1 2 DR. N. JUSTIN MARSHALL (Orcid ID : 0000-0001-9006-6713) 3 4 5 Article type : Original Article 6 7 8 Cardinalfishes (Apogonidae) show visual system adaptations typical of 9 nocturnally and diurnally active fish. 10 11 Martin Luehrmann (ML)1, Karen L. Carleton (KC)2, Fabio Cortesi (FC)1, Karen L. Cheney 12 (KLC)1,3, N. Justin Marshall (JM)1* 13 14 1Queensland Brain Institute, The University of Queensland, Sensory Neurobiology Group, 15 Brisbane, QLD 4072, Australia 16 2Department of Biology, The University of Maryland, College Park, MD, 20742, USA 17 3School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, 18 Australia 19 20 *Corresponding Author: Prof. Justin Marshall 21 Address: Queensland Brain Institute; University of Queensland 22 Brisbane | QLD 4072 | Australia 23 Fax number: +61 (0)7 33654522 24 Email: [email protected] Manuscript 25 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/MEC.15102 This article is protected by copyright. All rights reserved 26 Short running title: Visual pigment diversity in fish active during the day and at night 27 Abstract 28 Animal visual systems adapt to environmental light on various timescales. In scotopic 29 conditions, evolutionary time-scale adaptations include spectral tuning to a narrower light 30 spectrum, loss (or inactivation) of visual genes, and pure-rod or rod-dominated retinas. Some 31 fishes inhabiting shallow coral reefs may show activity during the day and at night. It is 32 unclear whether these fishes show adaptations typical of exclusively nocturnal or deep-sea 33 fishes, or of diurnally active shallow-water species. Here, we investigated visual pigment 34 diversity in cardinalfishes (Apogonidae). Most cardinalfishes are nocturnal foragers, yet they 35 aggregate in multispecies groups in and around coral heads during the day, engaging in social 36 and predator avoidance behaviours. We sequenced retinal transcriptomes of 28 species found 37 on the Great Barrier Reef, assessed the diversity of expressed opsin genes and predicted the 38 spectral sensitivities of resulting photopigments using sequence information. Predictions were 39 combined with microspectrophotometry (MSP) measurements in seven cardinalfish species. 40 Retinal opsin expression was rod opsin (RH1) dominated (>87%), suggesting the importance 41 of scotopic vision. However, all species retained expression of multiple cone opsins also, 42 presumably for colour vision. We found five distinct quantitative expression patterns among 43 cardinalfishes, ranging from short-wavelength-shifted to long-wavelength-shifted. These 44 results indicate that cardinalfishes are both well adapted to dim-light conditions and have 45 retained a sophisticated colour vision sense. Other reef fish families also show both nocturnal 46 and diurnal activity while most are strictly one or the other. It will be interesting to compare 47 these behavioural differences across different phylogenetic groups using the criteria and 48 methods developed here. 49 Introduction 50 Colour vision is mediated by comparing sensory inputs of distinct spectral classes of 51 photoreceptors defined by their morphology and the part of the wavelength spectrum to which 52 each is maximally sensitive (λmax). The outer segments of photoreceptors contain two 53 molecular building blocks of photopigments: the chromophore (often a Vitamin-A derived, 54 light sensitive molecule),Author Manuscript and an opsin (a transmembrane protein) to which the chromophore 55 is covalently bound (Yokoyama 2008; Hauser and Chang 2017). Vertebrate visual opsins are 56 divided into five classes dependent on their evolutionary history, photoreceptor specificity This article is protected by copyright. All rights reserved 57 and the light spectrum range to which they tune the photopigment to (Hunt and Collin 2014). 58 Rhodopsin (RH1) is found in rods that are used in dim light vision. SWS1 (UV), SWS2 (violet- 59 blue), RH2 (blue-green) and LWS (yellow/red) are found in cones and facilitate colour vision 60 (Yokoyama 2000). Over evolutionary and shorter timescales, the repertoire of visual opsins 61 can adapt to the light environment inhabited by a species and/or other ecological demands via 62 gene deletion, gene duplication, gene conversion and differential gene expression. 63 Furthermore, pigment sensitivities may be tuned via changes to the amino acid sequence of 64 the opsins due to single nucleotide polymorphisms (SNPs) (reviewed in Carleton, 2009; 65 Hauser & Chang, 2017; Yokoyama, 2008). 