DOI: 10.5061/dryad.bcc2fqz7v
title: Data from: A test of community assembly rules using foliar endophytes from a tropical forest canopy

description: Community assembly theory assumes that ecological communities are
 spatially delimited into patches. Within these patches, coexistence
 results from environmental filtering, competition, and immigration. Truly
 delineated communities exist in laboratory studies of microbial cultures
 in Petri dishes, yet empirical tests conducted in continuous environments
 often use patches defined by convention as opposed to realised boundaries.
 Here we perform a test of ecological community assembly rules using foliar
 endophyte communities from a tropical rainforest, where leaves are
 considered as patches for both fungal and bacterial communities. We
 determined the diversity of fungal and bacterial endophytes using
 environmental DNA sequencing of 365 top-canopy leaves, collected from 38
 host trees belonging to 22 different species across a 4-hectare research
 plot. Three leaves were collected from three or more branches within each
 tree crown. We tested the effect of host tree species and their level of
 phylogenetic relatedness on community composition as well as the
 contribution of geographic distance between leaves to endophyte community
 diversity.  Endophyte diversity significantly differed
 across host tree species, as did community composition. Within certain
 endophytic orders (Xylariales, Rhizobiales) species assemblages
 significantly differed across host tree species, but this trend was weaker
 or non-existent in other orders known to contain pathogens and saprotrophs
 (Polyporales, Solirubrobacterales). Phylogenetically related host tree
 species displayed more similar endophyte communities than expected by
 chance, but geographically close trees did not. Consistent with the
 finding of host-specificity, nearby leaves tended to host more similar
 communities than distantly positioned ones. These findings
 demonstrate that foliar endophytes are structured by dispersal across
 small spatial scales, but at the scale of the canopy they display patterns
 of neutral filtering, with only a small part of variation described by
 host tree differences. Endophyte communities thus act as a model system in
 evoking the rules predicted by theoretical community ecology.
description: Data collection took place at the Nouragues Ecological Research
 Station in French Guiana, within an area of pristine lowland tropical
 rainforest protected since 1996. This area is characterised by a high
 level of floral diversity, with over 1700 recorded angiosperm species
 (Sabatier, 1990, van der Meer and Bongers, 1996). The reserve experiences
 mid-day temperatures of 26 °C and rainfall of 2861 mm per year
 (Réjou-Méchain et al. 2015). It experiences two dry seasons, a longer
 dry season between September and mid-November, and a shorter dry season in
 March Fieldwork described here took place in August
 2017. Sampling was conducted in a 4-ha permanent sampling plot adjacent to
 the Pararé research station (4°02’ N, 52°41’ W) located on the Arataye
 tributary of the Approuague River. Access to the canopy was made possible
 by the ‘Canopy Operational Permanent Access System’ (COPAS), consisting of
 a trio of 45 m high pylons set up at the vertices of an equilateral
 triangle with edges 180 m in length, equipped with a mechanically operated
 harness which allows for the displacement of an individual anywhere within
 the upper canopy across the research plot (Fig. 1).
 <i>Sample design and preparation</i>
 Leaves were collected from the upper crown of 38 trees, of 22
 different species, selected to represent a phylogenetically diverse range
 of species (Fig. 2 & S.I. 1). A stratified sampling design was
 conducted for each tree: three leaves were collected from each of a
 minimum of three branches positioned across the crown. For those trees
 with a large crown, 4 or 5 branches were selected accordingly, so that
 sampling accurately represented the total spread of the crown. In total
 this resulted in 365 leaves Leaves were immediately
 transported to a field laboratory where they were surface sterilised in
 order to ensure that sampling consisted of only endophytes. This
 sterilisation included several steps, first mechanical cleaning and
 scraping with toothbrushes. Secondly, following from the protocol detailed
 by Arnold et al. (2000), leaves were soaked in diluted bleach (0,525%) for
 2 min before transfer into ethanol (70%) for a further 2 min. This method
 is the standard in eDNA extraction for endophyte research (e.g. Izuno et
 al. 2016, Haruna et al. 2018), proving efficient in removing the maximum
 amount of epiphytic fungi and bacteria from the leaf surface. Finally,
 leaves were dried with tissue paper, and stored in Ziploc bags containing
 silica gel before transfer to the lab for DNA extraction.
 <i>DNA Isolation, PCR amplification and
 sequencing</i> Two 0.5 cm<sup>2</sup>
 pieces of each leaf were placed into a 96 well plate, and a 3mm aluminium
 bead was added to each well. To facilitate the grinding step, the sealed
 plates were placed in a -80 °C freezer for at least an hour before
 grinding using a Tissue Lyser (<i>Qiagen, Germany</i>) for 45
 sec at 30Hz, followed by a centrifugation for 30 seconds at 6000 x g. DNA
 was extracted from samples using a NucleoSpin Plant II (96) kit
 (<i>Macherey-Nagel, Düren, Germany</i>) according to the
 manufacturer protocol modifying only cell lysis time to 1 h and elution
 volume to 100µL PCRs were performed using two primers;
 ITS1 nuclear rDNA primers to target fungi (Fwd: ITS5
 GGAAGTAAAAGTCGTAACAAGG (Epp et al. 2012) and a modified version of Rev:
 5.8S_Fungi CAAGAGATCCGTTGTTGAAAGTK, Taberlet et al. 2018), and 16S rDNA
 (V5-V6) primers to target bacteria (Bact01 primers - Fwd:
 GGATTAGATACCCTGGTAGT and Rev: CACGACACGAGCTGACG (Fliegerova et al. 2014)).
