DOI: 10.5061/dryad.qz612jmrc
title: Bacterial communities and soil chemistry from ten established invasions of Lupinus polyphyllus in southwestern Finland, 2020

description: Plants host microorganisms that can facilitate their success in becoming
 invasive. Established plant invasions might thus provide useful insights
 into potential changes in plant-associated microbiomes over the course of
 the invasion process. Here, we investigated the endophytic bacterial
 communities of the invasive herbaceous legume Lupinus polyphyllus, which
 is able to form mutualistic associations with N-fixing bacteria. More
 specifically, we examined the alpha diversity (observed bacterial taxa
 richness and Shannon diversity) and composition of bacterial communities
 in roots and nodules sampled from core and edge locations within 10
 established invasion sites (&gt;10 years old) in southwestern Finland.
 Moreover, we compared the alpha diversity and structure of bacterial
 communities in the rhizosphere and bulk soil between core and edge
 locations within these invasion sites. We found that roots and nodules had
 distinctive endophytic bacterial communities, with roots having 24% higher
 bacterial alpha diversity (Shannon diversity) than nodules. In nodules,
 the dominant bacteria were assigned to the family Bradyrhizobiaceae, which
 includes N-fixing bacteria. Soil bacterial communities, instead, were
 shaped by soil type, with bulk soil hosting up to 27% higher alpha
 diversity (richness and Shannon diversity) than rhizosphere soil; however,
 there was no apparent difference in their community composition. Soil
 bacterial communities were only weakly associated with soil chemistry.
  Endophytic and soil bacterial communities did not differ between
 core and edge locations within the established invasions. Our findings
 suggest that L. polyphyllus may not induce dramatic changes in the
 bacterial communities with which it associates over the course of the
 local invasion process.
description: <strong>Root and soil sampling</strong>
 We carried out root and soil sampling in 10 sites with
 established invasions of <em>L</em>.
 <em>polyphyllus</em> over four days (29 June–2 July) in the
 summer of 2020 when the species was flowering. All of these sites were
 close to Turku in southwestern Finland (latitude: 60.357–60.521,
 longitude: 22.168–22.740; distances 1.7–32.7 km apart). They featured
 sandy moraine or clay soil and were located in wastelands, road verges,
 former fields, or forest understoreys. The exact age of<em>
 </em>the invasions is unknown, but the species has been present in
 each location since at least 2010 when we visited the populations
 for<em> </em>the first time (Ramula, 2014). The size of these
 invasions vary between 65-2400 m<sup>2</sup> (mean ± SD =
 617.4 ± 736.4 m<sup>2</sup>) and currently, they contain
 hundreds of individuals (mean<em> </em>cover = 59.7%, range:
 2%–96%). In each invasion site, we haphazardly sampled 10 flowering
 individuals from the core and edge locations, respectively, with plant
 density typically being lower in the latter (mean ± SD = 3.3 ± 1.1 for
 core and 2.0 ± 0.9 for edge). We dug up each sampled plant, used a spade
 to gently remove soil close to its root system, cut a ca. 10-cm piece of
 root containing nodules, and placed it in a plastic ziplock bag, resulting
 in a total of 200 root samples (10 plants × 2 locations × 10 invasion
 sites). In addition, we collected soil samples from the ectorhizosphere
 (hereafter rhizosphere) of three plants (at a depth of 10–15 cm) and bulk
 soil (at a depth of &gt;20 cm) in the core and edge locations; these
 samples were pooled within each location per invasion site and stored in a
 plastic bag. Altogether, we had 40 soil samples (2 locations (core and
 edge) × 2 soil types (rhizosphere and bulk) × 10 invasion sites). To avoid
