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The visual ecology of Holocentridae, a nocturnal coral reef fish family with a deep-sea-like multibank retina

The visual systems of teleost fishes usually match their habitats and lifestyles. Since coral reefs are bright and colourful environments, the visual systems of their diurnal inhabitants have been more extensively studied than those of nocturnal species. In order to fill this knowledge gap, we conducted a detailed investigation of the visual systems of the coral reef fish family Holocentridae (squirrelfish and soldierfish). In addition to their nocturnality, this family is particularly interesting for dim-light vision studies due to its ecological and evolutionary connection to deeper habitats. Results showed that the visual system of holocentrids is well adapted to their nocturnal lifestyle with a rod-dominated retina. Surprisingly, rods in all species were arranged into 6-17 well-defined banks, a feature most commonly found in deep-sea fishes, that may increase the light sensitivity of the eye and/or allow colour discrimination in dim-light. Holocentrids also have the potential for dichromatic colour vision during the day with the presence of at least two spectrally different cone types: single cones expressing the blue-sensitive SWS2A gene, and double cones expressing one or two green-sensitive RH2 genes. Some differences were observed between the two subfamilies, with Holocentrinae having a slightly more developed photopic visual system than Myripristinae. Moreover, retinal topography of both ganglion cells and cone photoreceptors showed specific patterns for each cell type, likely highlighting different visual demands at different times of the day, such as feeding. Overall, their well-developed scotopic visual systems and the ease of catching and maintaining holocentrids in aquaria, make them ideal models to investigate teleost dim-light vision and more particularly shed light on the function of multibank retina and its potential for dim-light colour vision.

. For example, in damselfishes (Pomacentridae), there is a strong correlation between herbivory and LWS expression, as long-wavelength sensitivity may help in detecting the far-red reflectance of chlorophyll found in algae and phytoplankton (Stieb et al. 2017). UV-sensitivity (SWS1 expression) on the other hand may be beneficial in intraspecific communication (Stieb et al. 2017) and in the case of the Great Barrier Reef anemonefish, Amphiprion akindynos, the co-expression of both SWS1 and SWS2B (violet-sensitive) in the temporal retina may also help in detecting conspecifics situated in front of the fish (Stieb et al. 2019). Intraretinal variability in the distribution and density of photoreceptors and ganglion cells (i.e. retinal topography), has also been shown to vary with the habitat structure (Collin and Pettigrew 1988a, b), behavioural ecology (Stieb et al. 2019, Luehrmann et al. 2020 and/or ontogeny (Tettamanti et al. 2019) of coral reef fishes.
While vision in diurnal reef fishes has received substantial attention, the visual systems of nocturnal reef fishes remain understudied. From the few studies available, nocturnal reef fishes seem to have developed similar adaptations to other teleosts living in low-light environments (turbid/murky waters or the deep-sea) in order to enhance the sensitivity of their eyes. These include: large eyes and pupillary apertures (Pankhurst 1989, Schmitz andWainwright 2011), a smaller focal length resulting in higher retinal illumination (McFarland 1991, Shand 1994b), a tapetum lucidum (a mirror-like layer at the back of the eye that enhances photon capture, (Nicol et al. 1973), a rod-dominated retina (Munz andMcFarland 1973, Luehrmann et al. 2020), longer and denser rods (Pankhurst 1989, McFarland 1991, Shand 1994a, and an increase in the summation ratio of rods onto bipolar and ganglion cells (Shand 1997). However, these observations are limited to few species and the opsin expression, retinal topography and most of the visual capabilities and visual ecology of nocturnal reef fishes remain relatively unknown with the exception of the Apogonidae, which have recently been investigated in greater detail (Shand 1997, Fishelson et al. 2004, Luehrmann et al. 2019, Luehrmann et al. 2020. To continue filling this knowledge gap, we focused our study on the visual systems of Holocentridae, a nocturnal coral reef fish family that contains 91 recognised species divided into two subfamilies, the squirrelfish (Holocentrinae) and the soldierfish (Myripristinae) (Fricke et al. 2020).
Holocentrids are found circumtropically and usually inhabit shallow coral reefs, although a few species, especially from the genus Ostichthys, occur in the deep-sea at depths of up to 640 m (Greenfield 2002, Greenfield et al. 2017. While the family is mainly active at night when they engage in feeding, they are also observed during the day hovering in or close to their refuges. Holocentrids are particularly wellknown for their vocalisation and auditory abilities, which they may use for courtship, aggression and/or predator avoidance, especially during crepuscular hours and to a lesser extend during the day (Winn et al. 1964, Carlson and Bass 2000, Parmentier et al. 2011. Their large eyes (Schmitz and Wainwright 2011) and ability to find their home after displacement (Demski 2003) indicate that vision also plays an important role in this family, although relatively little is known about their actual visual capabilities.
Genome mining in three species revealed that in addition to a single rod opsin, RH1, holocentrids possess several cone opsins: up to two SWS1 copies, two SWS2s, up to eight RH2 paralogs, and one LWS (Cortesi et al. 2015, Musilova et al. 2019). However, opsin gene expression and microspectrophotometry (MSP) in few species indicate that only a small subset of these opsins may be used in adult fishes (Losey et al. 2003, Musilova et al. 2019). These include a rod opsin pigment with a peak spectral sensitivity (λmax) ranging from 481 -502 nm, a short-wavelength pigment measured in single cones (most likely SWS2A-based) with a λmax ranging from 440 -453 nm, and one or two medium wavelength pigments measured in double cones (most likely RH2-based) with a λmax ranging from 506 -520 nm (Losey et al. 2003). Interestingly, their rod spectral sensitivity correlates with habitat depth, with deeper living holocentrids having shorter spectral sensitivities similar to those found in deep-sea fishes (λmax = 480 -485 nm), shallower living species having longer sensitivities, comparable to those observed in other shallow-water fishes (λmax = 500 -507 nm) and individuals living at intermediate depths having sensitivities somewhere in-between (λmax = 490 -495 nm) (Munz and McFarland 1973, Toller 1996, Yokoyama and Takenaka 2004. Furthermore, the holocentrid ancestor is predicted to have had an RH1 sensitive to ~ 493 nm λmax suggesting that the family first emerged at intermediate depths (~100 m or mesophotic depths; (Yokoyama and Takenaka 2004, Musilova et al. 2019). This putative deeper origin, in addition to their nocturnal lifestyle on the reef and the few species inhabiting the deep-sea, therefore make holocentrids particularly interesting for dim-light vision studies.
Using a range of techniques, including high-throughput RNA sequencing (RNAseq), fluorescence in situ hybridization (FISH), photoreceptor spectral sensitivity estimates, and retinal anatomy and topography, we set out to scrutinise the visual system and visual ecology of several species of shallow water holocentrids, with the following two aims in mind: 1) To extend our knowledge about the visual ecology of nocturnal coral reef fishes; and 2) To assess if the holocentrid visual system differs from other nocturnal coral reef fishes due to their atypical ecological and evolutionary ties to deeper habitats.

