four
 fertilised 2 m × 2 m plots from a pre-existing long-term nutrient addition
 experiment located ~1.5 km from Mount Caroline Nature reserve
 (31°46'56.43"S, 117°36'41.61"E; Fig. 1). The
 experiment was established in a grazed York gum woodland remnant in 2009,
 as part of the global Nutrient Network (NutNet) experiment (Borer
 <em>et al</em>. 2014). We also sampled four unfertilised plots
 from the same experiment, representing the ‘weakly invaded/enriched’ state
 that arose through historical sheep grazing, resulting in some plant
 invasion and N and P enrichment. To match the NutNet design, plots of 2 m
 × 2 m were established at both nature reserves for the reference and
 moderately invaded/N-enriched states. Plots were at least 20 m apart, with
 four replicate plots of each ground-layer state. Remnants of York gum
 woodlands are rare in the landscape; hence we chose the two closest nature
 reserves near the NutNet experiment. This was done to incorporate as much
 of the natural variation within Reference sites as possible. We note that
 a previous study found little variation in AMF communities among remnants
 of York gum woodlands across 200 km distance (Prober et al.
 2015). The experimental plots from NutNet were arranged
 in a randomised complete block design with four blocks. Nutrients were
 added annually in autumn to plots representing the highly
 invaded/NP-enriched state, as 10 g N per
 m<sup>2</sup>·yr<sup>−1</sup> of timed-release
 urea, 10 g P per m<sup>2</sup>·yr<sup>−1</sup> as
 triple superphosphate, and 10 g K per
 m<sup>2</sup>·y<sup>−1</sup> as potassium
 sulphate. These plots also received a once-off addition of other macro-
 and micronutrients in 2009: 100 g per m<sup>−2</sup> of a mix
 containing iron (15%), sulphur (14%), magnesium (1.5%), manganese (2.5%),
 copper (1%), zinc (1%), boron (0.2%), and molybdenum (0.05%). Weakly
 invaded/enriched plots were dominated by native plant species and highly
 invaded/NP-enriched plots were dominated by exotic plant species. The
 experimental plots had been open to livestock grazing since European
 colonisation (1860s) until 2015. <em>Sample
 collection and processing       </em> Sampling
 occurred in August 2021 during the growing season. In each plot, plant
 community composition and cover were recorded. Then, rhizosphere soil and
 roots were collected from two plants of the six most abundant plant
 species. Samples from the two plants per species were pooled for a total
 of six samples per plot. Soils were thoroughly mixed in a sealable bag,
 collecting a subsample for DNA analyses. Roots were immediately stored in
 98% ethanol pending processing. Soil and root samples were stored in 15 ml
 tubes and placed in dry ice during sampling and transport to the
 laboratory. Samples were stored at -80°C thereafter. Root samples were
 thoroughly cleaned with deionised water, and fine roots of &lt; 2 mm
 were retained. Clean root samples were split in two: one part for DNA
 analyses and one for root colonisation assessment. Two plots had only five
 plant species, resulting in 94 soil and 94 root samples (Table S1). No
 permit was needed to access and sample plots from the NutNet experiment.
 Permits to access and sample the two nature reserves were granted by the
 Department of Biodiversity, Conservation and Attractions of Western
 Australia (Licence: FT61000839; Regulation 4: CE006388).
 <em>Soil chemical analyses</em>
 Soil samples were sent to CSBP Laboratories (Bibra Lake, Western
 Australia) for nutrient analyses. Plant-available P and K were measured
 using the Colwell test (Colwell, 1963; Rayment & Higginson, 1992).
 Organic carbon (OC) was determined according to Walkley & Black
 (1934). Ammonium-N, nitrate-N, and total N were measured as per Searle
 (1984). Soil pH was measured in CaCl<sub>2</sub> in a solution
 ratio of 1:5 (Rayment & Lyons, 2012).
 <em>Root colonisation</em> Root
 subsamples allocated for measurement of root colonisation were cleared in
 1 M KOH and stained with ink in vinegar (5% v/v) as described by
 Vierheilig <em>et al.</em> (1998). Colonization by AMF,
 including Glomeromycotina-AMF and Mucoromycotina-AMF, was scored using the
 line intercept method (McGonigle <em>et al.</em> 1990). One
 hundred intercept points were scored for each sample, and the percentage
 of root length colonized by AMF was calculated.