66 67 Many diurnal animals have a broad palette of opsin genes which facilitate colour 68 vision and its associated behavioural tasks. On the other hand, in nocturnal animals and those 69 that live in dim photic environments, such as the deep-sea or caves, a subset of the original 70 opsin genes have been lost or are not expressed at a functional level. For example, nocturnal 71 owls have lost their violet/UV sensitive opsin (SWS1), which is found in diurnal raptors, 72 presumably because of the low incidence of short-wavelength light at night. Both the blue- 73 sensitive (SWS2) and red-sensitive opsins (LWS) experienced strong positive selection early 74 on during owl evolution, which may be linked to the shift from diurnal to crepuscular hunting 75 in their ancestor (Wu et al. 2016). In cetaceans, inactivated copies of SWS1 are common and 76 several cetacean lineages that dive to depths of over 100 m have convergently lost their LWS 77 opsin. Since mammals lost their SWS2 and RH2 opsins ancestrally, presumably as a 78 consequence of an evolutionarily prolonged nocturnal phase (Heesy and Hall 2010; Gerkema 79 et al. 2013), these cetaceans are effectively rod monochromats, despite exposure to broad 80 spectrum light during breathing intervals at the sea surface (Meredith et al. 2013; Schweikert 81 et al. 2016). 82 83 The visual systems of fishes range from pure-rod retinas containing only RH1 in some 84 deep-sea species (Partridge et al. 1992; Hunt et al. 2001; Davies et al. 2009; de Busserolles et 85 al. 2015; Musilova et al. 2018), to highly diversified duplex retinas containing both RH1 and 86 multiple different coneAuthor Manuscript opsins in many shallow-water fishes, facilitating colour vision in these 87 broad spectrum waters (Lythgoe 1979; Matsumoto et al. 2006; Carleton 2009; Hofmann et al. 88 2012; Marques et al. 2017). The opsin gene repertoire and their expression is primarily related This article is protected by copyright. All rights reserved 89 to the different ecological demands of a species. In freshwater cichlids, for example, it is 90 thought that the plastic expression of opsin genes in response to changes in the environment 91 has facilitated adaptive radiation (Kocher 2004; Carleton et al. 2005; Seehausen 2006; Terai 92 et al. 2006; Seehausen et al. 2008). The predominant ecological factor driving spectral 93 sensitivity adaptation is the light environment (Bowmaker et al. 1994; Lythgoe et al. 1994; 94 Hauser and Chang 2017). For example, in deep-sea fishes, the lack of UV and red light in 95 their environment has resulted in a loss of the LWS and SWS1 opsins, whereas their RH1 96 opsin is tuned to shorter wavelengths than in shallow-water fishes (Musilova et al. 2018). In 97 diurnal, shallow-water species, only a subset of the various cone opsin genes present in their 98 genomes are expressed at any one time (Carleton and Kocher 2001), and among several fish 99 clades, levels of expressed opsins differ depending on the spectral regimes that different 100 species inhabit (Shand et al. 2008; Fuller et al. 2010; Fuller and Claricoates 2011; Johnson et 101 al. 2013; Dalton et al. 2015; Carleton et al. 2016; Sakai et al. 2016). Finally, colour vision 102 may also be strongly influenced by behavioural tasks such as mate choice, foraging, and 103 predator avoidance (Seehausen et al. 2008; Pegram and Rutowski 2014; Sandkam et al. 2015; 104 Stieb et al. 2017). 105 106 The diversity of spectral sensitivities among coral reef fish, living in one of the most 107 colourful ecosystems on earth, is yet to be explained and probably has no simple answer (for 108 review see Marshall et al. 2018). Part of this diversity is known to be facilitated by differential 109 opsin gene expression and amino acid substitutions at opsin gene tuning sites, e.g., in 110 damselfishes (Pomacentridae) (Hofmann et al. 2012; Stieb et al. 2016; Stieb et al. 2017), 111 wrasses (Labridae) (Phillips et al. 2016), and dottybacks (Pseudochromidae) (Cortesi, Feeney, 112 et al. 2015; Cortesi et al. 2016). However, it is unclear how light environment and/or 113 behavioural tasks drive these processes. There are some clear trends and correlation with 114 increasing depth or water turbidity on and around reefs (Lythgoe 1979; Lythgoe et al. 1994) 115 and even a loose relationship between fish colours and spectral sensitivities (Marshall et al. 116 2003). None of these factors seem to account for the diversity between reef fish genera and 117 more comparative work is needed, especially with specific behavioural tasks in mind (Losey 118 et al. 2003; MarshallAuthor Manuscript et al. 2003). 119 This article is protected by copyright. All rights reserved 120 Here, we investigated the visual system evolution, in terms of opsin gene diversity and 121 gene expression, in cardinalfishes (Apogonidae). Cardinalfishes are small, abundant, and 122 highly diverse, mainly carnivorous fishes that live in shallow reef habitats (Helfman 1986; 123 Marnane and Bellwood 2002; Allen et al. 2003; Barnett et al. 2006). Most species are 124 nocturnal foragers, while social behaviours are carried out during the day (Kuwamura 1983; 125 Kuwamura 1985; Marnane and Bellwood 2002; Saravanan et al. 2013; Brandl and Bellwood 126 2014).