Sample collection and ocular tissue preservation
Nine species of holocentrids were investigated in the study. For each species, the number of individuals, their size, their location, and the specific experiments they were used for are listed in Table   S1. Most fishes were collected on the Great Barrier Reef around Lizard Island, Australia, under the Great Barrier Reef Marine Park Permit (G12/35005.1) and the Queensland General Fisheries Permit (140763) using spear guns on SCUBA in 2015 and 2016. Five specimens were obtained from the aquarium supplier Cairns Marine who collects fish from the Northern Great Barrier Reef (Cairns Marine Pty Ltd, Cairns, Australia). Each individual was anaesthetized with an overdose of clove oil (10% clove oil; 40% ethanol; 50% seawater) and killed by decapitation. Eyes were subsequently enucleated, the cornea and lens removed, and the eye cup preserved in different fixative solutions depending on the analysis (see below for details). All experimental procedures were approved by The University of Queensland Animal Ethics Committee (QBI/236/13/ARC/US AIRFORCE and QBI/192/13/ARC).

Histology
One eye of Sargocentron diadema, Neoniphon sammara and M. murdjan was enucleated in daylight conditions and fixed in a solution of 2.5% glutaraldehyde and 2% PFA in 0.1 M PBS. An extra individual of S. diadema was dark adapted for 2 h prior to euthanasia in the dark and one eye was enucleated and fixed as above. The retinas were dissected out of the eye cup and small pieces from different locations (dorsal, temporal, ventral, nasal, central) were processed and analyzed. Each piece of retina was post-fixed in 1-2 % osmium tetroxide in 0.15 M phosphate buffer (PB), dehydrated through an acetone series, and infiltrated with Epon resin (ProSciTech) using a Biowave tissue processor. Resin samples were then polymerized at 60°C for 48 h. Semi-thin transverse sections of the retinas (1 μm) were cut with a glass knife using a Leica EM UC7 Ultramicrotome and stained with an aqueous mixture of 0.5 % Toluidine Blue and 0.5 % borax. Sections were viewed with a Carl Zeiss Axio Imager compound light microscope and photographed using an Olympus DP70 digital camera. Retinal thickness, photoreceptor layer thickness and rod outer segment length were then measured from the photographs using ImageJ v1.52p. (National Institutes of Health, USA). An average of three measurements were taken for each parameter.
Transcriptome sequencing, quality filtering and de novo assembly The retinas from two Myripristinae (M. murdjan and M. violacea) and three Holocentrinae species (S. diadema, S. rubrum and S. spiniferum) were dissected out of the eye cup (one retina per species; all adult individuals) and preserved in RNAlater (Thermo Fisher Scientific) at -20 o C until further processing. In the laboratory, total RNA was extracted using the RNAeasy Mini or Midi Kit (Qiagen) including a DNAse treatment step according to the manufacturer's protocol. RNA integrity was assessed using Agilent's Eukaryotic Total RNA 6000 Nano Kit on an Agilent 2100 BioAnalyzer (Agilent Technologies), and the concentration was subsequently measured using the Qubit RNA HS assay (Thermo Fisher Scientific). Library preparation and sequencing was performed by the Queensland Brain Institute's sequencing facility. Sequencing libraries were constructed from 100-1,000 ng of total RNA using the TruSeq total stranded mRNA Library Prep Kit (Illumina, San Diego). Library concentrations were assessed using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) before being barcoded and