 cross-contamination, the spade and secateurs were wiped with ethanol after
 each sampling event. We transported all samples in a cold box to the
 laboratory and preserved the soil samples at -80<sup>o</sup>C
 for later use. In the laboratory, we rinsed the roots with tap water to
 remove the soil, and cut about 2 cm of each sample and some nodules for
 further analysis. These root and nodule samples were surface-sterilised in
 96% ethanol for 1 min and 3% NaClO for 3–5 min and rinsed with deionised
 water three times. The specimens were preserved in Eppendorf tubes at
 -80<sup>o</sup>C for DNA isolation. To
 characterise soil chemistry, we collected separate soil samples from the
 rhizosphere of <em>L. polyphyllus</em> in the core and edge
 locations of each invasion site (no bulk soil was sampled). Again, we
 sampled three flowering plants per location and pooled their rhizosphere
 soil (0.5 L soil in total), resulting in 20 soil samples (2 locations × 10
 invasion sites). The following soil properties were analysed: total
 nitrogen (N), nitrate
 (NO<sub>3</sub><sup>-</sup>), ammonium
 (NH<sub>4</sub><sup>+</sup>), calcium (Ca),
 phosphorus (P), potassium (K), magnesium (Mg), and pH. The soil analysis
 was carried out by Eurofins Agro in Finland
 (https://www.eurofins.fi/agro). <strong>DNA
 extraction</strong> We cut the root samples into
 pieces 2–4 mm in length using sterilised (in 70% ethanol) blades. We then
 crushed the nodules and root pieces (up to 150 mg) using tweezers
 sterilised in 70% ethanol for DNA extraction. For the soil samples, we
 used 300 mg of each sample for DNA extraction. For all samples (roots,
 nodules, soil), we extracted DNA with the NucleoSpin Soil Kit
 (Macherey-Nagel GmbH Co. KG, Duren, Germany) following the provided
 protocol. To improve the DNA yield, root and nodule samples were placed in
 TissueLyser II (Qiagen) for the sample lysis step. The extracted DNA
 samples were preserved at -20<sup>o</sup>C for PCR and
 sequencing. <strong>Polymerase chain reaction
 (PCR)</strong> Locus-specific PCR and library
 preparation was done by the company Bioname Ltd, Finland (www.bioname.fi).
 The ribosomal bacterial 16S v8–v9 gene region was amplified using nested
 PCR and two primer pairs (799F [AACMGGATTAGATACCCKG; Chelius &
 Triplett, 2001] and 1492R [GGYTACCTTGTTACGACTT; Lane 1991]), and (1062F
 [GTCAGCTCGTGYYGTGA; modified from Ghyselink <em>et al.</em>,
 2013] and 1390R [ACGGGCGGTGTGTRCAA; modified from Zheng <em>et
 al</em>., 1996]). The secondary pair of the primers included a
 linker-tag to enable the subsequent attachment of NGS adapters. To
 increase amplicon library diversity, the secondary primer pair was used as
 four different versions so that they included heterogeneity spacers
 between the linker-tag and the locus-specific oligo. A blank PCR control
 was added to each PCR batch to evaluate the purity of reagents and the
 level of cross-contamination. In this project, the PCR controls were done
 in parallel in separate tubes because the DNA plates contained no empty
 wells. Initially, we carried out a pre-PCR using the primers 799F and
 1492R to amplify a longer fragment of the bacterial 16S region. This
 primer pair is used to exclude plant chloroplast amplification (Chelius
 & Triplett, 2001). The reaction setup followed Kankaanpää et al.
 (2020) and included 5 μL of 2×MyTaq HS Red Mix (Bioline, UK), 2.4 μL of
 H<sub>2</sub>O, 150 nM of each primer, and 2 μL of DNA extract
 per sample in a 10-μL volume. Cycling conditions for 799F and 1492R were
 as follows: 3 min at 95°C, 35 cycles of 45 sec at 95°C, 45 sec at 54°C,
 and 1 min at 72°C, ending with 5 min at 72°C. The nested PCR reaction
 included the locus-specific primer pair 1062F and 1390R. The PCR mixture
 from the pre-PCR round was used as a DNA template in this round of PCR.
