DOI: 10.5061/dryad.6wwpzgn78
title: Data for analyzing the microbiome of bromeliads and fly larvae inhabiting the tropical cloud forest of Mexico

description: Background. The tropical montane cloud forest contains high levels of
 epiphyte diversity. Epiphytic tank bromeliads play an important role in
 the functioning of these ecosystems and provide a microhabitat for many
 species of invertebrates. Microbial ecology theory suggests that the
 environment serves as a source of microbes for animals, but the
 contribution of this factor to the composition of an animal microbiome
 varies. In this study, we examined the extent to which tank bromeliads
 (Tillandsia multicaulis) serve as a source of microbes for fly larvae in a
 cloud forest fragment in central Veracruz, Mexico. Methods. We used 16S
 rRNA sequencing to characterize the bacterial communities in the organic
 matter within bromeliad tanks and in the whole bodies (surface and gut) of
 larvae from two fly taxa (Austrophorocera sp. and Copestylum sp.) that
 inhabit these bromeliads. To assess the contribution of bromeliads to the
 microbiome of the fly larvae, we conducted fast expectation-maximization
 microbial source tracking (FEAST) analysis. Results. The bacterial
 communities in bromeliad tanks were primarily composed of Pseudomonadota,
 Acidobacteriota, Bacteroidota, Verrucomicrobiota, and Spirochaetota.
 Similarly, communities of the fly larvae contained Pseudomonadota,
 Bacteroidota, Bacillota, and Actinomycetota. Bromeliad tanks exhibited the
 highest bacterial richness, followed by Copestylum and Austrophorocera
 larvae. Beta diversity analyses indicated that bacterial communities
 clustered by species. We found a modest contribution of bromeliads to the
 fly microbiome, with nearly 30% of the larvae microbiome originating from
 the organic matter deposited in the tanks. Conclusions. Our data suggest
 that the microbiome of Diptera larvae, which inhabit tank bromeliads
 during their larval stage, is nourished to some extent by the bacterial
 communities present in the organic matter within the tank.
description: Data was generated from 16S rRNA sequencing from two sample
 types: organic matter contained in the tank of bromeliads
 (<em>Tillandsia multicaulis</em>) and from larvae of two fly
 taxa (<em>Austrophorocera</em> sp. and
 <em>Copestylum</em> sp.). Microbial reads were processed in
 QIIME2 and further analyses were conducted in R, using several
 packages.
description: # Microbiome data from bromeliads and fly larvae
 [https://doi.org/10.5061/dryad.6wwpzgn78](https://doi.org/10.5061/dryad.6wwpzgn78) ## Description of the data and file structure All files are found in the compressed folder 'bromeliads-flies-microbiome-working-files.zip'. The files containing the unweighted (**unw-distance-matrix.tsv**) and weighted (**we-distance-matrix.tsv**) UniFrac distance matrices are used to run beta diversity analyses. The three **.qza** files contain processed microbiome information and are required to create a phyloseq object in R. This phyloseq object is used for subsequent diversity and taxonomy analysis. To run FEAST analysis in R, we also included a specific metadata file (**1_metadata_for_FEAST.txt**) and a data frame (**2_feature_table_bromeliads_for_FEAST.txt**) containing the contribution (proportions) of each bromeliad microbiome to the fly larvae microbiome. Additionally, we have provided three **.csv** files (**bromeliads_df.csv; 3_feast_graph.csv; and centroid_bromeliads.csv**) to create the plots used in our study. ## Files and variables ### Files * **unw-distance-matrix.tsv**:  A file containing dissimilarity distances between samples based on the unweighted UniFrac metric * **we-distance-matrix.tsv**:  A file containing dissimilarity distances between samples based on the weighted UniFrac metric * **metadata_bromeliads.tsv**: Metadata containing categorical variables * **merged-table-final.qza**:  Table containing all ASVs calculated per sample * **bromeliads-rooted-tree.qza**:  The microbial phylogenetic tree used in different analysis * **bromeliads-taxonomy.qza:**  A file containing the taxonomy of each ASV * **bromeliads_df.csv**: A file that contains alpha diversity and microbial distances per sample * **1_metadata_for_FEAST.txt**:  Metadata containing the