Opsin gene mining, phylogenetic reconstruction and expression analyses
Two different strategies were used to mine visual opsin genes from the transcriptomes. First, assembled transcripts were mapped against the opsin gene coding sequences extracted from the genomes of N. sammara and M. jacobus (Musilova et al. 2019) using the medium sensitivity settings (30% max. mismatch between transcripts) in Geneious v.9.1.5 and v.11.0.2 (www.geneious.com). Because assemblies based on short-read sequences tend to overlook lowly expressed genes and/or may result in hybrid transcripts, a second, raw-read mapping approach was also taken, as described in detail in de Briefly, raw-reads were mapped against the reference datasets using medium-low sensitivity settings (20% max. mismatch between reads). Moving from single nucleotide polymorphism (SNP) to SNP along the mapped reads and taking advantage of paired-end information to bridge gaps between nonoverlapping regions, mapped reads were manually sorted into different copies or alleles. Extracted reads were de novo assembled and if the coding region was incomplete, the consensus was used as a template against which unassembled reads were re-mapped with customised low sensitivity settings (0 -2% max. mismatch between reads) to elongate the region of interest.
Holocentrid opsin genes were scored for similarity to publicly available opsin sequences using BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Their phylogenetic relationship was then confirmed using a reference dataset obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The combined opsin gene dataset was first aligned in MAFFT v.7.388 (Katoh and Standley 2013) using the L-INS-I algorithm and default settings in Geneious. jModeltest v.2.1.10 (using AIC for model selection; (Ronquist et al. 2012)) and MrBayes v.3.2.6 (Ronquist et al. 2012) were then run on the CIPRES platform (Miller et al. 2010) to select the most appropriate model of sequence evolution and to infer the phylogenetic relationship between genes, respectively. We used the GTR+I+γ model, with two independent MCMC searches (four chains each), 10 Million generations per run, a tree sampling frequency of 1,000 generations, and a burnin of 25% to generate the holocentrid opsin gene consensus tree.
Opsin gene expression was subsequently calculated by mapping the unassembled filtered reads against the extracted coding sequences of each species-specific opsin repertoire (threshold of 2-3% max. mismatch between reads; read count was normalised to the length of the coding sequence of each opsin), as detailed in de  and Tettamanti et al. 2019(Tettamanti et al. 2019. The expression of each cone opsin was calculated as the proportion of the total cone opsins expressed or, in the case of the rod opsin, as the proportion of RH1 compared to the total opsin expression. Because RH2 paralogs in the Holocentrinae showed high sequence similarity (> 96% pairwise identity), RH2-specific reads were extracted and sub-mapped against high variability areas (100 -200 bp in length). The proportional gene expression of RH2 paralogs was then re-calculated using normalised read counts from the sub-mapping approach.

Spectral sensitivity estimations
Spectral sensitivities of holocentrid visual pigments were estimated by first translating the newly extracted opsin gene sequences to amino acid sequences using the amino acid translate function in Geneious. Holocentrid opsin amino acid sequences were then aligned with bovine rhodopsin (NP_001014890.1) using MAFFT (v. 7.222) (Katoh et al. 2002). This allowed us to identify holocentrid specific opsin residues corresponding to known tuning and binding pocket sites according to the bovine rhodopsin protein structure (Palczewski et al. 2000). Holocentrid opsin protein sequences were then aligned with reference sequences of well-studied model systems with known visual pigment spectral sensitivities acquired through in vitro expression or MSP, and their visual pigment spectral sensitivities were inferred based on sequence differences to each primary reference sequence: (RH1, O. latipes RH1 (Matsumoto et al. 2006 (Parry et al. 2005). We focused on variable amino acid residues either at known tuning sites or at retinal binding pocket sites. Site effects were then either inferred for known substitutions or for substitutions that cause a change in polarity compared to the residue found in the primary reference sequence. Detailed methods including the tuning sites that were used and corresponding references can be found in the Supplementary Materials.

Fluorescence in situ hybridization (FISH)
After dark-adaptation for an hour, the eyes of two N. sammara and one M. berndti were enucleated and prepared for FISH following a customised protocol modified from Barthel and Raymond (Barthel and Raymond 2000). The vitreous was removed enzymatically, directly in the eye cup, by treatment with hyaluronidase (Sigma, 200 U/ml) and collagenase (Sigma, 350 U/ml) in 0.1 M PBS for up to 30 min at room temperature. The retinas were then briefly rinsed in 0.1 M PSB, dissected out of the eye cups and the retinal pigment epithelium was removed mechanically by PBS jetting. The retinal wholemounts were then pinned down in a petri dish and fixed overnight in a solution of 4% paraformaldehyde in 0.1 M PBS (100 nM PB with 5% sucrose), rinsed in PBS, transferred briefly (~10 sec) to 70% methanol, and stored in 100% methanol until further processing.
Following previously described methods (Raymond and Barthel 2004, Allison et al. 2010, Dalton et al. 2014, Dalton et al. 2015, dual-labelling FISH was performed on wholemount retinas or quadrants of retina for very large retinas. In brief, extracted total RNA (see transcriptome sequencing above for extraction details) was reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems). cDNA was then used to generate the probe template by standard PCR using the MyTaq TM HSRED DNA Polymerase (Bioline) and opsin specific primers (listed in Table S2)

Preparation of retinal wholemounts
Eyes were fixed in 4% PFA in 0.1 M PBS (pH = 7.4) for 48 h. Retinal wholemounts were then prepared according to standard protocols (Stone 1981, Coimbra et al. 2006, Ullmann et al. 2011. Radial cuts were performed in order to flatten the eye and subsequently the entire retina onto a glass slide. The orientation of the retina was kept by referring to the position of the falciform process that ends ventrally for Holocentrinae and naso-ventrally for the Myripristinae. The sclera and choroid were gently removed, and the retina was bleached overnight at room temperature in a solution of 3% hydrogen peroxide in 0.1 M PBS. For photoreceptor analysis, retinas were wholemounted (photoreceptor layer up) in 100% glycerol on a microscope slide. For ganglion cell analysis, the retinas were wholemounted, ganglion cell layer facing up, on a gelatinised slide and left to dry overnight in formalin vapour to improve fixation and cell differentiation (Coimbra et al. 2006, Coimbra et al. 2012. Wholemounts were then stained in 0.1% cresyl violet following the protocol of Coimbra et al. 2006(Coimbra et al. 2006) and finally mounted with Entellan New (Merck). Possible shrinkage during staining was considered negligible and if present confined to the retina margin, since the retinal wholemount was attached to the slide during the entire staining process (Coimbra et al. 2006).

