Supplementary Information 2 3 FIGURE S1

1 Supplementary Information 2 3 FIGURE S1. Proportion of individuals per species counted in each defined microhabitat 4 category across sites. N = number of sampling sites at which each species was found. n = 5 total number of individuals counted. Also see Figure 1 for microhabitat categorisation. 6

7 8 FIGURE S2. Topographic distribution of photoreceptor cells (total cones TC, double cones 9 DC, single cones SC) in two individuals of Ostorhinchus doederleini highlighting a low 10 intraspecific variability. Black lines represent isodensity contours and values are expressed in 11 densities x 103 cells/mm2. The arrows indicate the orientation of the retinas, V = ventral, T = 12 temporal. Scale bars = 1 mm. 13 T Individual 1 Individual 2

6 4.8 1 7.2 5.6 5.6 7.2 6.4 6.4 2 8 6.4 7.2 1 8 8.8 7.2 8 8.8 4 V 8 8.8 9.6 1 8.8 10.4 6 9.6 10.4 9. 11.2 1 12 11.2 10.4 11.2 1 4.4 1.2

0 1 12.8 1 5 14.4 14.4 12.8 12.8 6 .2 13.6

1 2 5

C .

2 1 14.4 1 13.6 6 12 T 13.6 .2 11.2 1 9.6 1 1 5.2

8 10.4 12 14.4 2 9.6 11.2 1 . 6 . 7 8.8 12.8 3 8.8 10.4 4 . 1 13.6 12.8 4 8 9.6 1 8.8 8 .8 11.2 1 8 2 8

7.2 8

6 1 0 .4

4.2 4.8 4.8 0 6 5.4 2

5.4 6 6.6 1 7.2 5.4 6.6 6.6 7.2 6 7 6 8.4 7.8 6.6 8 .2 7.2 7. 9 7.8 7.8 8.4 9 1 10.2 9 8.4 1.4 9.6 9.6 10 1 1 11. 10.8 9 .2 2. 2 4 0 6 12 4

8 . 11 1 10 C .8 10.8 1 10.8 2 12.6 10.2 9

D 9 7 9 7.8 1 9 0 12 8.4 . 9.6 4 8 . 1 6 1 1

11.4 8 7 1 . 7.8 4 . 8 6 .4 2 7 2 10.8 .2 0. 10.2 7.2 6.6 7.8 1 7.2 9.6 9.6 .6 6 6 6.6 9

8 .

4 6

1 5

1 3 0.8 0.8 1.2

5 1 . 1.2

1 2 1.4 1.2 1.4 1.2 3 1.4 1 1.6 1.4 1.8 1.6 1.6 1.6 1.8 2 2.4 1.8 2 2.6 2

2.2 2.2 2.8 2.2 5

2 2.4 . 3 3 3. 2 2.8 2

2.4 2 2 3 . 3 .6 2

2.2 2.8 3 5 .4 S 2.6

. 2 2 .2 1.8 3 3

1.8 1 .6 3 2.2 2

1.6 .2 3 2.8 2 5

1.6 2 . 1.4 2 .2 1.4 2.6 1 2 1 .4 2.2 1 1.2 .8

2 14 1 15 FIGURE S3. Type of retinal specialisation in the five cardinalfish species analysed in this 16 study plotted against the cladogram of the family. Cladogram made from the phylogeny 17 presented in Figure 5a. A = area, S = streak. 18

19 20 FIGURE S4. Retinal photoreceptor mosaic patterns found in cardinalfishes. (a) square 21 pattern, (b) chaotic pattern (after Collin and Shand 2003). (c) Light microscopy view of the 22 central-temporal part of the retina in O. doederleini. (d) Light microscopy view of the nasal 23 part of the retina in T. fucata. (e) and (f) Location of photoreceptor mosaic patterns shown 24 superimposed over total photoreceptor distribution maps from Fig. 4 (main manuscript). S = 25 square pattern, C = chaotic pattern. Black lines represent isodensity contours. Values are 26 expressed in densities x 103 cells/mm2. The arrows indicate the orientation of the retinas, V = 27 ventral, T = temporal. Scale bars = 1 mm.

