Host-microbiome associations in livebearing fishes adapted to toxic streams rich in hydrogen sulfide: Code for analyzing 16S rRNA dataset
Data files
Aug 06, 2025 version files 865.02 KB
Abstract
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.
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.
We sampled 8 populations of P. mexicana 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 P. mexicana. 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.