2.7. Phylogenetic Analysis and Divergence Time Estimation

To determine the phylogenetic position of S. forsteni, phylogenetic analysis was conducted using single-copy homologous genes from its genome and those of thirteen teleost fishes (O. niloticus, O. latipes, L. crocea, C. rostratus, Plectropomus leopardus, Archocentrus centrarchus, Labrus bergylta, C. undulatus, Notolabrus celidotus, D. rerio, Acanthochromis polyacanthus, A. ocellaris, Stegastes partitus) and one outgroup (Homo sapiens). The genomic data for these species, obtained from NCBI, were analyzed for homologous clustering using the OrthoFinder v2.5.4 [59]. Subsequently, based on the above results, the unique genes of S. forsteni were identified, and the single-copy homologous genes of each species were aligned using MAFFT v7.490 [60] to create a super-alignment matrix. A phylogenetic tree was then constructed using RAXML v8.2.12 [61] with default settings. In this study, divergence times for 14 species were estimated using a phylogenetic tree with specific time nodes using the Mcmctree program from PAML v4.9. This involved five calibration points: the divergence between Danio rerio and Homo sapiens (approx. 423.3–440.0 Mya), D. rerio and Sparisoma partitus (approx. 180.0–251.5 Mya), S. partitus and Ambloplites centrarchus (approx. 74.4–96.5 Mya), Labrus bergylta and Cichla undulatus (approx. 51.8–63.1 Mya), and Cichla rostratus and N. celidotus (approx. 83.2–142.9 Mya) [62]. Additionally, we conducted statistical comparisons of multi-copy orthologs, unique paralogs, other orthologs, and unclustered genes in the predicted gene libraries, across the species.