 To discriminate samples after sequencing, forward and reverse primers were
 synthetized with a combination of two-different 8-nucleotide tags per
 sample, following a double-indexing strategy (Binladen et al. 2007). As
 such, each PCR was amplified with a unique combination of tagged primers.
 Each PCR reaction was performed in a total volume of 20 µl and comprised
 10 µl of AmpliTaq GoldMaster Mix (Life Technologies, Carlsbad, CA, USA),
 5.84 µL of nuclease-free water Ambion<sup>TM</sup>
 (Invitrogen, Waltham, Massachusetts, USA), 0.25 µM of each primer, 3.2µg
 of BSA (Roche Diagnostic, Basel, Switzerland), and 2 µl of DNA template.
 Two PCRs per sample were performed under the following conditions:
 Polymerase reactivation 10 min at 95°C, followed by 35 (fungi) or 30
 (bacteria) cycles of 30 sec at 95°C, 30 sec at 55°C (fungi) or 57°C
 (bacteria) and 1 min at 72°C; followed by a final step of 7 min at 72°C.
 Two wells in each plate were filled with water, to act as PCR negative
 controls. Eight wells positioned randomly across each plate were left
 empty (with no PCR products) to act as sequencing controls (non-used tag
 combinations) Amplicons were pooled and libraries were
 prepared with the TruSeqNano PCR free Illumina kit and were sequenced on
 Paired-end (2X250 bp) in the Illumina Miseq platform (Illumina, San Diego,
 CA, USA) at the INRA Genotoul-GetPlaGe core facility (Toulouse, France)
 using the Paired-end MiSeq Reagent Kit V3 (Illumina, San Diego, CA, USA),
 following the manufacturer’s instructions
 <i>Sequence data curation</i> The
 generated reads were subject to a data-curation pipeline using scripts in
 R (R Development Core Team, 2013), and the OBITools package (Boyer et al.
 2016). Firstly, ‘Illuminapairedend’ was used for paired-end read assembly
 where an exact alignment algorithm assigns a quality score for each
 nucleotide position, generating a score for each read based on the number
 of mismatches, followed by ‘ngsfilter’ which removes primer and tag
 sequences and assigns reads to samples. Here, reads with any primers with
 more than two mismatches, and any tags with 1 or more mismatches are
 removed. ‘obiuniq’ then dereplicates only those sequences which are
 strictly identical. ‘obigreb’ removes sequences of low quality (containing
 Ns or with a pair alignment score below 50). Finally sequences with only
 one read across the global dataset (singletons) were removed, since these
 represent potential sequencing artefacts.‘sumaclust’ was then used to
 generate Operational Taxonomic Units (OTUs), where pairwise sequence
 dissimilarity using the raw number of mismatches was calculated, and where
 those sequences ³ 97% or more were assigned to the same OTU, a threshold
 commonly applied to delimitate proxies for during molecular analysis of
 fungi in particular (Nilsson et al., 2008; Coissac 2012). For each OTU,
 the sequence with the highest number of reads in each cluster was assigned
 as the seed sequence, used for subsequent taxonomic assignment.
 Taxonomic assignment of OTUs was conducted using reference
 databases with the ‘ecotag’ function, which uses global alignment of
 sequences against full-length references. For fungi the reference database
 was obtained by running an in-silico PCR with the ecoPCR program (Ficetola
 et al., 2010) on Genbank (release 197; ftp://ftp.ncbi.nlm.nih.gov/genbank)
 using the primer pairs employed here. Taxonomic assignment yielding the
 highest similarity score was kept, with similarity scores for fungi
 ranging from 0.5 to 1 with sequences in ncbi. For Bacteria, taxonomic
 assignment was conducted using the SILVA database (Quast et al. 2012),
 with similarity parameters kept at 0.97, higher than fungi since bacterial
 sequence databases are much more complete than for fungi (Truong et al.,
 2017). This approach allowed for the elimination of a certain number of
 chimeras which are likely to have formed during PCR. In particular,
 taxonomic identification facilitated later analysis, by distinguishing
 between orders known to contain OTUs corresponding to endophytic fungi
 (Xylariales, Capnodiales, Chaetothyriales, Phyllachorales, Diaporthales,
 Sordariales, Pleosporales, Hypocreales) against orders which are known to
 contain generalist saprotrophs (Polyporales, Eurotiales, Corticiales).
 Similarly, bacterial endophyte orders which have strict relationships with
 plants (e.g. Rhizobiales, Acetobacterales, Sphingomonadales) could be
 compared with orders known to contain more generalist bacteria (e.g.
 Solirubrobacterales, Pseudonocardiales, Acidobacteriales).
description: Here we provide the sequence files post datacuration using the
 Obitools pipeline (ITSpoolTOT for fungi,
 SECIL3_NoIndex_R1R2_ngsfilt_n50_ali_uniq_noS_cl97 for bacteria).  We also
 provide the sample file which associates this information with the leaves
 these OTUs were generated from (Sample_Info_blanks4 for fungi,
 Sample_Info_blanks3 for bacteria)