1 Microhabitat partitioning correlates with opsin gene expression in coral reef 2 cardinalfishes (Apogonidae) 3 4 Martin Luehrmann (ML) 1, Fabio Cortesi (FC) 1, Karen L. Cheney (KLC) 1,2, Fanny de Busserolles 5 (FbB)1, N. Justin Marshall (JM) 1 6 7 1Queensland Brain Institute, The University of Queensland, Sensory Neurobiology Group, 4072, 8 Brisbane, QLD, Australia 9 2School of Biological Sciences, The University of Queensland, 4072, Brisbane, QLD, Australia 10 11 Corresponding Author: Dr Martin Luehrmann 12 Sensory Neurobiology Group, Queensland Brain Institute, University of Queensland, Brisbane | 13 QLD 4072 | Australia, Fax number: +61 (0)7 33654522 14 Email: [email protected] 15 16 Keywords 17 Microhabitat partioning, opsin gene expression, fish, cardinalfish, LWS, RH2, SWS2, vertebrate 18 visual system evolution, eye size, retinal topography 19 20 Headline: Visual adaptation to microhabitats in reef fish Author Manuscript This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1365-2435.13529 This article is protected by copyright. All rights reserved 1 2 DR MARTIN LUEHRMANN (Orcid ID : 0000-0002-4060-4592) 3 DR KAREN CHENEY (Orcid ID : 0000-0001-5622-9494) 4 5 6 Article type : Research Article 7 Editor : Christine Miller 8 Section : Evolutionary Ecology 9 10 11 Microhabitat partitioning correlates with opsin gene expression in coral reef 12 cardinalfishes (Apogonidae) 13 14 Martin Luehrmann (ML) 1, Fabio Cortesi (FC) 1, Karen L. Cheney (KLC) 1,2, Fanny de Busserolles 15 (FbB)1, N. Justin Marshall (JM) 1 16 17 1Queensland Brain Institute, The University of Queensland, Sensory Neurobiology Group, 4072, 18 Brisbane, QLD, Australia 19 2School of Biological Sciences, The University of Queensland, 4072, Brisbane, QLD, Australia 20 21 Corresponding Author: Dr Martin Luehrmann 22 Sensory Neurobiology Group, Queensland Brain Institute, University of Queensland, Brisbane | 23 QLD 4072 | Australia, Fax number: +61 (0)7 33654522 24 Email: [email protected] 25 26 Keywords 27 microhabitat partioning, opsin gene expression, fish, cardinalfish, LWS, RH2, SWS2, vertebrate 28 visual system evolution, eye size, retinal topography 29 Abstract 30 Author Manuscript 31 1. Fish are the most diverse vertebrate group, and they have evolved equally diverse visual 32 systems, varying in terms of eye morphology, number and distribution of spectrally distinct 33 photoreceptor types, visual opsin genes and opsin gene expression levels. This article is protected by copyright. All rights reserved 34 2. This variation is mainly due to adaptations driven by two factors: differences in the light 35 environments and behavioural tasks. However, while the effects of large-scale habitat 36 differences are well described, it is less clear whether visual systems also adapt to 37 differences in environmental light at the microhabitat level. 38 3. To address this, we assessed the relationship between microhabitat use and visual system 39 features in fishes inhabiting coral reefs, where habitat partitioning is particularly common. 40 4. We suggest that differences in microhabitat use by cardinalfishes (Apogonidae) drive 41 morphological and molecular adaptations in their visual systems. To test this, we 42 investigated diurnal microhabitat use in 17 cardinalfish species and assessed whether this 43 correlated with differences in visual opsin gene expression and eye morphology. 44 5. We found that cardinalfishes display six types of microhabitat partitioning behaviours 45 during the day, ranging from specialists found exclusively in the water column to species 46 that are always hidden inside the reef matrix. 47 6. Species predominantly found in exposed microhabitats had higher expression of the short- 48 wavelength sensitive violet opsin (SWS2B) and lower expression of the dim-light active rod 49 opsin (RH1). Species of intermediate exposure, on the other hand, expressed opsins that are 50 mostly sensitive to the blue-green central part of the light spectrum (SWS2As and RH2s), 51 while fishes entirely hidden in the reef substrate had a higher expression of the long- 52 wavelength sensitive red opsin (LWS). 53 7. We also found that eye size relative to body size differed between cardinalfish species, and 54 relative eye size decreased with an increase in habitat exposure. 55 8. Retinal topography did not show co-adaptation with microhabitat use, but data suggested co- 56 adaptation with feeding mode. 57 9. We suggest that, although most cardinalfishes are nocturnal foragers, their visual systems – 58 and possibly those of other (reef) fishes – have also adapted to the light intensity and the 59 light spectrum of their preferred diurnal microhabitats. 