Keywords
Coral reef fish, ontogeny, phylogeny, gene expression, opsins,
colouration
Abstract
Damselfishes (Pomacentridae) are widespread and highly abundant on
tropical coral reefs. They exhibit diverse body colouration within and
between the ~250 species and across ontogenetic stages. In
addition to human visible colours (i.e., 400-700 nm), most adult
damselfishes reflect ultraviolet (UV, 300-400 nm) colour patches. UV
sensitivity and UV colour signals are essential for feeding and form the
basis for a secret communication channel invisible to the many UV-blind
predatory fish on the reef; however, how these traits develop across
ontogenetic stages, and their distribution across the damselfish family
is poorly characterised. Here, we used UV photography, phylogenetic
reconstructions of opsin genes, differential gene expression analysis
(DGE) of retinal samples, to investigate the development of UV vision
and colour patterns in three ontogenetic stages (pre-settlement larval,
juvenile, and adult) of eleven damselfish species. Using DGE, we found
similar gene expression between juveniles and adults, which strongly
differed from larvae. All species and all stages expressed at least one
UV-sensitive sws1 opsin gene. However, UV body colour patterns
only started to appear at the juvenile stage. Moreover,Pomacentrus species displayed highly complex UV body patterns
that were correlated with the expression of two sws1 copies. This
could mean that some damselfishes can discriminate colours that change
only in their UV component. We demonstrate dramatic shifts in both UV
sensitivity and UV colouration across the development stages of
damselfish, while highlighting the importance of considering ontogeny
when studying the coevolution of visual systems and colour signals.
Introduction
Animals use colour vision for critical tasks such as feeding, mating,
predator avoidance and navigation (Cronin et al., 2014). To perform
these tasks efficiently in highly variable light environments, such as
those on tropical coral reefs, fishes have evolved visual systems to
perceive short-wavelengths of light (ultraviolet UV, ~
350 nm) up to longer-wavelengths (red, ~ 600 nm)
(reviewed by Cortesi et al., 2020). Damselfishes (Pomacentridae), one of
the most prevalent reef fish families, display a high species diversity,
a variety of ecologies, and differ widely in colouration and morphology
(Allen, 1991). Damselfish visual systems also vary significantly as they
differ in visual gene (opsin) expression and structure (Hofmann et al.,
2012; Stieb et al., 2024, 2023, 2019, 2017, 2016). A notable feature of
damselfishes is their ability to perceive UV wavelengths, facilitated by
a UV-transmitting lens and cornea (Siebeck and Marshall, 2007, 2001),
and a UV-sensitive photoreceptor that expresses the
short-wavelength-sensitive 1 (sws1 ) opsin gene (Job and Bellwood,
2007; Mitchell et al., 2024, 2023; Powell et al., 2021; Siebeck et al.,
2010).
In teleost fishes, five different types of visual opsin genes are found.
Rod photoreceptors express the rhodopsin (rh1 ). In contrast, cone
photoreceptors express sws1 and sws2 , mid-wavelength
sensitive rhodopsin-like 2 (rh2 ), and long-wavelength sensitive
(lws ) opsin genes (Carleton et al., 2020; Musilova et al., 2019).
Opsin genes in teleosts have experienced a dynamic evolutionary history
affected by gene duplications, gene losses, sequence modifications, and
gene conversion (Carleton et al., 2020; Cortesi et al., 2015; Mitchell
et al., 2021; Musilova et al., 2021, 2019; Musilova and Cortesi, 2023).
The latter describes a process by which an unequal crossing over during
meiosis results in a unidirectional transfer of genetic information and
consequential identical residues in different genes (Chen et al., 2007;
Holliday, 1964). Fishes can also change the expression of opsin genes
over development or shorter timescales, and some species have been found
to co-express multiple opsins in the same photoreceptor (Cortesi et al.,
2016; Dalton et al., 2014; Savelli et al., 2018; Torres-Dowdall et al.,
2021). Moreover, some fishes can also convert their chromophore from
A1-derived to A2-derived using the
CYP27C1 enzyme, switching the sensitivity of visual pigments to longer
wavelengths (Enright et al., 2015). These processes allow visual
adaptations to different light environments, behaviours, and ecologies
across generations or within the lifetime of a species (Carleton et al.,
2020).
