Fig. 8
Functional differences of SPP120 according to fertilization types and molecular evolution of SPP120 (a) The N-terminal region of SPP120 was checked in several species (N = 6 in NofSPP, N = 5 in NlcSPP). The ratio of sperm motility before and after the addition of proteins or Ca2+ (in percentage) is shown. Spermatozoa immobilization and aggregation were significantly stronger in oral fertilization than in the other types (NofSPP120: Phylogenetic generalized least squares P < 0.001, Additional file 22: Table S5; NlcSPP: Tukey’s HSD P < 0.05). (b) Maximum likelihood phylogenetic tree of SPP120 and codon sites under positive selection within the five groups formed by SPP120 copies (mouth brooder groups 1, 2, and 3, Ectodini, and substrate brooders). Functional SPP120 copies among the five groups were applied in molecular evolutionary analyses (codon site model and branch-site analyses) regarding non-synonymous/synonymous mutations (ω) using CODEML in PAML [56]. Two models (null hypothesis: neutral and hypothesis: positive selection) were run for each analysis and their log-likelihoods were compared to assess if there was positive selection or not. Black bars indicate positively selected sites in codon site analyses and red bars indicate positively selected sites in branch site analyses. Species indicated in red were used as the foreground in branch site analyses to examine accelerated evolution