DOI: 10.5061/dryad.69p8cz967
title: Pelvic spine reduction affects diet but not gill raker morphology in two polymorphic brook stickleback (Culaea inconstans) populations

description: Pelvic spine polymorphism occurs in several species in the stickleback
 family (Gasterosteidae). Given parallel selection driving similar
 phenotypic polymorphisms in multiple stickleback species, we sought to
 determine the extent of parallelism in the ecological consequences of
 pelvic spine reduction. Based on a metabarcoding analysis of brook
 stickleback gut contents in two polymorphic populations, we found a shift
 towards a planktonic diet was associated with pelvic spine reduction.
 These results contrast with those found in threespine stickleback where
 pelvic spine reduction is associated with a shift towards a benthic diet.
 Hence, we found non-parallel consequences of spine polymorphism across
 species. Furthermore, a change in gill raker morphology has been
 consistently implicated in the change in diet in pelvic-reduced threespine
 stickleback. But we found no evidence of any difference in gill raker
 morphology associated with pelvic spine polymorphism in brook stickleback.
description: Brook stickleback were collected in Alberta, Canada, from Muir
 Lake (UTF-8 encoded WGS84 latitude and longitude: 53.627659,
 -113.957524) and Shunda Lake (52.453899, -116.146192).
 <strong>For gut content metabarcoding:</strong> Fish
 guts collected from 95 fish in 2019 were shipped to the Canadian Centre
 for DNA Barcoding at the University of Guelph for membrane-based DNA
 extraction and amplicon sequencing. This metabarcoding approached used
 five PCR primer sets designed to amplify a barcode region of the
 cytochrome c oxidase subunit I (COI) gene in arthropods, mollusks,
 annelids, amphipods, and microalgae (details of COI amplification can be
 found at
 http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_Amplification.pdf). A
 second round of PCR amplification added Ion Torrent sequencing adapters
 and a unique multiplex identifier (MID) sequence to the 5’ end of
 amplicons for each sample. Amplified COI fragments were then pooled and
 single-end sequenced on an Ion Torrent PGM sequencing platform (Thermo
 Fisher, Waltham, MA, USA). The resulting sequence data was automatically
 de-multiplexed by the PGM Torrent Browser (Thermo Fisher, Waltham, MA,
 USA). <strong>For gill rakers:</strong>
 Fish that had been bleached and stained with alizarin red for
 morphological analyses were used to investigate variation in gill raker
 morphology associated with different pelvic phenotypes. We removed the
 first branchial arches from both sides of each fish with forceps and
 scissors, cleared away all the gill filaments, and photographed the gill
 rakers in a plastic weighing boat using a Nikon SMZ-75T Greenough-type
 stereo microscope with a mounted Imaging Source camera and NIS-Elements D
 Software (©2021 Nikon Corporation). We counted the number of gill rakers
 on the left and right gill arches, and we measured the length of the
 second through fourth gill raker from the epibranchial-ceratobranchial
 joint on the ceratobranchial, which were consistently the three longest
 gill rakers on each arch.
description: <strong>For gut content metabarcoding:</strong> We
 removed primer and adapter sequences from the de-multiplexed reads, and
 discarded reads that consisted of only primer or adapter sequence using
 CUTADAPT v2.3. We used DADA2 in R v4.2.2 to trim and filter reads,
 estimate error rates for the filtered and trimmed amplicon dataset,
 combine identical reads into unique sequences, and infer biologically
 meaningful (as opposed to spurious) amplified sequence variants (ASVs). We
 exported the list of ASVs across all samples to a fasta file and used the
 JAMP pipeline in BOLDigger to identify the best-fitting taxonomic
 identification for each ASV. We used the PHYLOSEQ package to create a data
 object in R that allowed us to associate the abundance of each taxon
 identified in each fish’s gut with other sample data (i.e. lake, sex,
 size, and pelvic phenotype). We used the DESeq2 package in R to conduct
 Wald tests of the Log2 fold-differences in the abundances of each
 eukaryote family in the diets of brook stickleback.