Damselfishes express different sets of these visual opsin genes, with
some species only expressing three and others expressing up to six cone
opsin copies (Mitchell et al., 2021; Stieb et al., 2024, 2023, 2019,
2017, 2016). In adult damselfishes, single cones have been found to
express the short-wavelength-sensitive opsin genes (sws1 andsws2b ), and the double cones (two fused single cones; Walls,
1942) to express the mid- and long-wavelength-sensitive genes
(rh2 s and lws ). The adults of some, but not all,
damselfish species have also been found to tune opsin gene expression
over short periods (weeks to months), with depth, and between seasons to
adapt to changes in their light environments (Luehrmann et al., 2018;
Stieb et al., 2016). Moreover, species-specific ecologies and
colouration seem to influence opsin gene expression: longer wavelength
sensitivity occurs in herbivorous damselfishes and is more pronounced in
species with red colouration, while shorter wavelength sensitivity
correlates with UV body colour patterns (Stieb et al., 2024, 2023, 2019,
2017, 2016).
Behavioural studies have found that these small fishes use UV signals to
communicate with con- and hetero-specifics (Mitchell et al., 2023;
Siebeck et al., 2010). Because larger predatory fish are UV-blind, this
has led to the hypothesis that damselfishes use UV vision and
colouration as a ‘private communication channel’ (Siebeck, 2004; Siebeck
et al., 2010; Stieb et al., 2017). Moreover, in freshwater fishes, UV
patterns correlate to sexual selection (Macías Garcia and de Perera,
2002; Smith et al., 2002). Some damselfish species, particularly
anemonefishes, also express multiple sws1 copies (Mitchell et
al., 2021; Stieb et al., 2024). The sws1 gene duplication seems
to have occurred independently at least twice in the damselfish family.
One duplication occurred in the Pomacentrinae subfamily and has been
dated to the last common ancestor of the Pomacentrus ,Neopomacentrus and Amphiprion genera (termed Pomacentrinae
3, 4 and 5 in McCord et al., 2021 (Stieb et al., 2024). A second,
species-specific duplication was discovered in the genome ofChromis chromis (Musilova et al., 2019). The two Pomacentrinae
copies cluster in short (sws1α ; λmax 356-362 nm)
and longer sensitive (sws1β ; λmax 368-370 nm)
clades depending on changes in key amino acid sites of the opsin protein
at positions 114 and 118 (Mitchell et al., 2021; Stieb et al., 2024).
However, how widespread the expression of these copies is and their
function remained unclear.
While there have been several studies on adult damselfish vision (e.g.,
Luehrmann et al., 2018; Siebeck et al., 2010, 2010; Stieb et al., 2017),
very little is known about the visual systems of earlier developmental
stages. Studying ontogenetic changes in coral reef fish vision is of
great importance to better understand how they visually adapt to face
diverse challenges encountered through their life stages (e.g. predation
and competition for food and shelter, see Anderson, 2001; Carr et al.,
2002; Hixon and Carr, 1997). During the pelagic larval stage,
damselfishes are transparent and feed on zooplankton (Sampey et al.,
2007). Most zooplankton either reflect or absorb UV light, and it has
been shown that fishes, including salmonids (Flamarique, 2000),
zebrafish (Baden, 2021), cichlids (Jordan et al., 2004), and larval
damselfish (Job and Shand, 2001), use UV vision to spot them against the
UV-lit background in the water column. Once damselfishes settle on the
reef and metamorphose into juveniles, they show various feeding
ecologies and colour patterns, likely to contain UV colours. Often,
damselfish colours and patterns, at least in the human visible, change
again when they turn into adults (Frédérich and Parmentier, 2016).
