DOI: 10.5061/dryad.brv15dv6h
title: Data from: Parallel and non-parallel changes of the gut microbiota during trophic diversification in repeated young adaptive radiations of sympatric cichlid fish

description: Background: Recent increases in understanding the ecological and
 evolutionary roles of microbial communities has underscored their
 importance for their hosts’ biology. Yet, little is known about gut
 microbiota dynamics during early stages of ecological diversification and
 speciation. We sequenced the V4 region of the 16s rRNA gene to study the
 gut microbiota of extremely young adaptive radiations of Nicaraguan Midas
 cichlid fish (Amphilophus cf. citrinellus) from two crater lakes to test
 the hypothesis that parallel divergence in trophic ecology is associated
 with parallel changes of the gut microbiota. Results: Bacterial
 communities of the water and guts were highly distinct, indicating that
 the gut microbiota is shaped by host-specific factors. Across individuals
 of the same crater lake, differentiation in trophic ecology was associated
 with gut microbiota differentiation, suggesting that diet, to some extent,
 affects the gut microbiota. However, differences in trophic ecology were
 much more pronounced across than within species whereas similar patterns
 were not observed for taxonomic and functional differences of the gut
 microbiota. Across two crater lakes, we could not detect evidence for
 parallel changes of the gut microbiota associated with trophic ecology.
 Conclusions: Similar cases of non-parallelism have been observed in other
 recently diverged fish species and might be explained by a lack of clearly
 differentiated niches during early stages of ecological diversification.
description: Specimens of
 the <i>Amphilophus</i> cf. <i>citrinellus</i>
 species complex were caught during field trips to Nicaragua in 2014 and
 2015. All specimens were sacrificed by applying an overdose of MS-222.
 Then, whole guts were dissected, cleaned and stored in absolute EtOH at
 -20°C until DNA extraction. Muscle tissue of the same specimens was
 collected in absolute EtOH and stored at -20°C for stable isotope
 analyses. Four technical replicates of water samples were collected along
 the shores of the four lakes in 2018. Briefly, for each replicate 500 ml
 of lake water were filtered through a cellulose nitrate filter (ø 25 mm,
 pore size 1 µm) and filters were stored in Longmire’s solution  at -20°C
 until DNA extraction. DNA from midgut tissue was extracted using the
 commercial QIAamp DNA Stool Mini Kit according to the manufacturer’s
 protocol (Qiagen, Hilden, Germany). DNA from water samples was extracted
 from cellulose nitrate filters using a QIAGEN DNeasy Blood &
 Tissue kit. All DNA extractions and PCR amplifications were performed
 under sterile conditions in a laminar flow hood to minimize contamination
 risk. Fish gut and water samples were separately pooled in an equimolar
 manner and size selection was performed on a Pippin Prep device (Sage
 Science, Beverly, MA). The quality of the pooled libraries was assessed
 using a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). Both
 libraries were paired-end sequenced, each in one lane of the Illumina flow
 cell. For the fish guts, we sequenced 2x250 bp on an Illumina HiSeq 2500
 platform at TUCF Genomics (Tufts University). Water samples were sequenced
 2x150 bp on an Illumina HiSeq X-ten at BGI Genomics. We obtained a
 total of 62,728,287 (median: 238,073 reads/specimen) and 111,949,556
 (median: 6,825,739) raw sequencing reads that could be unambiguously
 assigned to a specific sample for fish guts and water samples,
 respectively. Illumina adapters were removed and reads were trimmed with
 Trimmomatic v0.36. As there was no overlap between forward and reverse
 reads for water samples and the sequence quality of forward reads was
 higher, we used 135 bp of the forward reads for all analyses. The
 demultiplexed and trimmed reads were imported into the open-source
 bioinformatics pipeline Quantitative Insights Into Microbial Ecology
 [QIIME2] to analyze microbial communities of fish guts and water samples.
 Briefly, sequence quality control was done with the QIIME2 plugin
 <i>deblur</i>. A phylogenetic tree of bacterial taxa was
 produced with FastTree 2.1.3. Bacterial species richness and bacterial
 community composition [weighted UniFrac, unweighted UniFrac, Bray-Curtis
 dissimilarity] were calculated and taxonomy was assigned using vsearch
 against the SILVA 132 ribosomal RNA (rRNA) databases at a 97% similarity
 threshold. Statistical analyses were performed in R v3.2.3.