Overview of experimental design and methods

A) Schematic of the experimental design. Replicate groups of juvenile clownfish (Amphiprion percula) housed as size-matched pairs (n = 30 pairs) and size-matched solitary individuals (n = 30 individuals). Standard length (SL) was measured at the beginning and end of the five-week experiment, and individuals were denoted as dominant (P1), subordinate (P2), and solitary (S) at the end of the experiment based on their emergent social positions. B) Feeding behavior scoring method for pairs and solitary fish. During feeding, pairs were separated by a divider to prevent competition, divider was included in solitary tanks as a sham control, and individuals were provided with equal amounts of pre-portioned food daily. Feeding behavior was quantified weekly from video recordings by counting the number of bites taken by each fish over a 5-minute period starting from the first bite. Initially, paired fish were denoted as P-max and P-min based on the total number of bites taken, because individuals could not be reliably identified due to their similar size and lack of distinguishing markings. From week 3 onwards, bites taken were scored separately for dominant (P1) and subordinate (P2) fish, in pairs where fish could be reliably identified based on clear size differences. C) Agonistic behavior scoring method for pairs. Agonistic behaviors (aggression, submission) were quantified during a five-minute observation period from video recordings of paired interactions. Aggression was quantified by the number of chases, headbutts, and bites, while submissive behavior included flees and shakes. Initially, total aggression and submission were measured at the tank level, because fish were similar in size and lacked distinguishing markings. From week 3 onward, agonistic behaviors were scored separately for dominant (P1) and subordinate (P2) fish, in pairs where individuals could be reliably identified due to the emergence of clear size differences. In the accompanying illustrations, the focal fish is shown in orange, with its partner depicted in black and white. D) Gene expression profiling for pairs and solitary fish. At the end of the experiment (week 5), whole-body samples were collected from a subset of individuals [n = 15 per social role: dominant (P1), subordinate (P2), solitary (S)]. To maximize detection of gene expression patterns associated with social roles, replicates with the largest size differences between dominant and subordinate fish were selected for transcriptomic profiling. The resulting RNA-seq data were used to compare molecular signatures across emergent social roles. In all panels, the timeline at the top indicates when (week 1 – week 5) and how (calipers, videos, tissue samples) data was collected.

A) Growth over time. Standard length (mm) of individuals of each social position (pair rank 1 = P1; pair rank 2 = P2; solitary = S) over time with mean and standard error of mean (SEM). A.1) Overall growth (mm) over 5 weeks of each social position are shown with mean (dashed lines) and SEM. B) Feeding behavior over time. Individual food intake, measured in number of bites of size-matched pairs (P-max and P-min) vs solitary (S) individuals over time with mean and SEM. B.1) Food intake for a subset of replicates in which dominant (P1), subordinate (P2), and solitary (S) individuals could be reliably identified due to the emergence of clear size differences (week 3, n = 5; week 4, n = 17; week 5, n = 19), shown over time with mean ± SEM. C) Agonistic behavior of pairs over time. Aggression (the total number of chases, bites, and headbutts) and submission (based on the total number of flees and shakes) within pairs over time with mean and SEM. C.1) Aggression and C.2) Submission for a subset of pairs in which dominant (P1) and subordinate (P2) individuals could be reliably identified based on clear size differences (week 3, n = 5; week 4, n = 17; week 5, n = 19), shown over time with mean and SEM. Data points shown jittered on all figures for visual aid, but collected at the same time.

Overall gene expression profiles showed no significant differences between P2 and S individuals

A) Principal Component Analysis (PCA) of all differentially expressed genes (DEGs) across social positions, with PERMANOVA results indicating a significant effect of social position. B) Venn diagram illustrating DEGs from pairwise comparisons of social positions (FDR-adjusted p-value of 0.05). In the pairwise comparisons, P2 and S were compared to P1 individuals. Orange upward arrows and corresponding numbers represent significantly upregulated DEGs, while blue downward arrows and numbers indicate downregulated DEGs (Supplementary Materials Table 4).

Gene ontology (GO) enrichment of the brown module associated with growth and ossification

An exhaustive list of GO enrichment results for genes assigned to the brown WGCNA module (Fig. 5). Bubble size reflects numbers of genes associated with each term, color indicates GO category. Terms are ordered by adjusted p-value significance on the x-axis (–log₁₀ scale), with more significant terms on the right.

P1 individuals showed upregulation of genes associated with growth and ossification

A) Average eigen gene values across social positions for the brown module. B) Heatmap of significant DEGs (FDR p-value ≤ 0.05) of growth and ossification genes in the brown Biological Process (BP) and Molecular Function (MF) GO terms across the three social positions. Genes that are significantly differentially expressed between individuals of P1 vs P2 are denoted with an asterisk (*), those between P1 vs S with a double asterisk (**), and three asterisk (***) represents both comparisons. Genes shown in bold and italics indicate significance at an FDR adjusted p-value < 0.1.

P1 individuals showed downregulation of appetite suppression genes

Heatmap of significantly differentially expressed genes (DEGs) (FDR p-value ≤ 0.01) of appetite, TCA cycle and glycolysis pathways across the three social positions indicating genetic background of individuals (clutch ID). Genes that are significantly differentially expressed between individuals of P1 vs P2 are denoted with an asterisk (*), those between P1 vs S with a double asterisk (**), and three asterisk (***) represents both comparisons. Genes shown in bold and italics indicate significance at an FDR adjusted p-value < 0.1.