 All PCR reactions after the pre-PCR were carried out in two technical
 replicates, in which each replicate contained two heterogeneity versions
 of each primer. <strong>Library preparation and
 sequencing</strong> Library PCR followed, with
 minor modifications, the procedure of Vesterinen et al. (2016). We used a
 dual indexing strategy, in which each reaction (including technical
 replicates) was prepared with a unique combination of forward and reverse
 indices. All index sets were balanced perfectly so that each nucleotide
 position included either T/G or A/C, which ensures base calling for each
 channel during sequencing. For a reaction volume of 10 μL, we mixed 5 μL
 of MyTaq HS RedMix, 500 nM of each tagged and indexed primer (i7 and i5),
 and 3 μL of locus‐specific PCR product from the first PCR phase. For
 library preparation PCR, we used the following protocol: initial
 denaturation for 3 min at 98°C, then 12 cycles of 20 s at 95°C, 15 s at
 60°C, and 30 s at 72°C, followed by 3 min at 72°C. All the indexed
 reactions were then pooled and purified using magnetic beads (Vesterinen
 <em>et al</em>., 2018). Sequencing was done at the Turku
 Centre for Biotechnology (Turku, Finland), on an Illumina NovaSeq6000 SP
 FlowCell v1.5 PE 2x250 apparatus (Illumina Inc., San Diego, California,
 USA), run together with other samples, including a PhiX control
 library. <strong>Bioinformatics and diversity
 measures</strong> Our bioinformatics pipeline
 closely followed that in Kaunisto et al. (2020) as summarised
 below<em>. </em>In this study, we processed a total of 44 489
 226 raw reads (25 151 901 in PCR replicate 1 and 19 337 325 in PCR
 replicate 2). Paired-end reads were merged and trimmed for quality using
 64-bit VSEARCH version 2.14.2 (Rognes <em>et al</em>., 2016),
 and the primers were removed from the merged reads using the software
 CUTADAPT version 2.7 (Martin, 2011) with 20% rate for primer mismatches
 and strict length parameters: minimum length 285 bp, maximum length 320 bp
 (longer sequences clipped from 3’ end). After removing singletons and
 chimers, the unique reads were clustered into ZOTUs (zero-radius
 operational taxonomic units) with 32-bit USEARCH version 11 (Edgar, 2010).
 ZOTUs were mapped back to the primer-trimmed reads to construct a zotu
 table with the ‘usearch_global’ algorithm in VSEARCH (Rognes <em>et
 al</em>., 2016). The resulting sequence variants were assigned to
 taxa using custom databases with SINTAX (Edgar, 2010) in VSEARCH (Rognes
 et al. 2016) The databases were downloaded from
 https://drive5.com/usearch/manual/sintax_downloads.html with the following
 reference: 16S RDP training set rdp_16s_v18 (21k seqs).<strong>
 </strong>The reads from both PCR replicates were pooled ifreads were
 detected in both of them. We removed singletons (read count &lt;2) and
 only retained reads assigned as Bacteria. Bioinformatic
 processing resulted in 15 003 ZOTUs for subsequent analyses. Since the
 sampling design for soil differed from that for roots and nodules, we
 analysed these two data sets separately. After rarefying (8 104 sequences
 per sample for roots and nodules and 88 sequences for soil), we continued
 our analyses with a total of 13 478 ZOTUs for the root and nodule samples,
 and 1 141 ZOTUs for the soil samples. Low sequence counts for soil were
 due to the inadequate dilution of the samples. To evaluate the diversity
 of these bacterial communities, we calculated two alpha diversity measures
 (observed ZOTU richness and Shannon index (H’)) separately for the root
 and nodule data and for the soil data using the R (R Core Team, 2023)
 packages “phyloseq” (McMurdie & Holmes, 2013) and “microeco” (Liu
 <em>et al</em>., 2021).
 <strong>References</strong>
 Chelius, M. K., & Triplett, E. W. (2001) The diversity of
 archaea and bacteria in association with the roots of <em>Zea mays
 </em>L. <em>Microbial Ecology</em>, 41,
 252–263. Ghyselinck, J., Pfeiffer, S., Heylen, K.,
 Sessitch, A., & De Vos, P. (2013) The effect of primer choice and
 short read sequences on the outcome of 16S rRNA gene based diversity
 studies. <em>PLoS One</em>, 8, e71360.
