Mitochondrial DNA sequences can identify the maternal species involved in hybridization events and provide behavioural clues of the matings. In this study, Mitochondrial DNA indicated hybrids were the offspring of female Coho salmon spawning with Chinook salmon males (all but two individuals from a reciprocal cross). This finding suggested two possible scenarios: accidental fertilization in crowded spawning grounds or heterospecific choice of mate when conspecifics were not available (e.g. differential abundance).

Two amplicon types for the characterization of salmonid were applied to the extracted DNA samples. Amplicons used were a ~270 bp segment of the chordate (16SChord) (Deagle et al. 2009), and a ~260 bp segment of the cytochrome oxidase I (COI) gene amplified with primers designed primarily for the amplification of salmonids (Thomas et al. 2017).

The 16SChord amplicons were multiplexed in one PCR reaction and the COI was performed in a separate reaction. All PCR amplifications were performed in 20 uL volumes using the Multiplex PCR Kit (Qiagen). Reactions contained 10 uL (2X) master mix, 0.25 uM of each primer, and 2 uL template DNA. Thermal cycling conditions were as follows: 95 °C for 15 min followed by 34 cycles of 94 °C for 30 s, 57 °C for 90 s and 72 °C for 60 s, and a final extension at 72 °C for 600 s.

Amplified 16SChord and COI amplicon samples were barcoded with unique, matching 10 bp Forward and Reverse tags with an edit distance of 5. The 16SChord and COI amplified samples were pooled by amplicon into single libraries. These were cleaned and concentrated using DNA Clean and Concentrate-5 columns (Zymo) prior to library preparation and indexing using the KAPA Low Throughput Library Preparation kit (KAPA) for Illumina platforms as per manufacturer’s instructions. The quantity of the indexed library pools was assessed using dsDNA High Sensitivity kit (Qubit) and quality and size assessed using the DNA 1000 Bioanalyzer chip (Agilent). Finally, the pools were combined into a single library of 1:1 and processed on a 301bp single end MiSeq V2 chip (Illumina) with a 5% phiX spike in. The data was de-multiplexed by library pool using the TruSeq^TM adapter sequences on the sequencer which produced fastq files.

FASTQ sequence files for each indexed library pool were imported into QIIME for demultiplexing by sample and sequence assignment to species (Caporaso et al. 2010). For a sequence to be assigned to sample it had to match the full forward and reverse primer sequences, and match the 10 bp primer tag for that sample (allowing for up to 2 mismatches in either primer or tag sequence). USEARCH was used to cluster OTUs with a similarity threshold = 0.99, a minimum cluster size = 3 and de novo chimera detection. For all DNA sequences successfully assigned to a sample, a Genbank BLAST search (Altschul et al. 1990; Edgar 2010) was done against our custom 16S or COI reference databases whereby an assignment was made based on the best match in the database (threshold BLASTN e-value < 1e-20 and a minimum identity of 0.9). Samples were excluded if they contained < 20 identified prey DNA sequences.

Deagle, B.E., Kirkwood, R., and Jarman, S.N. 2009. Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces. Molecular Ecology 18:2022– 2038

Thomas, A.C., Nelson, B.W., Lance, M.M., Deagle, B.E., and Trites, A.W. 2017. Harbour seals target juvenile salmon of conservation concern. Canadian Journal of Fisheries and Aquatic Sciences 74:907-921.

Caporaso, J.G., J. Kuczynski, J. Stombaugh, K. Bittinger, F.D. Bushman, E.K. Costello, N. Fierer, A.G. Pena, J.K. Goodrich, and J.I. Gordon. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7:335-336.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J., 1990. Basic local alignment search tool. Journal of molecular biology, 215(3), pp.403-410.

Robert C. Edgar, Search and clustering orders of magnitude faster than BLAST, Bioinformatics, Volume 26, Issue 19, 1 October 2010, Pages 2460–2461, https://doi.org/10.1093/bioinformatics/btq461

Dataset in text format.