28 29 TABLE S1. Range of standard length (SL) and horizontal eye diameter measured in sampled 30 cardinalfishes. 31 Species n SL (mm) Eye diameter (mm) Lens diameter (mm) Apogon crassiceps 3 26 - 30 3.1 - 3.3 1.25 - 1.55 Cheilodipterus* artus 4 66 - 96 6.95 - 10.5 2.95 - 3.40 macrodon 1 88 9 3.65 quinquelineatus 6 46 - 69 5.1 - 8.6 2.5 - 3.25 Fibramia thermalis 1 37 4 1.75 Fowleria variegata 2 35 - 48 4.1 - 5.6 1.9 - 2.2 Nectamia* fusca 6 35 - 58 6.1 - 10.1 2.6 - 4.2 savayensis 22 26 - 53 4.8 - 9.1 2.0 - 4.3 compressus 11 63 - 85 8.8 - 12.3 3.7 - 5.3 Ostorhinchus* cookii 21 28 - 63 4.3 - 8.1 3.95 - 1.8 cyanosoma 19 31 - 48 4.2 - 6.7 1.9 - 3.0 doederleini 15 33 - 52 4.9 - 6.4 2.0 - 2.95 holotaenia 3 26 - 41 3.9 – 5.1 1.8 – 2.1 nigrofasciatus 14 30 - 57 4.25 - 6.75 1.75 - 2.95 notatus 3 50 - 55 6.5 - 6.9 2.8 novemfasciatus 9 35 - 49 4.15 - 6.6 1.85 - 2.65 Pristiapogon* exostigma 12 40 - 77 5.1 - 8.4 2.0 - 3.75 fraenatus 1 53 5.95 2.2 Rhabdamia* gracilis 24 34 – 47 3.6 – 5.3 1.35 – 1.9 Sphaeramia* nematoptera 7 41 - 49 5.95 - 7.2 2.35 - 3.1 Taeniamia* fucata 5 37 - 60 4.25 - 7.85 1.6 - 3.6 zosterophora 12 40 - 56 5.05 - 7.8 2.05 - 3.2 Zoramia* leptacanthas 22 33 - 49 4.6 - 6.9 1.8 - 2.96 viridiventer 11 31 - 44 3.95 - 5.5 1.67 - 2.5 32 n = individuals measured 33 * Used to identify relative eye size differences on the genus level (see also Fig. 4c, Table S2) 34 35 TABLE S1. Results of post-hoc pairwise comparisons of relative eye size in the investigated 36 cardinalfish genera using Dunn’s post-hoc test and p-value adjustment according to Hochberg 37 (1995). Significant p-values are shown in bold. 38 Genus Cheilodipterus Nect. Osto. Pristi. Rhabd. Sphaer. Taeni. Nectamia z -1.912 p 0.051 Ostorhinchus z 1.03 4.688 p 0.199 0 Pristiapogon z 2.427 4.989 2.253 p 0.019 0 0.027 Rhabdamia z 5.242 9.307 6.92 2.655 p 0 0 0 0.012 Sphaeramia z -1.04 0.419 -2.121 -3.193 -5.614 p 0.2 0.357 0.036 0.0028 0 Taeniamia z 1.141 3.649 0.432 -1.5 -4.628 2.103 p 0.174 0.0006 0.359 0.102 0 0.0348 Zoramia z 2.113 5.51 2.018 -0.79 -4.373 2.976 0.984 p 0.035 0 0.041 0.251 0 0.005 0.2078 39 40 41 42 TABLE S2. Summary of counting parameters used to analyse the topographic density 43 distribution of photoreceptors and ganglion cells (one retina/count). 44 Species SL PR/GC Grid size Counting frame Schaeffer's (mm) (µm) (µm) CE R. gracilis 47.6 GC 330x330 60x60 0.040 T. fucata 60.5 PR 610x610 100x100 0.042 GC 600x600 80x80 0.037 O. cyanosoma 45 PR 550x550 80x80 0.035 45 GC 520x520 80x80 0.045 O. doederleini N/A PR 450x450 100x100 0.036 45 PR 400x400 100x100 0.039 O. notatus 50.4 PR 550x550 80x80 0.043 45 PR = Photoreceptors, GC = Ganglion cells, CE = Schaeffer’s coefficient of error 46 TABLE S4. Overview of photoreceptor and ganglion cell densities determined in various cardinalfish species. For T. fucata, photoreceptor and 47 ganglion cell counts were performed on either eye of the same individual. For O. cyanosoma, photoreceptor count and ganglion cell counts are from 48 different individuals. 