 Kankaanpää, T.,  Vesterinen, E., Hardwick, B., Schmidt, N. M., Andersson, T., Aspholm, P. E., Barrio, I. C., Beckers, N., Bêty, J., Birkemoe, T., et al. (2020)<em> </em> Parasitoids indicate major climate‐induced shifts in arctic communities. <em>Global Change Biology</em>, 26, 6276–2695. Kaunisto, K. M., Roslin, T., Forbes, M. R., Morrill, A., Sääksjärvi, I. E., Puisto, A. I., Lilley, T. M., & Vesterinen, E. J. (2020) Threats from the air: Damselfly predation on diverse prey taxa. <em>Journal of Animal Ecology</em>, 89, 1365-1374. Lane, D. J. (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, GoodfellowM (eds.), <em>Nucleic Acid Techniques in Bacterial Systematics</em>. NewYork: Wiley, 115–175. Liu, C., Cui, Y., Li, X., & Yao, M. (2021) Microeco: An R package for data mining in microbial community ecology. <em>FEMS Microbiology Ecology</em>, 97, fiaa255. McMurdie, P. J., & Holmes, S. (2013) Phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. <em>PLoS ONE</em>, 8, e61217. R Core Team. (2023) R: A Language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing. Available from: https://www.R-project.org/ Ramula, S. (2014) Linking vital rates to invasiveness of a perennial herb. <em>Oecologia</em>, 174, 1255-1264. Rognes, T., Flouri, T., Nichols, B., Quince, C., & Mahé F. (2016) VSEARCH: a versatile open source tool for metagenomics. <em>PeerJ</em>, 18, e2584 Vesterinen, E. J., Ruokolainen, L., Wahlberg, N., Peña, C., Roslin, T., Laine, V. N., Vasko, V., Sääksjärvi, I. E., Norrdahl, K., & Lilley, T. M. (2016) What you need is what you eat? Prey selection by the bat <em>Myotis daubentoniid</em>. <em>Molecular Ecology</em>, 25(7), 1581–1594. Vesterinen, E. J., Puisto, A. I. E., Blomberg, A. S., & Lilley, T. M. (2018) Table for five, please: dietary partitioning in boreal bats. <em>Ecology and Evolution</em>, 8, 10914–10937. Zheng, D., Alm, E. W., Stahl, D. A., & Raskin, L. (1996) Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. <em>Applied Environmental Microbiology</em>, 62, 4504–1453.
description: # Bacterial communities and soil chemistry from ten established invasions
 of Lupinus polyphyllus in southwestern Finland, 2020 \[Access this dataset
 on Dryad] doi:10.5061/dryad.qz612jmrc Content: This dataset consists of
 three data files (Bacteria.root.nodule.csv, Bacteria.soil.csv,
 Soil.chemistry.csv) that contain all variables used for the statistical
 analyses presented in Ramula, Mousavi, and Vesterinen "Root, nodule,
 and soil bacterial communities associated with the invasive
 nitrogen-fixing Lupinus polyphyllus", submitted for Ecology and
 Evolution. Experimental procedures: To examine the alpha diversity and
 composition of endophytic and soil bacterial communities in core and edge
 locations within established invasions, samples were collected within ten
 sites invaded by the herbaceous Lupinus polyphyllus (&gt;10 years old) in
 southwestern Finland. The distances between the ten sample sites varied
 from 1.7 to 32.7 km. In each invasion site, 10 haphazardly chosen
 flowering individuals were sampled from the core and edge locations,
 respectively, resulting in a total of 200 root samples (10 plants × 2
 locations × 10 invasion sites). In addition, soil samples were collected
 from the rhizosphere of three flowering plants (at a depth of 10–15 cm)
 and bulk soil (at a depth of &gt;20 cm) in the core and edge locations
 within invasion sites; these samples were pooled within each location per
 invasion site. Bacterial DNA was identified by sequencing the gene 16S
 rRNA, resulting in a total of 13 487 ZOTUs (zero-radius operational
 taxonomic units) for the root and nodule samples, and 1 141 ZOTUs for the
 soil samples after rarefying the data. To characterise soil bacterial
 diversity, bacterial richness (i.e., number of bacterial ZOTUs per sample)
 and Shannon diversity index were calculated based on the bacterial ZOTUs.