Distribution of the different neural cell types across the retina
Different types of analyses were performed for high-density cell types (that is, single cones, double cones and ganglion cells) and low-density cell types (triple cones). Following the protocols described in de Busserolles et al. 2014a,b (de Busserolles et al. 2014a, de Busserolles et al. 2014b), topographic distribution of single cones, double cones, total cones and ganglion cells were assessed using the optical fractionator technique (West et al. 1991) modified by Coimbra et al. 2009(Coimbra et al. 2009, Coimbra et al. 2012 for use in retinal wholemounts. Briefly, for each wholemount, the outline of the retina was digitized using a 5x objective (numerical aperture 0.16) mounted on a compound microscope (Zeiss Imager.Z2) equipped with a motorised stage (MAC 6000 System, Microbrightfield, USA), a digital colour camera (Microbrightfield, USA) and a computer running StereoInvestigator software (Microbrightfield, USA). Cells were randomly and systematically counted using a 63x oil objective (numerical aperture 1.40) and the parameters listed in Table S3 and S4. The counting frame and grid size were chosen carefully to maintain the highest level of sampling (~200 sampling sites) and achieve an acceptable Schaeffer coefficient of error (CE). The CE is a measure of the accuracy of the estimated total number of cells and is considered acceptable below 0.1.
Single cones and double cones were easily distinguished ( Figure 1) and counted separately and simultaneously using two different markers to generate data for single cones alone, double cones alone, and the two cell types combined (total cones). Due to the low number of single cones present in the retinas of all holocentrids, the analysis for the single cones was repeated using a larger counting frame (Table S4).
In Holocentrinae, ganglion cells were arranged in a single layer in the ganglion cell layer.
However, in Myripristinae, several ganglion cells were displaced and present in the inner nuclear layer ( Figure S1). Since it was not always possible to confidently identify the ganglion cells present in the inner nuclear layer from the other cell types (amacrine cells, bipolar cells), only ganglion cells present in the ganglion cell layer were counted in this study. In each species, ganglion cells present in the ganglion cell layer were easily identified from other cell types (displaced amacrine cells and glial cells) using cytological criteria alone (Hughes 1975, Collin andCollin 1988). As a result, amacrine cells and glial cells were excluded from the analysis and only ganglion cells were counted in this study.
Topographic maps were constructed in R v.2.15.0 (R Foundation for Statistical Computing, 2012) with the results exported from the Stereo Investigator Software according to Garza-Gisholt et al. 2014(Garza Gisholt et al. 2014). The Gaussian kernel smoother from the Spatstat package (Baddeley and Turner 2005) was used and the sigma value was adjusted to the distance between points, that is, grid size.
The distribution of the triple cones was mapped from one retina of S. rubrum using the Neurolucida software (MicroBrightField). The outline of the retinal wholemount was digitized using a 5x objective (numerical aperture, 0.13). The entire retina was then scanned in contiguous steps using a 20x objective (numerical aperture, 0.8), and each triple cone was marked. Results were exported from the Neurolucida software, and a dot map representing the location of each triple cone was constructed in R v.2.15.0 (R Foundation for Statistical Computing) using a customized script based on Garza-Gisholt et al. 2014(Garza Gisholt et al. 2014.

Spatial resolving power
The upper limit of the spatial resolving power (SRP) in cycles per degree was estimated for each individual using the ganglion cell peak density as described by Collin &Pettigrew 1989 (Collin andPettigrew 1989). Briefly, the angle subtending 1 mm on the retina (angle a) can be calculated as follows: Where f, the focal length, is the Matthiessen's ratio (i.e. the distance from the centre of the lens to the retina, (Matthiessen 1882)) times the radius of the lens. While in most teleosts the Matthiessen's ratio is close to 2.55, in holocentrids it is between 2.1 and 2.2 (McFarland 1991). Accordingly, we used a ratio of 2.15 in this study.
Knowing a, the peak density of ganglion cells (PDG in cells/mm) and the fact that two ganglion cells are needed to distinguish a visual element from its neighbour, the SRP in cycles per degree (cpd) can be calculated as follow:

Anatomy of the retina
Holocentrids show a typical vertebrate retina organised in several layers: photoreceptor, outer nuclear, inner nuclear and ganglion cell layer (Figure 2A, C). However, compared to the duplex nature of the photoreceptor layer in most vertebrates, i.e., one layer that contains both rod and cone cells, holocentrids possess a multibank retina composed of one layer of cones and several layers of rods. In the three species investigated (from three different genera and the two subfamilies) this multibank organisation was found across the entire retina ( Figure S2). However, the number of rod banks varied in different areas of the retina and between species (Figure 2, Figure S2). Up to six and seven banks could be identified in N. sammara and S. diadema (Holocentrinae), respectively, while in M. murdjan (Myripristinae) up to 17 banks were observed. The highest number of banks in the Holocentrinae was found in the temporal and central areas whereas in M. murdjan the highest number of banks was found in the ventral area ( Figure S2). Indicative of a higher rod photoreceptor density, the outer nuclear layer (ONL) in M. murdjan also showed an increased thickness compared to the ONL in Holocentrinae ( Figure   2). In all three species, the rod outer segment length appeared to be uniform across all banks but varied between species. Overall the higher the numbers of banks, the shorter were the outer segments (~15 µm in M. murdjan, ~21 µm in S. diadema and ~31 µm in N. sammara, Table S5). However, at least for S. diadema, the width of the outer segment seemed to increase from layer to layer, with the first layer (B1, most scleral layer) having the thinnest rods ( Figure 2B). Consequently, in S. diadema, the first layer of rods (B1) had the highest density of cells and the last layer (B7) had the lowest. It is notable that for several sections from all three species, the rod nuclei in the ONL were arranged in vertical lines, as illustrated for M. murdjan in Figure 2C.
Although rod-dominated, three types of cones (single, double and triple) were also found in the retinas of holocentrid species ( Figure S3). Double cones were the most frequent type, followed by single cones, while triple cones were rarely found. In Myripristis spp., double and single cones were not organised in a regular array or mosaic, but instead were arranged randomly throughout the retina ( Figure   1, Figure S4). In Holocentrinae, cone arrangements varied in different parts of the retina ( Figure S4). In general, double cones were organised in regular rows, except in the temporal retina where the arrangement was more squared (i.e., double cones were positioned at an angle) and in the central retina where there was no apparent organisation. Single cones appeared evenly spread out throughout the retina although no obvious general pattern was observed, except in the nasal retina of N. sammara where they were arranged in a square pattern. When present, single cones in Holocentrinae were always placed in the middle of four double cones, as seen in the classic teleost square mosaic ( Figure 1C, D).
The holocentrid retina also showed clearly discernible photoreceptor and retinal pigment epithelium (RPE) retinomotor movements. In the light-adapted state ( Figure 2D, E), the cones and the melanin pigment granules within the RPE were positioned in the most vitreal part of the photoreceptor layer at the level of the first rod bank ( Figure 2D, E). Conversely, in the dark-adapted state, cones were positioned at the level of the third rod bank and the melanin pigment granules migrated all the way to the top of the last rod layer ( Figure 2B). Therefore, in bright conditions, cones were first exposed to the incident light and rods were protected by the RPE, while in dim-light conditions, all rod layers were exposed to the light.