Skin reflectance measurements using spectrophotometers have commonly
been used to assess the colour of damselfishes, especially in the UV
(Marshall et al., 2019). However, these measurements do not contain
information about the spatial distribution of UV patterns. Only in two
sister species of damsefish (Pomacentrus amboinensis and P.
moluccensis ) (Siebeck et al., 2010) has UV photography been used to
assess the nature of UV patterns. In theseb, the UV patterns show a high
degree of complexity with differently shaped dotted and lined motifs on
the operculum and simpler motifs on the fins and the body that the fish
use to distinguish individuals, similar to a human fingerprint (Gagliano
et al., 2015; Siebeck, 2014, 2004; Siebeck et al., 2010).
In this study, we hypothesised that complex UV colour patterns with
variations in shape and structure are widespread in the damselfish
family. However, these patterns emerge only in later developmental
stages, reflecting the ontogenetic change in colour communications of
these species. Moreover, because several adult damselfishes have been
found to express two sws1 opsins with different absorption
maxima, it is possible that these fishes can distinguish between colours
that only differ in UV wavelengths. Colour discrimination between UV
wavelengths alone has been shown in butterflies (Finkbeiner and Briscoe,
2021) and in mantis shrimp (Thoen et al., 2014), and besides needing
multiple photoreceptors with different UV sensitivity, it also
necessitates UV colours that have dissimilar peaks in the spectral
curve. Hence, we predicted the expression of two sws1 opsins to
correlate with the occurrence of UV patterns that differ in structural
complexity and spectral reflectance. To test our hypotheses, we first
used standardised photography in the human visible (i.e., RGB) and UV to
correlate expression changes with shifts in ecology and colouration,
focusing on UV complex patterns, sws1 expression and considering
the damselfish phylogeny. We then used comparative transcriptomics to
investigate ontogenetic changes in opsin gene expression in eleven
damselfish species from two (Pomacentrinae, Chrominae) of the four
damselfish subfamilies (McCord et al., 2021).
Methods
Specimen collection
Eleven damselfish (Pomacentridae) species (subfamily Chrominae:
Chromis atripectoralis , Dascyllus aruanus ; subfamily
Pomacentrinae: Amphiprion akindynos , Chrysiptera
flavipinnis , Dischistodus perspicillatus , Neoglyphidodon
melas , Pomacentrus amboinensis , P. bankanensis , P.
chrysurus , P. nagasakiensis , and P. pavo ) were collected
from coral reefs around Lizard Island (14°40′S, 145°27′E), Northern
Great Barrier Reef (GBR), between 2019 and 2022. Collections were
conducted in the summer months (mid-November to early March) to avoid
seasonal variability in body colouration and gene expression. Animal
collection, husbandry and euthanasia followed procedures approved by The
University of Queensland’s Animal Ethics Committee (2016/AE000304 &
2022/AE000569). The collections were conducted under permits from the
Great Barrier Reef Marine Park Authority (G17/38160.2) and Queensland
General Fisheries (207976).
We sampled three life stages per species: larval, juvenile, and adult
specimens as, in a few species, significant morphological changes occur
between these stages (e.g. N. melas )(Fig. S6-S16). Light traps
were used to collect the fish at the end of their larval phase before
settlement on the reef (Doherty, 1987). Light traps were placed 20-30 m
from the reef at sunset and collected at dawn. Specimens were sorted in
aerated tanks at the Lizard Island Research Station and processed the
same morning. Juvenile and adult fish were collected on the reefs at
depths of 1-15 m both on SCUBA and snorkel with hand nets and an
anaesthetic clove oil solution (1/6 clove oil, 1/6 of 99% EtOH, and 4/6
of salt water), or using barrier nets. Fish were kept in flow-through
aquaria at the research station, where they were exposed to natural
sunlight for a maximum of 48 hours before further processing.
Photography in RGB and UV
Three individuals per life stage per species (and n = 2 sub-adults ofD. perspicillatus ) were tentatively photographed in both the
human visible (i.e., RGB) and the ultraviolet spectrum (we were able to
collect only UV images of the larval stage for only six species) using
two Nikon D810 cameras, one of which had its original filters removed to
allow for full-spectrum sensitivity (Anderson Camera Repairs, Brisbane).