 To characterise soil chemistry, separate soil samples were collected from
 the rhizosphere of L. polyphyllus in the core and edge locations of each
 invasion site (no bulk soil was sampled). Again, three flowering plants
 were sampled per location and their rhizosphere soil was pooled (0.5 L
 soil in total), resulting in 20 soil samples (2 locations × 10 invasion
 sites). The following soil properties were analysed: total nitrogen (N),
 nitrate (NO3-), ammonium (NH4+), calcium (Ca), phosphorus (P), potassium
 (K), magnesium (Mg), and pH. The soil analysis was carried out by Eurofins
 Agro in Finland
 ([https://www.eurofins.fi/agro](https://www.eurofins.fi/agro)). Date of
 data collection: June-July 2020 (range: 2020-07-29 - 2020-07-02) ##
 Description of the data and file structure: ### Bacteria.root.nodule.csv
 Description: This data file contains bacterial communities in roots and
 nodules sampled within 10 invasion sites of the legume Lupinus polyphyllus
 in southwestern Finland, 2020. Definitions of all variables in the file:
 Site: ten invasion sites of L. polyphyllus in alphabetics (range: A-J)
 Location: sampling location within each invasion site with two levels
 (Core = core location, Edge = edge location) Tissue.type: plant tissue
 used for the extraction of bacterial DNA with two levels (Root, Nodule)
 Replicate: a replicate code per location at each site (range: 1-10 L.
 polyphyllus individuals sampled) Sample.ID: a unique sample code used for
 genetic libraries (380 samples in total) Richness: number of ZOTUs oberved
 per sample Shannon: Shannon deiversity index (H’) calculated from ZOTUs
 ZOTU1-ZOTU9491: columns containing the relative abundances of bacterial
 zero-radius operational taxonomic units (ZOTUs), 13478 ZOTUs in total.
 Note that the ZOTUs are not shown in consecutive numbers from smallest to
 largest. ### Bacteria.soil.csv Description: This data file contains soil
 bacterial communities sampled in core and edge locations within 10
 invasion sites of the legume Lupinus polyphyllus in southwestern Finland,
 2020. Definitions of all variables in the file: Site: ten invasion sites
 of L. polyphyllus in alphabetics (range: A-J) Location: sampling location
 within each invasion site with two levels (Core = core location, Edge =
 edge location) Soil.type: soil type used for the extraction of bacterial
 DNA with two levels (Bulk, Rhizosphere) Sample.ID: a unique sample code
 for genetic libraries (38 samples in total) Richness: number of ZOTUs per
 sample Shannon: Shannon diversity index (H’) calculated from ZOTUs
 ZOTU1-ZOTU9513: columns containing the relative abundances of bacterial
 zero-radius operational taxonomic units (ZOTUs), 1141 ZOTUs in total. Note
 that the ZOTUs are not shown in consecutive numbers from smallest to
 largest. ### Soil.chemistry.csv Soil chemistry in core and edge locations
 within 10 invasion sites of the legume Lupinus polyphyllus (n=20 soil
 samples) in southwestern Finland, 2020. Definitions of all variables in
 the file: Site: ten invasion sites of L. polyphyllus in alphabetics
 (range: A-J) Location: sampling location within each invasion site with
 two levels (Core = core location, Edge = edge location) pH: soil pH Ca:
 soil calsium content (mg/L) P: soil phosphorus content (mg/L) K: soil
 potassium content (mg/L) Mg: soil magnesium content (mg/L) NO3: soil
 nitrate content (mg/L) NH4: soil ammonium content (mg/L) N.total: soil
 total nitrogen content (mg/L) ## Software Microsoft Excel can be used to
 view Bacteria.root.nodule.csv, Bacteria.soil.csv, and Soil.chemistry.csv.
 The data files can be used independently.