 <em>DNA extraction and sequencing</em>
 Root samples allocated for DNA analyses were cut into 5 mm
 pieces, homogenised, and ground with beads. DNA was extracted from 20 mg
 of root and 250 mg of soil material using the DNeasy Plant Pro kit and
 DNease PowerSoil Pro kit, respectively, (Qiagen, Carlsbad, USA). PCR
 amplification and sequencing was performed by the Australian Genome
 Research Facility. For each sample, 15 ng DNA were used to amplify the 18S
 rRNA gene using the AMF primer set AMV4.5NF and AMDGR (Sato <em>et
 al.</em>, 2005). These primers accurately retrieve a wide range of
 AMF taxa, including both Glomeromycotina and Mucoromycotina subphyla
 (Orchard <em>et al.</em>, 2017; Albornoz <em>et
 al.</em>, 2022). Thermocycling was completed with an Applied
 Biosystem 384 Veriti and using Platinum SuperFi II mastermix (Life
 Technologies, Australia) for the primary PCR. Thermocycling consisted of
 an initial denaturation at 98℃ for 30 s followed by 30 cycles of 98℃ for
 10 s, 60℃ for 10 s and 72℃ for 30 s. The final extension was at 72℃ for 5
 min. The first stage PCR was cleaned using magnetic beads, and samples
 were visualised on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index
 the amplicons was performed with the same conditions with Platinum SuperFi
 II mastermix (Life Technologies, Australia). The resulting amplicons were
 cleaned using magnetic beads, quantified by fluorometry (Promega
 Quantifluor) and normalised. The eqimolar pool was cleaned a final time
 using magnetic beads to concentrate the pool and then measured using a
 High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was
 diluted to 5nM and molarity was confirmed using a Qubit High Sensitivity
 dsDNA assay (ThermoFisher). This was followed by sequencing on an Illumina
 MiSeq (San Diego, CA, USA) with a V3, 500 cycle kit (2 x 250 base pairs
 paired-end).
 <em>Bioinformatics</em> Following
 sequencing, adapters were trimmed using ‘cutadapt’ (Martin, 2011),
 retaining only sequences that contained primers. After trimming, further
 quality checks and sequence processing were done in VSEARCH v2.14.1 with
 default parameters (Rognes <em>et al.,</em> 2016). Trimmed
 paired-end sequences were merged with a minimum of 10 bp overlap. Merged
 sequences were filtered using a maximum error rate of 0.1 and a minimum
 length of 200 bp. Filtered sequences were dereplicated at 100% identity
 and singletons were discarded. Unique sequences were clustered into
 zero-radius operational taxonomic units (zOTUs), chimeras were detected
 (<em>denovo)</em>, and a zOTU abundance table was produced
 using the UNOISE3 algorithm in USEARCH v11 (Edgar, 2016). Finally, zOTUs
 were queried against the SILVA SSUref v138.1 database (Quast <em>et
 al.</em>, 2013). Taxonomy was assigned to zOTUs with a threshold of
 &gt; 95% match and query cover of &gt; 90%. Sequences matching
 Glomeromycotina or Mucoromycotina-AMF were classified as AMF.
 <em>Statistical analyses</em> Two
 samples failed to amplify DNA, resulting in 186 samples for statistical
 analyses. The initial denoised zOTU abundance table was rarefied to the
 smallest sequencing depth (4,927 sequences) to avoid sequencing depth bias
 (Dickie, 2010) (Fig. S1). All data were analysed, and
 figures were created, in R (R Core Team, 2016). To visualise variation in
 plant and AMF communities among ground-layer state, non-metric
 multidimensional scaling with Bray Curtis dissimilarity of the
 log-transformed matrix was used. To visualise species contributions to the
 ordination, two-way tables were constructed with the ‘inkspot’ function
 from the <em>rioja</em> package (Juggins and Juggins, 2020).