49 Species Micro- Total Peak PR Total Peak DC Total Peak SC % Total Peak GC Lens ⌀ SRP habitat number cells/mm2 number cells/mm2 number cells/mm2 SC number cells/mm2 (mm) (cpd) spec PR DC SC GC R. gracilis 1 ------368,305 23,056 2.0 7.1 T. fucata 3 559,756 12,300 370,176 9,500 188,781 4,700 33 334,976 8,281 3.6 7.3 O. cyanosoma 3 585,384 15,468 449,262 12,188 134,219 4,063 23 430,478 19,843 2.7 8.5 O. doederleini 4 430,389 18,800 347,214 15,500 79,054 4,100 18 - - - - 414,087 18,400 327,404 14,500 79,020 4,300 19 - - - - O. notatus - 616,786 16,562 449,208 12,969 167,456 4,843 27 - - - - 50 DC = double cone, SC = single cones, PR = total photoreceptors, ⌀ = diameter, SRP = spatial resolving power, cpd = cycles per degree 51 52 53 TABLE S5. Dataset used for phylogenetic least squares analysis (PGLS). Activity period (N = nocturnal; D = diurnal). Feeding mode (B = benthivore, 54 P+B = benthivore and planktivore, P = planktivore). Microhabitat (specialisation group: 1, 2, 3, 4, 5, 6; based on Figure 2). Proportional opsin gene 55 expression for a given gene relative to total single cone opsin (SWS2B, SWS2Aα, SWS2Aβ), total double cone opsin (RH2B, RH2A, LWS), and total 56 opsin (RH1) expression (taken from (Luehrmann et al. 2019). N/A = data not available. 57 Species Predictor variables Dependent variables Proportional opsin gene expression (%) Activity Feeding Microhabitat Relative period mode partition eye size SWS2B SWS2Aα SWS2Aβ RH2B RH2A LWS RH1 Apogon crassiceps N9,10 B3 N/A 0.346 5.3 94.7 0 0 100 0 98.1 melas D12 N/A N/A N/A 0 30 70 0 71.6 28.4 96.3 Cheilodipterus artus D12 P1 3 0.492 0 37.7 62.3 14.7 84.6 0.7 93.6 macrodon D12 P1,2 4 0.491 2.3 56.1 41.7 4.4 93.4 2.2 95.1 quinquelineatus N10 B+P1,2 3 0.470 0.2 47.8 51.9 1.4 95.4 3.2 91.8 Fibramia thermalis D9 N/A 2 0.384 0.4 41.5 58.1 1.9 81.6 16.4 90.6 Fowleria variegata N9 NA N/A 0.422 0.5 35.6 64.0 0 78.2 21.8 93.1 Nectamia fusca N3 P3 6 0.527 0 6.3 93.7 0.2 60.3 39.6 94.2 savayensis N4 P3 6 0.503 0 8.9 91.1 0 70.6 29.4 94.0 Ostorhinchus angustatus N3 N/A N/A N/A 0 23.2 76.8 0.3 99.0 0.6 91.0 compressus N10 P5 4 0.543 0 38.3 61.7 7.8 92.1 0.1 90.0 cookii N10 B6 4 0.48 0.9 50.8 48.4 2.0 90.8 7.2 94.3 cyanosoma D12 B+P1,2 3 0.457 0 24 76 18 81.3 0.7 92.3 doederleini N10 B+P1,2 4 0.469 0 40.5 59.5 1.5 94.1 4.4 94.0 nigrofasciatus N11 N/A 5 0.456 0 65.8 34.2 3.3 96.2 0.5 91.1 notatus N10 N/A N/A 0.479 0 26.1 73.9 3.1 96.8 0 92.9 novemfasciatus N10 N/A N/A 0.440 1.5 49.0 49.5 9.0 89.8 1.2 87.0 Pristiapogon exostigma N10 B+P1,2 5 0.447 0.4 99.6 0 0.1 99.9 0 94.5 fraenatus N12 N/A N/A 0.449 0 100 0 1.8 98.2 0 92.0 Species Predictor variables Dependent variables Proportional opsin gene expression (%) Activity Feeding Microhabitat Relative period mode partition eye size SWS2B SWS2Aα SWS2Aβ RH2B RH2A LWS RH1 cf. mirifica N10 N/A N/A N/A 0.8 22.7 76.5 0 90.1 9.9 95.9 Rhabdamia gracilis D4 P4 1 0.389 70.1 29.9 0 40.2 59.8 0 64.1 Sphaeramia nematoptera N7 P8 N/A 0.494 0 42.8 57.2 0.4 85.6 14.0 92.0 Taeniamia fucata N10 P1 3 0.449 0.1 17.6 82.3 1.4 96.6 2.0 91.0 zosterophora N10 P9 3 0.470 0 47.9 52.1 0.9 98.6 0.5 92.5 Zoramia leptacantha N10 P6 3 0.469 1.8 98.2 0 7.8 92.1 0.1 93.7 viridiventer N10 B+P1 2 0.423 40.6 59.4 0 13.8 86.2 0 89.8 58 1 - Barnett et al. (2006), 2 - Marnane and Bellwood (2002), 3 - Myers (1999), 4 - Kuiter and Tonozuka (2001), 5 - Job and Shand (2001), 6 - Frédérich et al. (2017), 7 - Lieske et al. 59 (2002), 8 - Nakamura et al. (2003), 9 - Allen et al. (2003), 10 - Paxton et al. (1989), 11 - Randall and Lachner (1986), 12 - Brandl and Bellwood (2014). 60 TABLE S3. PGLS model estimates for correlation of ecological and/or environmental 61 predictor variables and dependent variables. P-values are Bonferroni corrected for repeated 62 hypothesis testing, and statistically significant values are shown in bold. Microhabitat 63 comparison, n = 17 species; Activity period, n = 26 (opsin gene expression), n = 23 (relative 64 eye size); Feeding mode, n = 17; Relative eye size, n = 23. Dependent Predictor Pagel’s λ R2 Df F-Stat P-value (adj.) Relative eye size Microhabitat partition 0.0 0.676 5,11 7.66 0.02* Activity period 1.0 -0.039 1,21 0.169 0.686 Feeding mode 1.0 0.278 2,14 4.078 0.322 SWS2B Microhabitat partition 0.0 0.721 5,11 9.283 0.009** Activity period 0.754 -0.016 1,24 0.618 1 Feeding mode 0.896 -0.128 2,14 0.093 1 Relative eye size 0.0 0.227 1,21 7.474 0.087 SWS2Aα Microhabitat partition 1.0 0.438 5,11 3.491 0.313 Activity period 0.995 -0.033 1,24 0.193 1 Feeding mode 1.0 -0.044 2,14 0.664 1 Relative eye size 1.0 -0.023 1,21 0.507 1 SWS2Aβ Microhabitat partition 1.0 0.337 5,11 2.629 0.675 Activity period 1.0 -0.041 1,24 0.011 1 Feeding mode 1.0 -0.029 2,14 0.772 1 Relative eye size 1.0 0.096 1,21 3.335 0.575 RH2B Microhabitat partition 0.0 0.489 5,11 4.056 0.198 Activity period 0.776 0.133 1,24 4.842 0.264 Feeding mode 1.0 -0.019 2,14 0.852 1 Relative eye size 0.816 -0.043 1,21 0.100 1 RH2A Microhabitat partition 0.0 0.744 5,11 10.31 0.006** Activity period 0.963 0.159 1,24 5.74 0.173 Feeding mode 1.0 0.059 2,14 1.502 1 Relative eye size 0.991 -0.033 1,21 0.291 1 LWS Microhabitat partition 0.0 0.761 5,11 11.17 0.004** Activity period 1.0 -0.032 1,24 0.225 1 Feeding mode 1.0 -0.133 2,14 0.061 1 Relative eye size 1.0 -0.028 1,21 0.393 1 RH1 Microhabitat partition 0.0 0.858 5,11 20.37 <0.001*** Activity period 0.941 -0.042 1,24 0.000 1 Feeding mode 1.0 -0.050 2,14 0.621 1 Relative eye size 0.943 0.004 1,21 1.083 1 65 Df = degrees of freedom, P-values * < 0.05 , ** < 0.01, *** < 0.001 66 Supplementary References 67 Allen GR, Steene RC, Humann P, Deloach N. 2003. 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