Each camera was equipped with either a Nikon AF-S Nikkor 50 mm F/1.8G or
a Nikon Micro-Nikkor 60 mm F/2.8 lens, depending on the size of the
subject. The full-spectrum camera was constrained to ultraviolet
wavelengths with a Schott UG11 visible-blocking filter and a Newport
FSQ-BG39 blue bandpass filter. See the supplements (Figs. S3 and S4) for
spectral sensitivity curves.
Each fish was placed in a small glass aquarium and allowed to acclimate
for 2-3 min before being photographed by both cameras in sequence.
Lighting was provided by two Nikon Speedlight SB-26 flashes placed at
opposite corners of the aquarium, pointing down and inwards at the
subject at 45°. Both speedlights were stripped of their filters and
diffusers for full spectrum illumination (Fig. S5). Particularly for
bigger fish, a large aperture was used to increase the depth of field
and ensure the whole individual was in focus. Image J v1.53k was
subsequently used to convert raw images to black and white. Processed
images were then visually scored for the presence and type of UV
patterns: i) no UV reflectance/colour present, ii) simple UV pattern
defined as either uniform stripes or uniform UV body reflectance (see
Fig. 1C), iii) spatially complex UV patterns defined as facial or body
patterns with intricate, differently shaped dotted and lined motifs (see
Fig. 1A and S11-S15, Table S10).
Spectral reflectance
measurements
To assess whether the damselfish UV patterns would differ in a spectral
curve, i.e., if one individual uses different UV colours, we focused onP. amboinensis , for which complex UV (facial) patterns had
previously been described (Siebeck, 2004; Siebeck et al., 2010) and were
easily accessible. Spectral reflectance measurements of two adultP. amboinensis caught from the reefs around Lizard Island in 2005
and 2024 were obtained using an Ocean Optics (Dunedin, FL, USA) USB2000
spectrophotometer connected to a laptop computer running Ocean Insight
(https://www.oceaninsight.com) OOIBASE32 or SpectraSuite software.
Fishes were measured in the laboratory by removing them from the water
and placing them on a wet towel to facilitate handling. Spectral
reflectance curves measured this way do not significantly differ from
those measured in water (Marshall et al., 2003). We used a PX-2 pulsed
xenon light source (Ocean Insight) connected to a 400 μm UV-visible
fibre, and the colour reflectance was measured using a 100 μm UV-visible
fibre connected to the laptop. The bare end of the collecting fibre was
held at a 45° angle to prevent specular reflectance. A Spectralon 99%
white reflectance standard was used to calibrate the percentage of light
reflected at each wavelength from 350 – 750 nm. Measurements were taken
from the operculum and the centre of the body, where different types of
UV patterns occur (Siebeck et al., 2010; also see Fig. 1). Between 4 –
10 measurements per individual were taken from each location and
subsequently averaged.
RNA Extraction, Sequencing and Transcriptome
Assembly
Fish were first photographed (see below) before being sampled for RNA
sequencing (n = 3, samples per stage per species). Briefly, larvae were
anaesthetised and killed between 7 am and 11 am and subsequently stored,
as a whole, in RNAlater (Thermofisher) at –20 °C. Juveniles and adults
were anaesthetised and killed by decapitation between 1 pm and 5 pm;
retinas were removed from the eyecup and then stored in RNAlater at –20
°C.
Barcode DNA sequencing of the Cytochrome c oxidase I (COI) region
was used to identify larvae that could not be assigned to a species
based on morphology alone (n = 2 species). Briefly, DNA was extracted
from fin clips using a custom-made lysis buffer (30 µl of 50 mM NaOH)
and incubated at 90° C for 30 minutes. Subsequently, a neutralising
buffer was added to the solution (5 µL Tris-HCl 1M pH 8.0). The samples
were briefly vortexed and spun down; 2 µl of the supernatant was used to
run a polymerase chain reaction (PCR) using fish universal COI primers (Ward et al., 2005) (for detailed methods, see Supplementary
Methods). DNA was purified from the PCR product with the Monarch® PCR &
DNA Cleanup Kit (New England Biolabs, https://www.nebiolabs.com.au/) and
submitted for Sanger sequencing to the Australian Genomic Research
Facility (https://www.agrf.org.au/). COI sequences were assessed
using Geneious Prime v.2023.0.4 (https://www.geneious.com/), and
low-quality bases were removed after visual inspection. CleanedCOI barcodes were then uploaded to the Barcode of Life Data
System (http://v3.boldsystems.org/; Ratnasingham and Hebert, 2007) for
species identification using default settings.
Larval eyes were removed from the eyecup using forceps and then
homogenised using pestles (Interpath Services,
https://www.interpath.com.au/). Juvenile and adult retinas were
homogenised using glass beads (Sigma, https://www.sigmaaldrich.com/) and
a TissueLyser LT (Qiagen, https://www.qiagen.com/). Following
homogenisation, total RNA was extracted with the RNeasy Mini Kit
(Qiagen) following the manufacturer’s protocol. An optional DNase step
was performed to remove any trace DNA.
RNAseq library preparation using the NEBNext Ultra RNA library
preparation kit for Illumina (NEB; mRNA Poly-A enriched, non-stranded
library) and transcriptome sequencing were outsourced to Novogene
(Singapore, https://www.novogene.com/). The concentration and quality of
libraries were assessed by a Qubit dsDNA BR Assay kit (ThermoFisher)
before barcoding and pooling at equimolar ratios. The libraries were
then sequenced on a Hiseq2500 (PE150, 250–300 bp insert,
filtered and de novo assembled as per de Busserolles et al., 2017
and Tettamanti et al., 2019. Briefly, raw transcriptomes were uploaded
to the Galaxy Australia server (https://usegalaxy.org.au/) (Afgan et
al., 2018), filtered by quality using Trimmomatic (Galaxy version
0.36.6) (Bolger et al., 2014), then de novo assembled with
Trinity (default settings, paired-end, –min_kmer_cov = 4; Galaxy
version 2.9.1) (Haas et al., 2013).
Opsin gene mining and phylogenetic
reconstruction
To investigate developmental changes in damselfish fish vision in more
detail, we mined the damselfish retinal transcriptomes for opsin gene
sequences, following the protocols in de Busserolles et al., 2017 and
Tettamanti et al., 2019. Briefly, we used the opsin gene coding
sequences of the dusky dottyback, Pseudochromis fuscus (Cortesi
et al., 2016), as a reference against which to map the assembled
transcriptome of each individual in Geneious Prime. The P. fuscus opsin repertoire was chosen as a reference as the species is relatively
closely related to the damselfishes, and it possesses all orthologues of
the ancestral vertebrate opsin genes (Cortesi et al., 2016). Because
lowly expressed genes are often overlooked in short-read assemblies and
highly similar genes, such as the opsins, suffer from chimeric assembly
errors, we also used a second approach to verify our initial findings,
as per Musilova et al., 2019. Briefly, the raw transcriptome reads were
mapped against the mined damselfish opsin gene sequences (fine-tuning,
none; maximum gap per read, 10%; word length, 18; maximum mismatches
per read, 2%; maximum gap size, 12 bp; and index word length, 14).
Moving from single nucleotide polymorphism (SNP) to SNP, reads were
manually extracted, taking advantage of the paired-end information
between the SNPs. The extracted reads were then de novo assembled, and their consensus was extracted to form the entire coding
region of the opsin gene. Opsin identity was verified through BLAST
(https://blast.ncbi.nlm.nih.gov/) and by phylogenetic reconstruction
with a reference dataset of vertebrate opsins obtained from GenBank
(www.ncbi.nlm.nih.gov/genbank/) and Ensembl (www.ensembl.org).
The opsin gene phylogeny (Fig. S1) was obtained by first aligning the
damselfish opsin genes to the reference dataset using the L-INS-I
settings of the Geneious MAFFT plugin v1.5.0 (Katoh and Standley, 2013).
The two previously described sws1 copies of A. ocellaris (Mitchell et al., 2021) were added to the dataset to infer the identity
of the orthologues in other species. jModeltest v2.1.6 (Darriba et al.,
2012) was used to determine which model of sequence evolution was the
most appropriate based on the Akaike information criterion. The
phylogeny was then inferred using MrBayes v3.2.7a (Ronquist et al.,
2012) as part of the CIPRES platform (Miller et al., 2010) using the
following parameters: GTR+I+G model; two independent MCMC searches with
four chains each; 10 M generations per run; 1000 generation sample
frequency; and 25% burn-in.
