Social and associated behaviors in zebrafish. (A) Across lines, a two alternative-choice set-up was used to measure social preference and recognition abilities and (B) an open field test for measuring anxiety-driven thigmotaxis towards the periphery and edge-orienting. Regions of interest (ROI) were set within 1 standard body-length from target locations or stimuli. (C) Social tendency was measured by interaction preferences towards a shoal. Social (D) and non-social (E) discrimination tests were comprised of two phases: an acquisition phase, in which the focal fish was exposed to two unfamiliar items (two fish or two objects, respectively) followed (as indicated by arrow in D and E) by a probe-test phase, in which the focal fish had to discriminate between one of the previously seen items (fish or object) and a novel one; recognition in both the social (D) and non-social (E) context were measured by the ability to discriminate between a familiar and a novel stimulus. Males (full circles) and females (open circles) of all lines (5D, AB, LEO, TL, TU, Wik) exhibited above chance (dashed line) preference for shoal over an empty tank (social tendency, F) and discrimination between a novel and familiar stimulus in both a social (conspecific; G) and non-social (object; H) context (bars indicate 95% CI). Behavioral measures exhibited different degrees of correlation (r), illustrated in the cladogram as degrees of association (I), based on which factor analysis revealed three principal components (PC): PC1 aggregates social tendency and social and object exploration corresponding to a motivational component of sociality; PC2 aggregates thigmotaxis and (i.e., proportion time in periphery) and edge-orienting (distance to wall) measured in the open field test, corresponding to an anxiety component; PC3 aggregates object and social discrimination, corresponding to a general-domain cognitive component

Phenotypic correlation matrices. Phenotypic correlation matrices for the behavioral components of General Inspection, General Recognition and Anxiety across six different zebrafish laboratory lines (i.e., populations). Color code represents correlation (r) values

Genetic clustering and behavioral associations. (A) Hierarchical clustering of genetic distances (Jaccard distance) between the sampled individuals indicates the occurrence of five major clusters that overall match the six wild type lines used (pink cluster: TU; gold cluster: 5D; green cluster: AB; blue cluster: Wik), with LEO and TL included in the purple cluster but subsequently segregated from each other in two lower order clusters. (B) Venn diagrams representing the number of SNPs the General Inspection component shares with its constitutive behaviors (social tendency, social exploration and object exploration) and the Anxiety component. (C) Chromosome mapping of the SNPs that are significantly associated with the General Inspection component and its constitutive behaviors, following the color code used by the Venn diagrams, and with the position of each SNP on each chromosome is given in bp. SNPs, single nucleotide polymorphisms

Illustrative examples of SNPs associated with the General Inspection component. (A) D2b; (B) GnRH2; (C) 5HTR1aa; (D) 5HTR2d1; (E) 5HTR3a; (F) Nlgn2a; (G) Nlgn2b; (H) Nr4a3; (I) Tsc2; (J) MECP2; (K) NPY. Individuals of the different lines are represented by different colors according to color code indicated in the figure legend. SNPs, single nucleotide polymorphisms