Pelvic spine reduction affects diet but not gill raker morphology in two polymorphic brook stickleback (Culaea inconstans) populations

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.

Methods
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).

For gut content metabarcoding: 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).

For gill rakers: 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.