 Replicate plots were ordered on the x-axis by ground-layer states, while
 each individual zOTU was placed on the y-axis. Two-way tables summarise
 and present the raw community data as a powerful way of visualising the
 distribution of taxonomic units along environmental, spatial, or temporal
 gradients. To test for differences in community
 compositions among ground-layer states and source material (i.e., soil vs
 roots), permutational multivariate analysis of variance were performed
 using ‘adonis2’ within the <em>vegan</em> package (Oksanen
 <em>et al.</em>, 2017). To test which vegetation and soil
 variables correlated with plant and AMF communities among ground-layer
 states, vectors of maximum correlation were calculated with the
 vector-fitting procedure using ‘envfit’ (9,999 permutations) within the
 <em>vegan</em> package (Oksanen <em>et al.</em>,
 2017). A biplot was drawn on the ordination to display the relationships
 between explanatory variables and ordination axes. Because floristic
 variables strongly covaried (Fig. S2), principal component analyses (PCA)
 were used to distil covariates to PCA axes (Fig. S3). An ‘exoticPCA1’
 (i.e., first axis of a PCA between exotic plant cover and richness) and a
 ‘nativePCA1’ (i.e., first axis of a PCA between native plant cover and
 richness) indices were created to represent the difference in cover and
 richness for exotic and native plants, respectively.
 Differences in plant cover, plant richness, rarefied zOTU AMF
 richness (hereafter ‘AMF richness’), and soil chemistry among ground-layer
 states were evaluated. These differences were analysed using linear mixed
 effect models using ‘block’ as random effect. To test how environmental
 attributes associated with transition among ground-layer states related to
 AMF richness and community composition, structural equation models (SEM)
 were built using the <em>lavaan</em> R package (Rosseel,
 2012). To meet <em>lavaan</em>’s requirements, ground-layer
 state was transformed to an ordinal variable based on exotic plant
 abundance and levels of nutrient enrichment (reference = 0; weakly
 invaded/enriched = 1; moderately invaded/N-enriched = 2; highly
 invaded/NP-enriched = 3). To include a proxy of community composition in
 the SEMs, the first axis of a PCA of the log-transformed AMF community
 matrix was used. Due to high covariation among most soil variables (Fig.
 S2), models were simplified to only include variables with <em>a
 priori</em> knowledge of being involved in state transitions in
 eucalypt woodlands. These variables were OC, nitrate-N, ammonium-N, and
 available P, which are hypothesised to promote exotic annuals and AMF
 richness (Prober and Wiehl, 2012; Prober <em>et al.</em>,
 2014; Albornoz <em>et al.</em>, 2023). Neither nitrate-N nor
 ‘nativePCA1’ were selected in the best model. We
 acknowledge that feedbacks between soil chemistry and plants are likely.
 However, in our SEMs, we chose soil chemistry as the driver for exoticPCA1
 for two reasons: first, there is a priori knowledge that N enrichment
 promotes plant invasions in these ecosystems (Prober et al. 2012). Second,
 the highly invaded/NP-enriched state was created by deliberate nutrient
 addition, meaning it was demonstrated experimentally that nutrient
 addition promoted the observed plant invasions, not the other way
 around. Comparisons of AMF richness and communities
 between native and exotic plant species across treatments (i.e., Plant
 provenance × Ground-layer state) were not possible due to the absence of
 sufficient native plant species in the highly invaded/NP-enriched state,
 and exotic plant species from the reference state. Further, for most
 samples, no root material was left after prioritising the subsample for
 DNA analyses, leaving 31 (32%) samples for scoring colonisation. Hence,
 AMF richness and root colonisation comparisons between native and exotic
 plants are presented using ground-layer state as a random
 effect.
description: # Arbuscular mycorrhizal communities respond to nutrient enrichment and
 plant invasion in phosphorus-limited eucalypt woodlands
 [https://doi.org/10.5061/dryad.c866t1gfw](https://doi.org/10.5061/dryad.c866t1gfw) This data was used to conduct all analyses of the linked article. It contains treatments allocated to each sample, as well as all soil chemistry, vegetation, and arbuscular mycorrhizal fungi (AMF) variables ## Description of the data and file structure Three data files are provided: a raw community matrix of AMF (i.e. prior rarefaction), a raw community matrix of plants, and a data frame with the other is the environmental data frame with all soil chemistry, vegetation cover and richness, and AMF richness. Please refer to the published article for methodology on how these data were collected. All three data files are connected by the "sample" column from each data file. Each data file is in excel form and has two sheets: one with the data, and one with the metadata that explains each column. Missing data code: NA