DOI: 10.5061/dryad.4qrfj6qd1
title: Data from: Assembling microbial communities: a genomic analysis of a natural experiment in neotropical bamboo internodes

description: Microbes participate in ecological communities, much like multicellular
 organisms. However, microbial communities lack the centuries of
 observation and theory describing and predicting ecological processes
 available for multicellular organisms. Here, we examine early bacterial
 community assembly in the water-filled internodes of Amazonian bamboos
 from the genus Guadua. Bamboo stands form distinct habitat patches within
 the lowland Amazonian rainforest and provide habitat for a suite of
 vertebrate and invertebrate species. Guadua bamboos develop sealed,
 water-filled internodes as they grow. Internodes are presumed sterile or
 near-sterile (ie, containing small loads of endosphere-associated
 microbes) while closed, but most are eventually opened to the environment
 by animals, after which they are colonized by microbes.  We find
 that microbial community diversity increases sharply over the first few
 days of environmental exposure, and taxonomic identity of the microbes
 changes through this time period as is predicted for early community
 assembly in macroscopic communities. Microbial community taxonomic
 turnover is consistent at the bacteria phylum level, but at the level of
 Operational Taxonomic Units (OTUs), internode communities become
 increasingly differentiated through time. We argue that these tropical
 bamboos form an ideal study system for microbial community ecology due to
 their near-sterile condition prior to opening, relatively consistent
 environment after opening, and functionally limitless possibilities for
 replicates. Given the potential importance of opened internode habitats as
 locations of transmission for both pathogenic and beneficial microbes
 among animals, understanding the microbial dynamics of the internode is a
 key conservation concern for the insect and amphibian species that use
 this microhabitat.
description: <em>Sample Collection</em> We
 collected our samples at Los Amigos Biological Station in southern Peru
 starting on 30 November 2016. Los Amigos is located in lowland tropical
 rain forest adjacent to the Madre de Dios river, an Amazon river
 tributary. We collected water samples from eight bamboo stalks of the
 species <em>G</em><em>uadua weberbaueri </em>that
 were distributed across two plots in upland terra firme forest (Figure
 1A). We sampled four stalks from each plot. We determined the following
 eligibility for internode sampling: the internode must be 1 to 2 meters
 above the ground, contain water, and be closed. We opened the walls of
 bamboo internodes using a bleach-sterilized blade to cut a 20 – 40 mm
 notch horizontally near the top of the node, above the water level. Then
 we pulled back a section of the internode’s external skin using the edge
 of the blade (Figure 1B). We chose this method because it mimics the
 actions of foraging brown capuchin monkeys (Davidson et al. 2006; Jacobs
 and von May 2012). At the time of opening, we collected
 0.5mL of water from each bamboo internode using sterilized transfer
 pipettes (Figure 1B). We preserved these samples in 1.5mL of CTAB buffer.
 This buffer has been used in similar studies of microbial communities in
 other phytotelmata (e.g., pitcher plants; (Bittleston et al. 2016)). Our
 subsequent sampling occurred 24 hours after opening, 48 hours after
 opening, and 168 hours after opening. 
 <em>Nucleic Acid Extraction:</em>
 We extracted the total metagenomic DNA from the samples by
 incubating 200 microliters of CTAB and water mixture with 400 microliters
 of buffer ATL from Qiagen’s DNEasy Blood and Tissue kit and 10 microliters
 of proteinase K. We incubated the samples at 56 ̊C for 24 hours. We then
 performed a phenol-chloroform-isoamyl alcohol extraction following Barker
 1998. We chose the phenol-chloroform approach to minimize contamination by
 plant secondary metabolites that could inhibit PCR or sequencing
 reactions. We included a negative control in the extraction and processed
 and sequenced the control along with our samples. Samples were amplified
 using primers specifically developed to target the V3-V4 hypervariable
 region of the bacterial 16S rRNA gene (Kozich et al. 2013). We ran 25 PCR
 cycles with an annealing temperature of 55 C. Sequencing libraries were
 created with the NEBNext DNA Ultra prep kit 2 (cat# E7645L) with barcoded
 dual-index primers. Purified and eluted DNA amplicons
 were stored at 4 ̊C before being sent for sequencing. We standardized all
 PCR products using a Qubit measurement and pooled equimolar quantities of
 each separate PCR well for the final run. Each sample, taken from a single
 bamboo on a sampling day, was individually barcoded prior to DNA
 sequencing. Libraries were sequenced using the Illumina MiSeq platform.
 Data from this project can be found on NCBI’s Short Read Archive under
 BioProject PRJNA766623. <em>Bioinformatic
 pipeline</em>: We used the program mothur
 v.1.48.0 to identify OTUs (Schloss et al. 2009). Following the mothur
 miseq SOP, we made contigs from our sequences, removed fragments that were
 not within 8 base pairs of our 292 base pair target length, and removed
 sequences with ambiguous base calls or homopolymers over 8 bp long. We
 removed sequences that did not align within an 80% similarity cutoff to a
 16S bacteria sequence in the SILVA v.138 database (Quast et al. 2012). We
 identified the unique sequences in the dataset and removed chimeras. We
 matched each of our sequences to the RPD Classifier database using the
 mothur classify.seqs and removed any sequence that aligned most closely
 with a non-bacteria reference. We then clustered sequences at a 0.03
 distance threshold. Finally, we found the consensus classification for our
 OTUs using the RPD Classifier database v18. The RPD Classifier lists some
 chloroplast sequences under their “Bacteria” kingdom designation, using
 the phylum-level labels “Cyanobacteria/Chloroplast.” Since we were likely
 to have bamboo DNA in our extractions, we removed any plant contamination
 from our sequences, resulting in the removal of two OTUs.
 We used a custom script in R 4.1.1 to parse the output
 ‘mcc.shared’ sample by OTU matrix and ‘mcc.0.03.cons.taxonomy’ file into
 an OTU-by-sample matrix in which each cell of the matrix represented the
 number of reads of an OTU in a specific bamboo-day sample (R Core Team
 2021). Both input files and the code used for processing are available
 Dryad repository. To account for possible contamination, we removed any
 OTU represented by more than 10 sequences in our negative control.
 Finally, we set all OTUs with a read depth of 10 or less to zero to
 account for index hopping during sequencing. To account for possible
 amplification failure, we removed all days of any bamboo stems that had
 zero OTUs during any day besides the first. After these steps, we retained
 seven stems, 19-5, 19-7, 19-8, 19-10, 24-3, 24-6, and 24-9.
description: # Assembling microbial communities: a genomic analysis of a natural
 experiment in neotropical bamboo internodes
 [https://doi.org/10.5061/dryad.4qrfj6qd1](https://doi.org/10.5061/dryad.4qrfj6qd1) ## Description of the data and file structure This repository contains two data files and an R script that calls them. The data files are 16S rRNA metabarcode data that has been processed through the Mothur pipeline. The R script performs all analyses and generates all figures from the paper. ### Files and variables #### File: fin.opti\_mcc.shared **Description:** a sample x OTU matrix from samples from water in experimentally opened bamboo internodes. The OTUs are from 16S rRNA metabarcode data that has been processed through the Mothur pipeline. #### File: fin.opti\_mcc.0.03.cons.taxonomy **Description:** Taxonomic identification of OTUs in the sample x OTU matrix #### File: bamboo\_code.R **Description:**  The R script performs all analyses and generates all figures from the paper. ## Code/software All analyses are implemented in the R scripting language ## Access information Other publicly accessible locations of the data: * 16S rRNA sequences are available from [https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA766623](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA766623)