Visual opsin genes and their expression
Transcriptomes from eight holocentrid species (four species per subfamily) showed that they predominantly express one rod opsin and either two (Myripristinae) or three cone opsins (Holocentrinae) within their retinas. Phylogenetic reconstruction identified these opsins to be RH1 (rod opsin; dim-light vision), SWS2A (blue-sensitive), and either RH2B-1 or two RH2A paralogs (RH2A-1 and RH2A-2; bluegreen-sensitive) for Myripristinae and Holocentrinae, respectively. We were also able to extract a partial RH2B-2 sequence from the M. murdjan transcriptome and found evidence for a third RH2A-3 copy in S. spiniferum ( Figure 3A; Figure S5).

Fluorescence in situ hybridization
FISH was performed on the retinas of two holocentrid species, N. sammara (Holocentrinae) and M. berndti (Myripristinae). In both species, SWS2A expression was limited to single cones while RH2 opsins (RH2B-1 for M. berndti; RH2A-1 and RH2A-2 for N. sammara) were expressed in the double cones only (Figure 4). Moreover, in N. sammara, the two RH2A paralogs were co-expressed in both members of the double cones (Figure 4). These expression patterns were consistent throughout the retina of each respective species.
For all Myripristinae, RH2B-1 was estimated to be maximally sensitive at 500 nm. In M. berndti, this estimate was similar the lower limit of the double cone spectral sensitivities measured by MSP (mean ± s.d. 506 ± 2.4 and 514 ± 1.4 nm; (Losey et al. 2003)  berndti only contained a single RH2 opsin gene. In Holocentrinae, there was no or only very little difference (± 1 nm) in the λmax estimates of the two RH2A paralogs (λmax = 513-514/518 nm). These estimates were comparable to the N. sammara double cone λmax measured by MSP (λmax = 512 ± 3.0 nm, (Losey et al. 2003), Table S8). Holocentrinae had a well-defined area temporalis that weakly extended along the horizontal meridian but did not reach the nasal area of the retina. Myripristinae, on the other hand, had a very large area centralis with a peak density in the ventral-temporal part of the retina. In addition, M. berndti and M. murdjan also had a horizontal streak ( Figure S8). The subfamilies also differed in their ganglion cell numbers and densities (Table 1). Holocentrinae had a higher total number of ganglion cells compared to Myripristinae, with an average of 830,000 cells and 580,000 cells, respectively. Ganglion cell peak densities were also much higher in Holocentrinae compared to Myripristinae, with densities ranging from 9,510 to 23,786 cells/mm 2 and 2,150 to 5,990 cells/mm 2 , respectively. Consequently, although Myripristinae usually had bigger lenses (Table S3), Holocentrinae had higher visual acuity estimates. Most Holocentrinae had an estimated spatial resolving power (SRP) around 7 cycles per degree with the highest acuity recorded for N. sammara at 11 cycles per degree while all Myripristinae had an estimated SRP of around 4 cycles per degree (Table 1).

b) Total cone distribution
Similar to the ganglion cell distribution, total cone topography differed between each subfamily ( Figure 5). Myripristinae had a strong horizontal streak, slightly oblique in orientation, with a peak cell density in the central to temporal area. Holocentrinae, on the other hand, had two areas located temporally and nasally, as well as a weak horizontal streak. Moreover, their peak cell density was found in the temporal area ( Figure S9) with the exception of S. rubrum where the peak cell density was found nasally. Figure S9D) between the one found for the remaining Myripristinae ( Figure S9A-C) and the Holocentrinae ( Figure   S9E-I). Interestingly, for all Myripristinae, peak cell densities and topography patterns of total cone photoreceptors did not match the ones found for ganglion cells ( Figure 5, Figure S8, S9). In Holocentrinae, peak cell densities of total photoreceptors and ganglion cells matched pretty well even though the topography patterns were slightly different between cell types; the ganglion cell pattern was mainly defined by an area temporalis while the total photoreceptor pattern was characterised by two areas and a weak horizontal streak. Similar to the ganglion cell numbers, Holocentrinae had more cones than Myripristinae with numbers ranging from 586,815 to 984,127 cells and 308,075 to 637,051 cells, respectively (Table 2).

