Sampling and preparation of gut microbiome samples
Colonies of Cyprinodon pupfishes were collected from two hypersaline lakes on San Salvador Island, Bahamas (Crescent Pond and Osprey Lake) and Lake Cunningham, Bahamas in March, 2018 and were reared in aquaria at the University of California, Berkeley. Additional generalist populations were collected in May, 2018 from Fort Fisher Estuary in North Carolina. Cualac tessellatus eggs were provided by the Zoological Society of London and reared in the lab to produce a large second generation used for the four samples in this study. All samples, except for the recently collected NC population, came from first or second-generation captive-bred individuals reared in aquaria (40–80 L) according to species and location at 5–10 ppt salinity (Instant Ocean synthetic sea salt) and between 23 to 30°C. Individuals used for this study were first fed once daily ad libitum with a single commercial pellet food (New Life Spectrum Cichlid Formula, New Life International, Inc., Homestead, FL), containing 34% crude protein, 5% crude fat, and 5% crude fiber, for one month without exposure to any other food or tankmates. All animal care and experiments were conducted under approved protocols and guidelines of the University of California, Berkeley Institutional Animal Care and Use Committee (AUP-2018-08-11373).
In total, forty fishes were euthanized in an overdose of MS-222 and the entire intestinal tissue was immediately excised (Cyprinodontidae do not possess stomachs; Wilson and Castro, 2010) for DNA extraction. Standard length and gut length were measured for all samples (Table S1). Five individuals (F2 generation) from each of three species (C. variegatus , C. brontotheroides , and C. desquamator ) in both lake populations from San Salvador Island were sampled (n = 30 total). In addition, we included the following pupfish species as outgroups to our study: C. laciniatus (F1 generation; Lake Cunningham, New Providence Island, Bahamas; n = 4), C. variegatus (F0 generation; Fort Fisher, North Carolina, United States; n = 2) plus liver tissue as a tissue control, andCualac tessellatus (long-term captive colony; San Luis Potosí, Mexico, n = 4).
Each gut was divided into proximal and distal regions for all San Salvador Island samples to compare microbial composition between these regions. All outgroup samples used whole intestines. In addition, the microbial community was isolated from aquaria water in two tanks which contained F2 individuals of Osprey Lake C. variegatus and Crescent Pond C. variegatus , and used as controls (n = 2). The Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley also generated three controls, including a positive control and two no template controls (NTC). Microbial DNA extractions were performed in batches (stored on ice) immediately after intestinal dissections with the Zymobiomics DNA Miniprep Kit (Zymo Research, Irvine, CA).