60 Introduction 61 Animal visual systems are functionally diverse, with differences at the morphological and 62 the molecular level. In fish, this diversity is mainly driven by differences in the availability of light 63 (Hauser and Chang, 2017; Land and Nilsson, 2002), but can also be due to differences in habitat 64 complexity (Collin andAuthor Manuscript Shand, 2003; Hughes, 1977) or specific behavioural tasks, e.g. foraging or 65 sexual selection (reviewed in Hauser and Chang, 2017; Price, 2017). In aquatic environments, 66 differences in light mainly arise from wavelength selective light absorption and scattering due to 67 depth, the size of suspended particles, and the reflectance of such particles or the substrate 68 (Lythgoe, 1979). For example, the deep-sea has a blue-shifted light environment and consequently, This article is protected by copyright. All rights reserved 69 deep-sea species generally possess photoreceptors that are maximally sensitive to blue light (~ 480 70 nm) (Partridge et al., 1992). 71 72 Morphologically, visual systems may differ in eye size, shape, and at the retinal level in 73 functional type, number and/or distribution of neural cells including photoreceptors. Morphological 74 changes to boost sensitivity in low-light conditions, for example, may include rod-dominated 75 retinas, increased relative eye size, or a higher photoreceptor-to-ganglion cell summation ratio (de 76 Busserolles and Marshall, 2017; Kelber and Roth, 2006; Warrant, 2004). 77 78 At the molecular level, the part of the electromagnetic spectrum to which a photoreceptor is 79 maximally sensitive (λmax) may vary, primarily due to changes in its photopigments (Hunt and 80 Collin, 2014). Photopigments are molecules comprised of opsins - membrane-bound proteins with 81 receptor function - to which a vitamin A-derived chromophore is covalently bound. While in 82 vertebrates only two chromophore types (Vitamin A1 or A2) occur, opsins are more variable. They 83 are classified according to their λmax values, their phylogeny, and their specificity to 84 morphologically distinct photoreceptor types (Hunt et al., 2014). Opsins may alter photoreceptor 85 λmax via i) variations in their amino acid sequences, or ii) via differential expression of the different 86 opsin genes (reviewed in Carleton et al., 2016). In vertebrates, five opsin classes are found: rod- 87 specific rhodopsin (RH1) used for scotopic vision, and four cone specific classes used for photopic 88 and colour vision: SWS1 (short-wavelength-sensitive 1, ultraviolet); SWS2 (short-wavelength- 89 sensitive 2, violet/blue); RH2 (rhodopsin-like 2, blue-green/green); LWS (long-wavelength- 90 sensitive, yellow/red)(Hunt et al., 2014). In percomorph fishes, gene duplication has resulted in a 91 more diversified repertoire consisting of rod-specific RH1, single cone specific SWS1, SWS2B 92 (violet), SWS2A and SWS2A (blue), and double cone specific RH2B (blue-green), RH2A 93 (green), and LWS (Cortesi et al., 2015; Hunt and Collin, 2014). 94 95 In fish, visual systems adapt to large-scale lighting differences due to habitat depth, type 96 (e.g. reef vs. open ocean), or season (Lythgoe, 1979; Lythgoe et al., 1994; Muntz, 1982). However, 97 fish vision may also be tuned to light differences between habitats on smaller scales for species 98 sharing the same general habitat at similar depths. For example, in some African cichlids, opsin 99 gene expression differsAuthor Manuscript depending on the associated substrate (e.g. rock vs sand) (Sabbah et al., 100 2011). The photoreceptor spectral sensitivities of surfperch living among California’s kelp forests 101 are tuned to light in structurally distinct parts of that general habitat (e.g. canopied vs not-canopied) 102 (Cummings and Partridge, 2001). However, although suggested (Lythgoe, 1979; Marshall et al., 103 2003), it remains to be tested whether this phenomenon may be acting on an even smaller – This article is protected by copyright. All rights reserved 104 microhabitat – scale, and thus contributing to the remarkable diversification of colour vision among 105 fishes living on coral reefs, one of the most diverse ecosystems on earth, where species based 106 habitat partitioning, at times within a single coral head, is particularly common (reviewed in 107 Williams, 1991). 108 109 Here, in order to control for potentially confounding factors like phylogenetic constraint, we 110 focused on a group of closely related reef fishes with remarkable visual system diversity, the 111 cardinalfishes (Apogonidae) (Fishelson et al., 2004; Luehrmann et al., 2019). These fishes are