c) Double cone distribution
Double cones were the main cone photoreceptor type found in the holocentrid retina accounting for around 87% of all cones ( Table 2). As a result, the double cone topography matched the total cone topography for all species (Figure 5, Figure S9, S10).

d) Single cone distribution
Single cones only accounted for around 13% of the total cone population in the holocentrid retina (Table 2). The difference in single cone topography between the two subfamilies was the most pronounced of all neural cell types ( Figure 5, Figure S11). In Myripristinae, the strong streak seen in the total and double cone topographies nearly disappeared and was replaced by a small area temporalis. In Holocentrinae, the single cone pattern was quite similar to the total and double cone patterns, with the two areas (temporal and nasal) and a weak streak. However, single cones were also more present in the ventral part, and interspecific variability was greater for this subfamily compared to the double cones ( Figure   S11). With the exception of N. sammara, for which the single cone peak density was in the nasal area while the double cone peak density was in the temporal part, all Holocentrinae species had their single and double cone peak densities in the temporal part of the retina.

e) Ratio of double to single cones
The mean double to single cone ratio (DC/SC ratio) across the retina varied between species from 4:1 in S. rubrum to 9:1 in M. berndti, with most species having a ratio of 7:1 ( Table 2). The topography of DC/SC ratio was similar for all species with a higher DC/SC ratio in the dorsal part of the retina ( Figure   5, Figure S12).

f) Triple cones
Triple cone density and distribution was assessed for one individual of S. rubrum, the species for which the most triple cones were observed across the retina. Triple cones for this individual only represented 0.5% of the total cone population. While triple cones were present across most of the retina, they were more concentrated along the horizontal meridian similar in pattern to the total cone distribution ( Figure S3B and S9H).

The holocentrid multibank retina
The most striking feature of the holocentrid visual system is its multibank retina. While anecdotally mentioned by McFarland 1991(McFarland 1991, the multibank aspect of the holocentrid retina was not properly assessed or described. Multibank retinas are mostly found in teleost species that live in dim-light conditions, mainly deep-sea fishes and a few nocturnal shallow or freshwater species, including several species of Elopomorpha (Gordon et al. 1978, Shapley et al. 1980, Hess et al. 1998, Bozzano 2003, Omura et al. 2003, Taylor and Grace 2005, Taylor et al. 2015, the bastard halibut, Paralichthys olivaceus (Omura and Yoshimura 1999), and the torrentfish, Cheimarrichthys fosteri (Meyer-Rochow and Coddington 2003). Within the elopomorph representatives, two are also reef associated: the moray eel, Muraena helena, and the conger eel, Ariosoma balearicum (Hess et al. 1998).
Therefore, the presence of a multibank retina in Holocentridae, a nocturnal coral reef fish family with a strong connection to the deep-sea environment , Greenfield et al. 2017, while certainly unusual, is not so surprising.
Similar to the majority of teleosts with multibank retinas, rods in holocentrids are organised in well-defined banks. In other species the number of banks may vary in different parts of the retina (Locket 1985) and/or during ontogeny with banks added as the fish grows Wagner 1998, Omura et al. 2003). While the number of banks seems to vary across the holocentrid retina, it is currently not known if banks are added ontogenetically, and at what stage/age the multibank starts to develop in the first place. Shand 1994(Shand 1994b, studied the gross retinal structure of holocentrid larvae at settlement stage (i.e., after metamorphism) and did not report a multibank retina. This suggests that the extra banks are added later, although a more in-depth study of the development of the holocentrid retina is needed to confirm this.
The number of banks found in the holocentrid retina (6 to 17) is high compared to other species.
All shallow and freshwater species and most deep-sea fishes with multibank retinas only have two to six banks. To date, only five deep-sea species are known to possess more than six banks, with a record of 28 banks found in the deep-sea bigeye smooth-head, Bajacalifornia megalops (Locket 1985, Denton and Locket 1989, Landgren et al. 2014). However, in B. megalops, this extremely high number of banks is constrained to the fovea while the rest of the retina has two to three banks (Locket 1985). Therefore, holocentrids, and especially species from the genus Myripristis, are part of a small group of fish with the highest number of rod banks. Since the three species analysed in this study are shallow-water representatives of the family, a comparison with the retinal structure of the deep-sea holocentrids would be of particular interest. In any case, the fact that the multibank is so well developed in shallow water holocentrids suggests that this specialisation is functional and most likely used in dimlight conditions.
Although multibank retinas are common in deep-sea fishes and elopomorphs, their function is still poorly understood. Amongst the several theories put forward, two non-mutually exclusive hypotheses are commonly stated: 1) multibank retinas enhance the sensitivity of the eye by increasing the density and length of the rods , Warrant et al. 2003; 2) they enable rod-based colour discrimination by changing the light chromatically as it passes through the different banks (Denton and Locket 1989). Support for either hypothesis is lacking, mostly because of the difficulty to access live deep-sea specimens to conduct physiological and/or behavioural experiments. Therefore, holocentrids may offer an ideal model to address these questions as they can be readily accessed and perform well in captivity. Their large eyes (Schmitz and Wainwright 2011), short focal length (McFarland 1991), roddominated retina (this study), extremely high RH1 expression compared to other coral reef fishes [i.e. > 95% in holocentrids (this study) vs 50-70% in damselfishes , Stieb et al. 2019) and diurnal apogonids (Luehrmann et al. 2019)], and their rod spectral sensitivity tuned to their respective depth range (Toller 1996), indicate a visual system well-adapted to dim-light vision and a need for increased sensitivity. Therefore, multibank retinas in holocentrids are likely to contribute toward enhancing the overall light sensitivity of their eyes. However, a use in colour vision during crepuscular hours or at night is also a possibility (Denton and Locket 1989).