The same settings were used to infer the phylogeny of the sws1 gene clade. However, in this case, we only used the first and the fourth
exons for the alignment, as they carried the strongest phylogenetic
signal. Gene conversion between sws1 copies had confounded the
signal of the remaining exons (see below) (Fig. S2). After confirming
the identity of the sws1 copies, they were plotted against the
most recent damselfish phylogeny (McCord et al., 2021) to reconstruct
the evolutionary history of the gene in the Pomacentridae family.
Sws1 gene conversion
We used GARD (Genetic Algorithm for Recombination Detection) (Kosakovsky
Pond et al., 2006), with default settings to search for patterns of gene
conversion between the sws1 paralogs (n = 16, for nine
Pomacentrinae species). Domains between breakpoints, i.e., the sections
of putative sequence exchange, were subjected to phylogenetic
comparisons to identify the most likely ancestry for each section.
Sws1 spectral-sensitivity predictions
Amino acid comparisons at known tuning sites were used to estimate the
spectral sensitivities of damselfish SWS1-based visual pigments
following the methods in Mitchell et al., 2021 and Stieb et al., 2024.
Briefly, SWS1 amino acid sequences of the eleven damselfish species were
aligned to bovine rhodopsin (BRH; PBD accession no.1U19) as a reference.
We focused on the SWS1 amino acids at BRH sites 114 and 118 as these
sites have previously been shown to evolve in tandem and to confer peak
spectral sensitivity shift of ~10 nm (Stieb et al., 2024).
SWS1 copies with BRH sites A114 and A118 were classified as
shorter-wavelength sensitive (λmax 356-362 nm), and
copies with BRH S114 and S118 were classified as longer-wavelength
sensitive (λmax 368-370 nm), as per Stieb et al., 2024.
These estimations assume a visual pigment with an
A1-based chromophore. A1 is the dominant
chromophore in coral reef fishes (Toyama et al., 2008), and based
on the retinal transcriptomes, none of the species investigated in this
study expressed cyp27c1 (data not shown), the enzyme needed to
convert A1-based chromophores to
A2-based chromophores.
Opsin gene expression and
analysis
Opsin gene expression was calculated by mapping the filtered
transcriptome reads for each individual to the species-specific opsin
coding regions as per Tettamanti et al., 2019. The number of mapped
reads (R) was normalised to the length (bp) of the opsin gene (i) to
which they were mapped against:
\begin{equation}
R_{i,\ \ \ \text{normalized}}=\frac{R_{i}}{\text{bp}_{i}}\nonumber \\
\end{equation}The proportional gene expression (p) for single (pSC)
and double cone (pDC) opsins out of the total normalised
expression for each cone type (TSC; TDC)
was then calculated using the following equations:
\begin{equation}
p_{i,\ \text{SC}}=\frac{R_{i,\ \text{normalized}}}{T_{\text{SC}}}\text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }p_{i,\ \text{DC}}=\frac{R_{i,\ \text{normalized}}}{T_{\text{DC}}}\nonumber \\
\end{equation}The proportional gene expression of the rod opsin (prod)
was calculated by comparing it to the total normalised opsin expression
(Topsin):
\begin{equation}
p_{\text{rod}}=\frac{R_{\text{rod},\ \text{normalized}}}{T_{\text{opsins}}}\nonumber \\
\end{equation}Expression plots were generated in Rstudio v1.4.1106 (Allaire, 2012),
using a customised R script (R version 4.1.0, Ihaka and Gentleman,
1996).
