Identifying candidate sex-determining gene
To identify candidate sex-determining genes for both perches, we tested whether the scaffolds containing the sex-determining genes known from teleost fish also harbour sex-linked DArT SNPs or the Macquarie perch sexing region detected using WGS data. We searched our InterProScan5 output for amh , anti-Mullerian hormone, amhr,anti-Mullerian hormone receptor, bcar1 , breast cancer anti-resistance, brca2 , breast cancer type 2 susceptibility protein, dmrt1 , doublesex- and mab-3 -related transcription factor 1, dmY, dm-W, dmW, gsdf, gonadal soma derived growth factor, sdY and SOX (Bao et al., 2019; Graves, 2013; Rodríguez-Marí et al., 2011) and Gene Ontology term GO:0007530 (biological process: sex determination) and its child terms (https://www.ebi.ac.uk/QuickGO/term/GO:0007530). To confirm gene identity of the candidate sex-determining genes, we searched for similar sequences using Blastn (Zhang, Schwartz, Wagner, & Miller, 2000) in NCBI nucleotide database, retaining hits with annotations, an e-value <10-10, query coverage >99% and percent identity >90%. Finally, we tested the Macquarie perch sexing region for two genomic signatures of a cis-regulatory element (i.e. a DNA sequence that regulates tissue- and time-specific candidate gene expression): evolutionary conservation of non-coding regions, and the presence of multiple transcription factor binding sites (i.e. ~10bp-long degenerate sequences recognized by transcription factors) (Hardison & Taylor, 2012). We queried the 600-bp region containing the sexing region (GenBank accession SEMN01000633, bases 93001-93600) against the NCBI nucleotide database (using Blastn, retaining all hits with e-value <10-10). We also used CiiiDER (Gearing et al., 2019) and the JASPAR database of transcription factor binding profiles (Fornes et al., 2020) to search for potential binding-site locations in ~246-bp-long X- and Y- specific sequences (haplotypes) encompassing the sexing region. We investigated whether some transcription factor binding sites are specific to Macquarie perch X- or Y- haplotypes and if so, whether they are present in golden perch and Murray cod sequence (outgroups). A potential gain of a binding site on Y-haplotype was inferred if the site was present on Y- but absent on X-haplotype and both outgroups, whereas a potential loss of a binding site was inferred if it was absent on Y-haplotype but present on X-haplotype and in both outgroups.