1 Supplemental Figures and Tables: 2 3 Figure S1. Data on the visual system of Neolamprologus brichardi used to build visual 4 models 5 A) Relative opsin expression of N. brichardi determined by RNAseq. B) Normalized 6 transmittance of N. brichardi crystalline lens. C) Normalized absorbance of SWS1, RH2B and 7 RH2A. D) Estimated visual sensitivities incorporating the crystalline lens filtering medium. E) 8 Normalized downwelling and F) sidewelling irradiance at two depths. Increasing water depth 9 differentially reduces available longer light wavelengths. Refer to Figure 2. 10 11 Figure S1 (continued). 12 G) 95% confidence intervals of spectral reflectance in dominant and subordinate individuals. 13 Only horizontal facial stripe spectra do not overlap between dark and pale fish, other colors 14 overlap to various degrees. Colored curves refer to color patches in individuals with dark 15 horizontal stripe, while light grey curves refer to equivalent patches in individuals with pale 16 horizontal stripe. H) Chromatic contrast (mean ± SEM) is achieved by adjacency of color 17 elements and is not influenced by darkness of horizontal stripe. I) Achromatic contrast (mean ± 18 SEM) is achieved by paling of the horizontal stripe and has a differential influence on adjacent 19 and non-adjacent color elements. Refer to Figure 2. 20 21 Figure S2. Horizontal facial stripe provides reliable information on aggressive intent. 22 A) and B) Winners are larger and fight more aggressively than losers. Lines connect fish dyads. 23 C) and D) Principal components analysis of spectral data and signal experimental manipulation. 24 Principal Components 1 and 2 explain 96.5% of the variance (90.8% and 5.7%, respectively) and 25 clearly separate different colors. D) Zoom in at the melanistic area of the plot shows that 26 ‘Eyeliner’ is similar to dark melanic stripes of dominant fish, while ‘Wound Snow’ is similar to 27 pale melanistic stripes of subordinates. See also Table S1 for clustering of Principal 28 Components. E), F) and G) Reaction norms of amount of aggression incurred by individuals with 29 out-of-equilibrium signals and controls. Retaliation costs are highest for individuals with 30 artificially darkened stripe (‘bluffers’, E and F), followed by individuals with paled stripe 31 (‘Trojans’, E and G), and then controls (i.e. reliable signalers, F and G), which were the ones that 32 received the lowest amount of aggression. Lines connect the same individual, tested twice with 33 two different treatments. Refer to Figure 4. 34 35 36 Table S1. Cluster analysis of principal components of spectral data Clusters Color patch 1 2 3 4 5 Eyeliner 0 1 0 0 0 Wound Snow 1 0 0 0 0 Vertical facial stripe (D) 0 10 0 0 0 Vertical facial stripe (P) 0 10 0 0 0 Horizontal facial stripe (D) 1 9 0 0 0 Horizontal facial stripe (P) 10 0 0 0 0 Lachrymal stripe (D) 8 2 0 0 0 Lachrymal stripe (P) 10 0 0 0 0 Head (D) 9 1 0 0 0 Head (P) 10 0 0 0 0 Blue (D) 0 0 10 0 0 Blue (P) 0 0 10 0 0 Branchiostegal (D) 0 0 0 0 10 Branchiostegal (P) 0 0 1 0 9 Yellow (D) 0 0 0 9 1 Yellow (P) 0 0 0 9 1 37 Five clusters were identified. ‘Eyeliner’ clusters together with black melanistic stripes (cluster 2), 38 while ‘Wound Snow’ clusters with pale horizontal stripe, lachrymal stripe and head (cluster 1). 39 D: dark. P: pale. Refer to Figures 4 and S2C. 40 Supplemental Experimental Procedures 41 Choice of species 42 The Princess of Burundi, Neolamprologus brichardi (Teleostei: Cichlidae), is a small (up to 8 cm 43 in standard length) fish native to Lake Tanganyika, eastern Africa. Together with N. pulcher, it 44 has emerged as a model system in studies on the evolution of cooperative breeding behavior [1]. 45 Recently, substantial genomic and transcriptomic resources have become available for N. 46 brichardi [2]. Such resources make this species an excellent system for the study of speciation, 47 evolution of cooperative behavior and communication also from a genetic perspective. 48 Neolamprologus brichardi performs the complete range of behaviors observed in the wild under 49 lab conditions, which makes it an optimal species for behavioral studies [3]. Phylogenetic 50 relationships have been recently studied [4], which led some authors to synonymize it with N. 51 pulcher. The two species differ in their facial pigmentation patterns and are thought to behave 52 similarly. We adopt the pre-synonymy taxonomy because it highlights the differences in facial 53 pigmentation pattern, which are the focus of our study. This should not create taxonomic 54 confusion and favors the accumulation of clear information for each of the pigmentation 55 phenotypes. Like most other species of the Tanganyikan cichlid tribe Lamprologini, N. brichardi 56 is sexually monochromatic, i.e. coloration patterns are identical between males and females [5]. 