Colour vision under bright-and dim-light conditions
Colour vision relies on the opponent processing of light caught by a minimum of two differently tuned photoreceptors (Kelber et al. 2003). Since rods and cones usually function at different light levels and most vertebrates possess a single rod type, colour vision is often thought to be exclusively cone-based and limited to bright-light conditions. However, dim-light colour vision based on modified cones or on a combination of cones and rods does exist and has been demonstrated in geckos (Roth and Kelber 2004) and amphibians (Yovanovich et al. 2017), respectively. Moreover, several deep-sea fishes that possess multiple rod types , Musilova et al. 2019) and/or a multibank retina (Denton and Locket 1989) are likely candidates for purely rod-based colour discrimination. In the case of holocentrids, nocturnal colour vision may be achieved by their multibank retina, even with a single rod type, on the assumption that each bank acts as a spectral filter (Denton and Locket 1989). Under this scenario, each bank is assumed to have a different spectral sensitivity and colour vision is made possible by comparing the outputs of the different banks (Denton and Locket 1989). While this is possible in theory, only behavioural experiments combined with neurophysiological measurements will be able to attest whether holocentrids use their multibank retinas for dim-light colour discrimination.
In addition to their multibank retina, Holocentridae do possess several cone types and as such also have the potential for "classic" colour vision during daytime. Results from this study indicate that Holocentridae possess at least two spectrally different cones types and therefore are likely dichromats. All species investigated here possess single cones that express the SWS2A gene sensitive to the blue range of the spectrum, and double cones that express one or two RH2 genes, which confer sensitivity to the green range of the spectrum. While cone spectral sensitivity estimations made in this study were comparable to MSP measurements performed by Losey et al. 2003(Losey et al. 2003, some differences were observed, notably in M. berndti in which two different spectral sensitivities were found in double cones while only a single RH2B gene was expressed in the retinas of the fish from our study. The discrepancy between opsin gene expression and MSP data may be due to the different origin of the individuals studied; opsin gene expression was measured in fishes from the Great Barrier Reef while MSP was performed in fishes from Hawaii (Losey et al. 2003). Moreover, opsin gene expression, and by extension spectral sensitivities, provide a snapshot of the visual system of the animal at the time of sampling, a state that may change in teleost fishes over the course of the day (Johnson et al. 2013), between seasons (Shimmura et al. 2017) and at different habitat depths ). Therefore, it is possible that M. berndti individuals collected in February in Australia possess a different set of visual pigments to individuals collected in Hawaii in May-June. Notably, holocentrids do possess up to eight RH2 copies in their genomes (Musilova et al. 2019), but only a maximum of three were expressed at the same time in our dataset. Additional copies could therefore be used under different light settings and/or at different ontogenetic stages. Further in situ and experimental studies combining RNAseq and MSP will be needed to further explore this.
Unlike in other coral reef fish families that show high interspecific variability in cone opsin expression (Phillips et al. 2015, Luehrmann et al. 2020, interspecific variability was very low in holocentrids from the same subfamily and did not seem to correlate with habitat partitioning. Between the subfamilies, Holocentrinae had a higher proportion of cone opsin expression compared to Myripristinae, and expressed two RH2A paralogs compared to the single RH2B copy expressed in Myripristinae. Since the two RH2A genes in Holocentrinae were found to be co-expressed in both members of the double cones and are also predicted to have similar spectral sensitivities, the advantage of expressing two copies over a single RH2 gene remains to be investigated. Nevertheless, the higher cone opsin expression in Holocentrinae does suggest that they rely on their photopic visual system more than the Myripristinae. However, whether holocentrids can discriminate colour during the day remains to be investigated. The large size of their cones photoreceptors compared to diurnal species (Munz and McFarland 1973) may also increase sensitivity to lower light conditions and/or allow cone or a mixture of cone and rod-based colour vision in dim-light conditions (Hess et al. 1998). Consequently, their large cone photoreceptors and multibank retinas may enable holocentrids to perceive colour in a wide range of light intensities.