Differential gene
expression
To investigate differences in gene expression throughout ontogeny, we
first performed a Principal Component Analysis (PCA) using all samples
(n = 93). Based on the resulting three clusters (Fig. 2A), we selectedP. amboinensis, Chro. atripectoralis and A. akindynos as
representative species for more in-depth analyses of the top
differentially expressed genes (DEGs) between developmental stages. Due
to the unavailability of a high-resolution genome for any of the species
investigated, the filtered RNAseq reads were mapped to that of a close
relative, the false percula anemonefish, Amphiprion ocellaris (subfamily Pomacentrinae; NCBI accession number: GCF_022539595.1) (Ryu
et al., 2022) on the Galaxy Australia server. Mapping was performed
using HISAT2 v2.2.1 with default parameters (Kim et al., 2019) to create
a mapping-based estimation of transcript abundance. The function
htseq-count v0.9.1 from HTSEQ (Anders et al., 2015) was used to quantify
the number of mapped reads per gene based on the reference A.
ocellaris genome annotation. The function generate-count-matrix v1.0
was then used to create a read count data table.
Differential gene expression between larval, juvenile and adult fish was
inferred using DeSeq2 (Love et al., 2014), on the iDEP.96 platform
(http://bioinformatics.sdstate.edu/idep96/; Ge et al., 2018). Briefly,
the read count data was uploaded to iDEP.96 and matched automatically to
the zebrafish genome assembly GRCz11 (Danio rerio ;
GCF_000002035.6), and gene symbols were converted to ENSEMBL gene IDs
(https://asia.ensembl.org/index.html) for subsequent enrichment
analysis. Pre-processing included removing features with less than 0.5
counts per million across all samples and transforming the data with the
rlog algorithm from DESeq2. P. amboinensis filtered data was then
used to perform a PCA and to create a heatmap of the top 1000 variable
expressed genes, grouped into major clusters by k-means based on the
elbow method to infer the optimal number of clusters (Fig. 2B, C). An
enrichment pathway analysis of each cluster was then performed, and the
top two GO annotations, sorted by False Discovery Rate (FDR), were
selected to define the biological function of each cluster.
DESeq2 was used in the three species selected to find DEGs with a
p-adjusted value of < 0.05 and a minimum fold-change (FC) of
2. Three comparisons were made: larval vs. juveniles, larval vs. adults,
and juvenile vs. adults. The top 15 up- and down-regulated genes, as
determined by log fold-change of each comparison, were used to perform a
GO enrichment analysis in PANTHER via The Gene Ontology Resource (Tables
S1-9) (Ashburner et al., 2000; Thomas et al., 2022). In the majority of
cases, differentially expressed genes had Ensembl gene IDs allocated
(i.e., LOC followed by the NCBI gene ID, e.g. LOC111574217), and
corresponding gene orthologues from zebrafish or medaka (Oryzias
latipes ) were searched for using OrthoDB (Kuznetsov et al., 2023). If a
GO function was missing from a gene, AMIGO was used to infer the
function based on vertebrate orthologues (Carbon et al., 2009).
Results
Ontogenetic changes in UV colour and
patterns
UV and human-visible photography showed that none of the species had UV
patterns at the larval stage, and N. melas was the only species
with no UV colouration as an adult. Simple UV patterns were discovered
in juvenile and adult A. akindynos , Chro. atripectoralis ,Chry. Flavipinnis , D. aruanus , and N. melas juveniles. We identified complex UV patterns with variable stripes,
lines and dotted motifs in later ontogenetic stages of allPomacentrus species (P. amboinensis , P.
bankanensis , P. chrysurus , P. nagasakiensis , P.
pavo ), and D. perspicillatus (Fig. 1 for examples; Fig. S6-S16
for all species). In the Pomacentrus spp., the complex UV
patterns first appeared at the juvenile stage, and they maintained the
patterns after that, except for P. chrysurus, which did not show
the patterns as adults (Table S10; Fig. S14). For D.
perspicillatus , the complex UV patterns were initially only found in
the adult stage. Further analysis of sub-adult individuals (fish that
were judged to be in-between juveniles and adults based on intermediate
sizes, being tolerated within the territories of mature individuals, and
due to human-visible patterns in-between stages) revealed that the UV
patterns first emerge at this stage (Fig. S10). Spectral reflectance
measurements of the complex UV patterns on the body and operculum of two
individuals of P. amboinensis revealed differences in the shape
and the UV-peak of the reflectance curves, with peaks of the body being
around ~365nm and the facial patterns on the operculum
having reflectance closer to ~395nm (Fig. 1D).