57 The facial pigmentation of N. brichardi consists of two black melanic stripes, arranged in a 58 horizontal T-shape, surrounded by structural blue coloration, yellow pigmentation elements and 59 a white branchiostegal membrane. Another less conspicuous stripe is present in the pre-orbital 60 (lachrymal) area. The species has a beige body with fine orange elements in the posterior half 61 and white-fringed fins (see Figure 2 in main text). 62 Substantial data on life history and behavioral traits have been documented for these 63 species. The dominant, breeding couple has the peculiarity of being aided by up to 25 64 subordinate helpers in their tasks, and the social group is organized in a strict linear hierarchy [6– 65 8]. As a consequence of cooperative breeding and colony life, individuals repeatedly and 66 regularly interact [9] and it is known that vision plays a role in individual recognition of mates, 67 kin and neighbors [3, 10, 11]. Social groups of N. brichardi can be found on coastal rocky 68 substrates of Lake Tanganyika, usually between 3–50 m deep. The rocky substrate provides a 69 territory with shelters and breeding grounds where adhesive eggs are spawned [12, 13]. The 70 breeding male is always the largest individual of the group, usually followed by the breeding 71 female and subordinate helpers are the smallest [1]. Groups aggressively defend their territory 72 and dominant females have dominance behavior similar to dominant males, show high 73 testosterone levels and brain arginine vasotocin expression (a neuropeptide involved in 74 vertebrate territorial, reproductive and social behaviors) [14]. Group hierarchy is based on size, is 75 relatively stable over time and fish adopt one of two queuing strategies for breeding. Fish either 76 queue and breed in their natal group, or they disperse to a new group where they queue and breed 77 [1]. The mean distance between adjacent territories is 1.6 m and territories are clustered into 78 colonies [15]. These life history and behavioral traits create conditions for repeated interactions 79 among individuals. Most of them involve submissive behaviors, followed by aggressive 80 behaviors and only then territory maintenance (such as digging) and broodcare [16]. 81 82 Study animals 83 Neolamprologus brichardi were raised and kept under controlled captive conditions at the 84 Zoological Institute, University of Basel, Switzerland, on a 12:12 h light:dark regime, in tanks 85 with about 1.5 cm of sand, a foam filter, a heater and terracotta flowerpots as shelters. Fish were 86 fed a combination of newly hatched Artemia nauplii, commercial flakes and frozen cichlid food 87 once or twice a day. All experiments were authorized by the Cantonal Veterinary Office, Basel, 88 Switzerland (permit numbers 2317 & 2356). 89 90 Color reflectance spectra 91 Spectral reflectance measurements of N. brichardi facial patterns were taken using a USB4000 92 spectrophotometer (Ocean Optics Inc.) and DH-2000-DUV Mikropack deuterium-halogen light 93 source, connected to a laptop computer running Ocean Optics SpectraSuite software. Twenty 94 individuals were tranquilized using a solution of KOI MED® Sleep (KOI&BONSAI, 0.5% v/v 95 2-Phenoxyethanol) before being transferred to a shallow tray filled with sufficient water to fully 96 cover the fish. Because tranquilizing the fish before measuring their spectral reflectance may 97 induce a short term darkening of their skin pigmentation we took care to measure reflectance 98 after original conditions were re-established (~15 seconds). Spectral reflectance of various facial 99 color patches (Figures 2A, 2B, S1G) was measured with a 200 µm bifurcated optic UV⁄visible 100 fiber. The bare end of the fiber was held at a 45º angle to prevent specular reflectance. A 101 Spectralon 99% white reflectance standard was used to calibrate the percentage of light reflected 102 at each wavelength from 350–750 nm. At least ten measurements per facial pattern per 103 individual were taken and subsequently averaged. Spectra were assessed based on the 104 wavelength at which light was reflected and the shape of the reflectance curves, and classified 105 into previously established categories of reef fish colors [17]. 106 107 108 Visual system 109 To characterize the visual system of N. brichardi, we used published quantitative opsin data [2, 110 18] and amino acid sequences from eye RNAseq data [2] done on our stock of N. brichardi, and 111 collected new ocular transmission measurements from wild specimens. Our N. brichardi express 112 the UV-sensitive SWS1, and the two green-sensitive RH2A and RH2B opsin genes, which is a 113 common opsin expression palette in cichlid species, including the ones from Lake Malawi 114 (Figure S1) [19]. On comparisons of amino acid sequences of these three genes to the sequences 115 of their Lake Malawi relatives we found that there are only minor differences between species.