DOI: 10.5061/dryad.dz08kps7v
title: Host-microbiome associations in livebearing fishes adapted to toxic streams rich in hydrogen sulfide: Code for analyzing 16S rRNA dataset

description: Organisms inhabiting extreme environments must tolerate a variety of
 physiochemical stressors. In some cases, host-associated microbial
 communities facilitate survival of their hosts in extreme environments,
 but extremophile symbioses have not been identified in vertebrates. We
 used 16S rRNA amplicon sequencing to investigate commonalities and
 differences in the gut microbial communities of livebearing fish (Poecilia
 mexicana species complex, Poeciliidae) that have repeatedly colonized
 toxic sulfide streams in southern Mexico. We found shared core gut
 microbiomes across habitat types and drainages, but also differences in
 the microbiomes between sulfidic and nonsulfidic populations, both in
 terms of patterns of diversity and community composition. Most
 importantly, we documented convergent changes in microbiome composition
 across evolutionarily independent sulfide spring lineages. These patterns
 were consistent when we analyzed whole gut microbiomes and primarily
 host-associated microbiomes that excluded taxa that are commonly found in
 the environment. Our analyses also revealed several microbial taxa
 associated with sulfide spring colonization that have previously been
 implicated in symbioses and may influence the host’s tolerance to the
 extreme environmental conditions. Further research will need to test how
 host-associated microbes impact host function and fitness under extreme
 environmental conditions.
description: We sampled 8 populations of <em>P. mexicana</em>
 throughout the Río Grijalva basin in southern Mexico, dissected the
 intestines, preserved the whole intestines in ethanol, and used 16S rRNA
 amplicon sequencing to characterize the gut microbiomes. Fish were sampled
 from four river drainages (Tacotalpa, Ixtapangajoya, Pichucalco,
 Puyacatengo), and each drainage contained a sulfide-adapted and a
 sulfide-intolerant population of <em>P. mexicana</em>.
 Additionally, we collected water and sediment samples from all of the
 sites to characterize the environmental microbiomes, and we collected
 intestine samples from lab-raised fish following the same methods as in
 the field. The amplicon library was prepared following the Illumina MiSeq
 System 16S Metagenomic Sequencing Library Preparation protocol (Illumina),
 and it was sequenced on an Illumina MiSeq at the Kansas State University
 Integrated Genomics Facility. The sequences were processed using QIIME2,
 and the taxonomy count matrices are included here as input files for the R
 code.
description: # Host-microbiome associations in livebearing fishes adapted to toxic
 streams rich in hydrogen sulfide: Code for analyzing 16S rRNA dataset
 [https://doi.org/10.5061/dryad.dz08kps7v](https://doi.org/10.5061/dryad.dz08kps7v) ## Description of the data and file structure We sampled four population pairs of sulfide-adapted and sulfide-intolerant populations of livebearing fishes in the *P. mexicana* species complex throughout the Río Grijalva basin in southern Mexico to investigate patterns of gut microbial diversity between ecotypes and across river drainages. We additionally sampled environmental microbiomes from these sites, as well as gut microbiomes of *P. mexicana* raised in a laboratory setting. We aimed to understand whether sulfide-adapted lineages have commonalities in their gut microbiomes and identify microbes of interest for future research investigating potentially adaptive host-microbiome associations. ### Files and variables #### File: Labonly-merged-reads-level-6-taxonomy-rarefied-depth-18834.csv **Description:** This file contains the taxonomy counts for the laboratory samples, which includes sulfide-adapted and sulfide-intolerant fish that were raised in a common-garden environment for multiple generations and fed the same diet. These laboratory samples were analyzed together and rarefied to 18,834 reads. ##### Variables: * Each variable is the name of a microbial taxonomic group, down to the genus level. #### File: Pmexcomp-gut-and-environment-rarefied-depth-10405-level-6-taxonomy.csv **Description:** This file contains the taxonomy counts for the field samples, which includes sulfide-adapted and sulfide-intolerant fish from the Ixtapangajoya, Tacotalpa, Pichucalco, and Puyacatengo river drainages in southern Mexico (8 populations total). It also contains the taxonomy counts for water and sediment samples collected from each of the sites inhabited by fish. These field samples were analyzed together and rarefied to 10,405 reads. ##### Variables: * Each variable is the name of a microbial taxonomic group, down to the genus level. ## Code/software #### Software needed to run the analyses The analyses were run using R (version 4.2.0) in RStudio, which is a free and open program available online. The microbiome analyses were conducted using the R package *phyloseq* (McMurdie and Holmes 2013), and graphs were generated using the R package *ggplot2* (Wickham 2011). #### File: Pmex_microbiome_analyses9_for_manuscript_revision.Rmd **Description:** This file contains the code for analyzing all the gut microbiome and environmental microbiome samples from both the field and the laboratory. Several alpha and beta diversity metrics were used to analyze the microbiome samples, and the top microbial taxa driving differences between sulfidic and nonsulfidic fish were identified. The code also includes analyses for visualizing the microbial taxa that were shared and unique across the populations, both when including environmental microbes in the analysis and when removing environmental microbes from the analysis.