Specimens and sequencing

Two Ogcocephalus cubifrons specimens, designated Ocub1 (male) and Ocub2 (unknown sex), were obtained from an ornamental fish supplier, and euthanised by immersion in a 3 L bath containing a lethal dose of MS-222 (Tricaine, 100 mg/L, diluted in artificial seawater), in accordance with local animal welfare regulations. Thymus and testes were harvested immediately for RNA- and genomic DNA-extraction respectively. RNA was extracted from thymus tissue using the TRI Reagent (Merck/Sigma-Aldrich) method, as per the manufacturer’s instructions. RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit, and then sequenced on an Illumina NovaSeq 6000 (Ocub1) or NextSeq 500 (Ocub2) sequencer. Genomic DNA was isolated from testis tissue by digestion with proteinase K, and subsequent phenol-chloroform extraction. Genomic sequencing libraries were prepared using the Illumina TruSeq PCR-Free Library Prep Kit, and sequenced on an Illumina NovaSeq 6000 sequencer. RNA was sequenced from both Ocub1 and Ocub2 thymi; genomic sequencing was performed on Ocub1 only. All sequencing data has been deposited into the NCBI sequence read archive (SRA) and can be accessed under the BioProject ID PRJNA905091. To validate the species identity of the sequenced individuals, a complete Cytochrome c oxidase I (COI) gene sequence was assembled from both Ocub1 and Ocub2 sequence data, and used to search the BOLD Identification System species level barcode records [50]. The top three hits in both instances were to previously existing O. cubifrons barcodes, which exhibited 100–99.85% identity to the search sequences. To estimate genomic coverage we performed SPAdes[51] de novo assembly, followed by BUSCO[52] (version 5.4.2) assessment, which yielded the following output: C:47.5%[S:46.0%,D:1.5%],F:14.5%,M:38.0%,n:3640 (where C = complete, S = complete and single-copy, D = complete and duplicated, F = fragmented, M = missing and n = total BUSCO groups searched; the actinopterygii_odb10 lineage dataset was used). A similar approach was used to estimate transciptome coverage. Transcriptomes were assembled de novo using Trinity [53] (version 2.8.5), and the pooled thymus transciptome (Ocub1 and Ocub2) was subjected to BUSCO assessment, which yielded the following output: C:85.5%[S:25.0%,D:60.5%],F:2.9%,M:11.6%,n:3640. The de novo assemblies are available from the authors upon request.

In some instances, specific targets were validated by Sanger sequencing; in these cases, target amplicons were amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and the primers listed in Table 2. Amplicons were subsequently cloned into pJET1.2 using the CloneJET PCR Cloning Kit (ThermoFischer Scientific), and clones of interest were sequenced using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit, and reactions were analysed on an Applied Biosystems 3730xl DNA Analyzer.

PCR primers used in this study