categorical variables used for FEAST analysis * **2_feature_table_bromeliads_for_FEAST.txt**:  A data frame containing proportions of bromeliads as source of microbes to each fly sample * **3_feast_graph.csv**: A file that contains proportion data from FEAST analysis to create a plot * **centroid_bromeliads.csv**: A file that contains the calculated distances to the centroid; data used to create plots ### Variables **Description:** This file contains the categorical variables to be tested in alpha and beta diversity analysis and taxonomic analysis. * **metadata_bromeliads.tsv** SampleID: Sample identification BarcodeSequence: Empty cells LinkerPrimerSequence: Empty cells Country: Country where the study took place Location: State within country where the study took place Habitat: Forest type where samples were collected. Sample: Sample IDs used in the creation of the Venn diagram Individual: Code for each sample based on its origin SampleType: Type of sample; either fly larvae or bromeliads Species: Species type including bromeliads and the two fly taxa **Description**: This file contains the categorical variables to be tested for FEAST analysis. * **1_metadata_for_FEAST.tsv** SampleID: Sample identification Env: Type of environmental sample; either fly larvae or bromeliad phytotelma SourceSink: Samples set as "source" or "sink". Bromeliad phytotelma was considered the source and fly larvae were considered sink ID: Sequential numbers for each "sink" samples Species: Species type including bromeliads and the two fly taxa **Description**: This file contains categorical and numerical variables to plot changes in the proportion of microbial contribution from bromeliads to flies. * **3_feast_graph.csv** Host: Fly taxa that served as hosts of microbial communities. Source: Source of microbes; either bromeliad phytotelma or an unknown source Proportion: Contribution of the source to the microbiome of fly larvae (unit: proportions from 0 to 1) **Description**: This file contains categorical and numerical variables to plot changes in two metrics of alpha diversity. * **bromeliads_df.csv** IDSample: Sample identification Species: Species type including bromeliads and the two fly taxa ASVs: Amplicon sequence variants (ASVs) per sample. This is an estimation of microbial richness to assess alpha diversity (unit: observed numbers of ASVs; the higher the number the higher the amount of ASVs found in samples) PD: Phylogenetic diversity per sample (unit: numbers expressing diversity; higher the number the higher the PD) **Description**: This file contains categorical and numerical variables to plot differences in the distance-to-centroid per sample. * **centroid_bromeliads.csv** IDSample: Sample identification dist.uw: Unweighted UniFrac distances from each sample to the centroid of the cluster. This metric takes into account microbial presence only (unit: values ranging from 0 to 1). dist.we: Weighted UniFrac distances from each sample to the centroid of the cluster. This metric takes into account the relative abundance of microbes within the community (unit: values ranging from 0 to 1). Species: Species type including bromeliads and the two fly taxa ## Code/software **Description**: These two files provide the scripts to replicate all our analyses. File S1 contains the script to processes the raw sequences in QIIME2. File S2 contains the script to run alpha and beta microbial diversity analysis, taxonomic analysis, creation of plots, and FEAST analyses. File S1. The scripts used in this study to run QIIME2 sequencing processing File S2. The R scripts used in this study to run all statistical analyses Software versions: Quantitative Insights into Microbial Ecology 2 QIIME2 (qiime2-amplicon-2023.9) RStudio (4.3.2) Package versions: qiimer (0.9.4) vegan (2.6.8) permute (0.9.7) lattice (0.22.6) tidyverse (2.0.0) qiime2R (0.99.6) phyloseq (1.46.0) microbiomeMarker (1.9.0) RColorBrewer (1.1.3) data.table (1.17.0) ggpubr (0.6.0) cowplot (1.1.3) MicEco (0.9.19) FEAST (0.1.0) Rccp (1.0.12) RccpArmadillo (0.12.8.1.0) ggthemes (5.1.0) gridExtra (2.3) reshape2 (1.4.4) conflicted (1.2.0) ## Access information Other publicly accessible locations of the data: * Microbial sequences were deposited at Sequence Read Archive (SRA): accession number BioProject PRJNA1146765