Holocentrid visual ecology a) Ganglion cell topography and acuity
Retinal ganglion cell topography is a powerful tool in visual ecology, highlighting areas of high cell density and therefore high acuity in a specific part of the visual field. In teleost fishes, several studies have shown a strong link between the retinal topography pattern and the habitat and/or behavioural ecology of an animal (Collin and Pettigrew 1988a, b, 1989, Shand et al. 2000, Collin 2008, de Busserolles et al. 2014b). In holocentrids, ganglion cell topography showed very little intraspecific and interspecific variability within each subfamily. However, density and topography patterns differed between the two subfamilies. While both subfamilies share similar habitats (Gladfelter and Johnson 1983), they differ in feeding ecologies, with Holocentrinae mainly feeding on benthic crustaceans and species from the genus Myripristis (Myripristinae) feeding on large zooplankton in the water column (Gladfelter andJohnson 1983, Greenfield 2002). Accordingly, an area temporalis that extends into a weak horizontal streak may allow Holocentrinae to scan and detect crustaceans situated in front of them, on or close to the sea floor.
In Myripristinae, a large area temporo-ventralis that provides higher acuity in front and above of them, may instead facilitate the detection of zooplankton when seen against the background illumination. A similar ganglion cell topography and relationship with feeding mode was also observed in another nocturnal coral reef fish family, the Apoginidae (cardinalfishes) (Luehrmann et al. 2020).
In addition to the topography pattern, spatial resolving power (SRP) also differed between the two subfamilies with Holocentrinae having higher acuities than Myripristinae. However, this result has to be interpreted carefully since Myripristis spp had a large population of displaced ganglion cells that was not included in the analysis, potentially resulting in an underestimation of their SRP. Displaced ganglion cells have been described in many vertebrates and shown in several cases to be part of the accessory optic system (Simpson 1984). As such, all or some of these displaced ganglion cells are likely to have a different function to the ones found in the ganglion cell layer and may not contribute to visual acuity.
Future labelling and tract tracing experiments will be needed to elucidate the function of the displaced ganglion cell population in Myripristinae. Moreover, while displaced ganglion cells were not obvious in Holocentrinae, their presence/absence was not studied here and will need to be assessed further.
Regardless, holocentrid visual acuity was relatively low compared to diurnal reef fishes with similar eye sizes [e.g. the SRP of M. murdjan and S. rubrum is 4.38 and 7.35 cpd, respectively, vs an SRP in Lethrinus chrysostamus of 22 cpd, (Collin and Pettigrew 1989)]. However, Holocentrids did have a similar SRP than the nocturnal apogonids (~7 cpd, (Luehrmann et al. 2020)). Since apogonids are much smaller fish with much smaller eyes, this suggests a higher eye investment in visual acuity for apogonids compared to holocentrids. Conversely, the large eyes in holocentrids (Schmitz and Wainwright 2011) coupled with a short focal length (McFarland 1991), is likely an adaptation to increase the overall sensitivity of the eye rather than its acuity. b) Photoreceptor topography Photoreceptor cells constitute the first level of visual processing and as such their density and distribution provide important information about the visual demands of a species. Even though the holocentrid retina is rod-dominated, only the density and distribution of cone photoreceptors could be assessed in this study due to the presence of the multibank retina.
In diurnal teleost fishes that have a cone-dominated retina, cones are generally arranged in a regular pattern or mosaic. Conversely, nocturnal and bottom-dwelling fishes that have a rod-dominated retina, tend to have disintegrated cone mosaics (Engström 1963). Accordingly, holocentrids, and especially Myripristinae, were found to have a mostly disintegrated cone mosaic that fits with their nocturnal activity pattern. Holocentridae, however, did have a more organised cone arrangement, especially in the temporal part, the area with the highest visual acuity. This, in addition to their higher cone densities, cone opsin expression and visual acuity suggest that the Holocentrinae visual system is more adapted for photopic conditions than the visual system of the Myripristinae.
Similar to the ganglion cells, photoreceptor topography may be used to identify areas of the visual field that are ecologically meaningful for a species. While photoreceptor and ganglion cell topography usually match and have peak cell densities in the same region of the retina, variations do exist, and may indicate different visual demands in different parts of the visual field of the animal and/or at different times of the day (Stieb et al. 2019, Tettamanti et al. 2019. In Holocentrids, total cone and ganglion cell topographies differed, especially in Myripristinae. Since holocentrids are nocturnal fish and have a roddominated retina, it is likely that the topography and peak density of their rod photoreceptors matches that of their ganglion cells, as seen in some deep-sea fishes . This is supported by the highest number of banks being located in the area of the highest ganglion cell density in all three species. Unfortunately, limited information is available about holocentrid daytime activities. During that time, they appear to hover in or above their refuges and may partake in some social interactions such as courtship, aggression and/or predator avoidance (Winn et al. 1964, Carlson andBass 2000). Accordingly, a horizontal streak may allow them to scan a wide area of their visual field to look for possible intruders or conspecifics while staying within the safety of their refuges, as suggested for the highly territorial anemonefishes (Stieb et al. 2019). Moreover, a high density of cells in the nasal area, as seen in Holocentrinae, may also help in detecting predators coming from behind (Collin and Pettigrew 1988a).
For all holocentrids, but especially in the Myripristinae, single and double cone topography differed from one another, suggesting that the different types of cones may be used in different visual tasks. While it has been demonstrated that both single and double cones are used for colour discrimination in the coral reef Picasso triggerfish, Rhinecanthus aculeatus (Pignatelli et al. 2010), in many other species this is not clear . Since the spectral sensitivity of double cones often matches the spectral distribution of the ambient/background light they may be used in luminance detection tasks (McFarland 1991, Carleton et al. 2020. In holocentrids, this idea is supported by the ratio of double to single cones which was consistently higher in the dorsal retina. If double cones are indeed used in achromatic tasks, having a higher proportion in the dorsal retina, the area that samples light in the field of view below the fish where background illumination is lower, might assist in increasing sensitivity. However, behavioural tests will be needed to confirm this.

Conclusion
Holocentridae have a visual system that is well-adapted for their nocturnal lifestyle with large eyes, short focal length, rod-dominated retina, multibank retina, extremely high rod opsin expression, rods tuned to their preferred light conditions, few and lowly expressed cone opsins, few cone photoreceptors, and relatively low visual acuity. Moreover, the fact that the holocentrid ganglion cell topography correlates with their feeding mode, a task which in this family is exclusively conducted at night, further supports their heavy reliance on their scotopic visual system. The presence of at least two spectrally different cone types with their own topography patterns also indicates the use of their cone-based visual system during the day and the potential for dichromacy. Moreover, while interspecific variability was very low within the family, differences in visual adaptations could be seen between the two subfamilies at all levels with Holocentrinae having a slightly more developed photopic visual system compared to Myripristinae. Finally, what really sets the holocentrid family apart from other coral reef fishes is their well-developed multibank retina, an adaptation mostly found in deep-sea fishes, and their potential for colour vision in a wide range of light settings, especially under scotopic conditions. Future ontogenetic and behavioural analyses should therefore be conducted in order to understand the origin and function of the multibank retina, as well as to assess if this family is able to discriminate colours and under which light intensities. Additionally, investigation of other teleosts with intermediate depth ranges, such as mesophotic species, are likely to reveal interesting adaptations for dim-light vision.