 <strong>For gill rakers:</strong> We set contrasts
 among factors using the contr.sum function, and we used the CAR package in
 R to generate an ANOVA table with type III sums of squares.
description: # Pelvic spine reduction affects diet but not gill raker morphology in two
 polymorphic brook stickleback (Culaea inconstans) populations *** The data
 consist of two files - one is a zip archive of sequence data from
 metabarcoding of brook stickleback gut contents, and one is a csv file of
 measurements and counts of brook stickleback gill rakers. In addition,
 there is a separate "metadata" csv file associated with the
 metabarcoding dataset, which includes the phenotype, sex, size, and
 population information for each individual in the metabarcoding dataset
 (this sample information is included as columns in the csv file for the
 gill raker dataset). ## Description of the data and file structure
 Mee\_etal\_CCDB\_Culaea\_guts\_metabarcoding.zip - This zip archive
 contains fastq files with the sequence information from 95 brook
 stickleback guts. Metadata\_culaea\_gut.csv - this csv file has all the
 sample information (e.g. sex, pelvic phenotype, length, population) for
 all 95 brook stickleback in the metabarcoding dataset. Columns in this
 file: Sample\_ID.IonCode: the unique sample ID associated with each
 individual in the sequence data file Sample\_ID: the unique sample ID
 assigned to each individual at the tim of capture Lake: the lake of origin
 for each individual Trap\_location: description of the access point for
 setting traps in each lake. "North" and "South" for
 Shunda Lake refer to direction relative to the boat launch. Habitat:
 whether the sample was collected in a trap set adjacent to shore
 ("Littoral") or in a trap floated &gt;50m from shore
 ("Pelagic") Year: the year the individual was sampled FL\_mm:
 the fish length in centimetres (FL stands for "fork length", but
 this is actually the "total length" because stickleback
 don't have a forked caudal fin) Sex: sex, as assigned at time of
 capture Pelvic\_code: the pelvic phenotype coded as 1 = spined, 2 =
 vestigial, 3 = unspined Pelvic\_pheno: the pelvic phenotype coded as
 "spined", "int" (= vestigial), or "unspined"
 Pelvic\_pheno2: the pelvic phenotype coded as "spined"
 (corresponding to Pelvic\_code = 1) or "reduced" (corresponding
 to Pelvic\_code = 2 or 3) Mee\_etal\_gill\_rakers.csv - this csv file has
 all the sample information and gill raker measurements for all the brook
 stickleback analysed in this study. Missing data is stored as blank cells.
 Columns in this file: Sample\_ID: the unique sample ID for each individual
 Sex: sex, as assigned at time of capture Lake: the lake of origin for each
 individual Year: the year the individual was sampled Habitat: whether the
 sample was collected in a trap set adjacent to shore
 ("Littoral") or in a trap floated &gt;50m from shore
 ("Pelagic") FL\_cm: the fish length in centimetres (FL stands
 for "fork length", but this is actually the "total
 length" because stickleback don't have a forked caudal fin)
 Pelvicpheno: the pelvic phenotype coded as 1 = spined, 2 = vestigial, 3 =
 unspined Reduced: the pelvic phenotype coded as "no" = not
 reduced (corresponding to Pelvicpheno = 1) or "yes" = reduced
 (corresponding to Pelvicpheno = 2 or 3) L\_rakers: the number of gill
 rakers on the left arch R\_rakers: the number of gill rakers on the right
 arch Rakers\_mean: the mean number of gill rakers (averaged across left
 and right arches) Rakers\_max: the maximum number of gill rakers (on
 either the left or right arch) L\_raker\_length\_1: length of the second
 gill raker (in micrometers) from the epibranchial-ceratobranchial joint on
 the left ceratobranchial L\_raker\_length\_2: length of the third gill
 raker (in micrometers) from the epibranchial-ceratobranchial joint on the
 left ceratobranchial L\_raker\_length\_3: length of the fourth gill raker
 (in micrometers) from the epibranchial-ceratobranchial joint on the left
 ceratobranchial L\_raker\_max: the length of the longest gill raker (in
 micrometers) on the left arch R\_raker\_length\_1: length of the second
 gill raker (in micrometers) from the epibranchial-ceratobranchial joint on
 the right ceratobranchial R\_raker\_length\_2: length of the third gill
 raker (in micrometers) from the epibranchial-ceratobranchial joint on the
 right ceratobranchial R\_raker\_length\_3: length of the fourth gill raker
 (in micrometers) from the epibranchial-ceratobranchial joint on the right
 ceratobranchial R\_raker\_max: the length of the longest gill raker (in
 micrometers) on the right arch Raker\_length\_max: the length of the
 longest gill raker (in micrometers) use: coded "yes" or
 "no" to indicate whether to use the sample in analyses or not
 notes: justification for "use" decision