Opsin gene mining and phylogenetic
reconstruction
Transcriptome mining and subsequent phylogenetic reconstruction revealed
that all damselfishes expressed a rod opsin (rh1 ) and at least
four cone opsins (lws , rh2a , rh2b and sws1 )
in their retinas (Fig. S1). Two species (P. amboinensis andP. nagasakiensis ) expressed a second green-sensitive rh2a copy, and Chry. flavipinnis expressed a second red-sensitivelws gene. Several species also expressed the violet-sensitivesws2b gene. Six species (A. akindynos , N. melas ,P. amboinensis , P. bankanensis , P. chrysurus , andP. nagasakiensis ) expressed two UV-sensitive sws1 copies.
The separate sws1 phylogeny based on exons 1 and 4 revealed that
the single sws1 expressed in D. auranus and C.
atripectoralis formed a sister clade to the sws1α andsws1β duplicates (Fig. 3). Also, sws1 in D.
perspicillatus and C. flavipinnis fell within the α clade; the two sws1 paralogs in the remaining Pomacentrinae
species could be assigned confidently to either clade.
Sws1 opsin evolution in
damselfish
Plotting the sws1 opsin genes onto the latest damselfish
phylogeny (McCord et al., 2021) revealed that the sws1 duplication most likely occurred in the Pomacentrinae ancestor (Fig. 3
and Fig. 4). The sws1β copy was most likely lost in the ancestor
of the most ancestrally derived Pomacentrinae clade (Cheiloprionini as
per Whitley, 1929; termed Pomacentrinae 1 in McCord et al., 2021; Tang
et al., 2021), containing D. perspicillatus and C.
flavipinnis .
Comparing the amino acids at the two major SWS1 tuning sites (BHR 114
and 118) revealed two distinct phenotypes: I) SWS1 orthologs with S114
and S118, predicted to be longer-wavelength shifted (~370
nm λmax), II) SWS1 orthologs with A114 and A118,
predicted to be shorter-wavelength shifted (~360 nm
λmax) (Fig. 3). SWS1 in the two Chrominae species,D. auranus and C. atripectoralis , and most SWS1β fall into
group I, whereas most SWS1α belong to group II (Fig. 3).
Gene conversion analysis between the Pomacentrinae sws1 paralogs
revealed widespread conversion affecting different segments of thesws1 genes, prompting us to rely only on exons 1 and 4 to resolve
the sws1 phylogeny (Fig. 3). The effects of gene conversion were
most pronounced in N. melas , where sws1β was being pulled
towards the sws1a clade, with the 114 and 118 tuning sites
exchanged between copies (Fig. 3).
Relative opsin gene expression through
ontogeny
Using bulk retinal transcriptomes, we found that the relative opsin gene
expression differed between species and ontogenetic stages (Table 1).
Rod opsin, rh1 expression was highest in larvae, except forD. perspicillatus and N. melas , in which the adults had
the highest expression. Regarding the cone opsins, most species and
stages predominantly expressed four to five genes (sws1α & β ,rh2b , rh2a , and lws ). However, C.
atripectoralis expressed three cone opsin genes independent of life
stage (sws1 , rh2b , and rh2a ), and P.
amboinensis expressed six (sws1α & β , rh2b ,rh2a-1 , rh2a-2 , and lws ). There were notable
differences in the expression of double-cone opsins with ontogeny.
Generally, larval fish had a higher expression of rh2b thanrh2a , with juveniles and adults exhibiting the opposite.Lws was lowly expressed or not expressed at all in most species
at the larval stage. However, four species (D. perspicillatus ,C. flavipinnis , P. bankanensis , and P. chrysurus )
showed a higher expression (≥ 2% of proportional DC expression) at this
stage. In the juvenile and adult stages, more species expressedlws . Notably, N. melas , A . akindynos , andP. amboinensis had > 3% proportional DC expression.
For the SC, there were significant differences in the expression ofsws1 paralogs throughout ontogeny for six species (P.
amboinensis, P. nagasakiensis, P. chrysurus, P. bankanensis, andN. melas ), transitioning from a higher expression of sws1α in the larval stage towards a greater expression of sws1β in the
later stages (Fig. 5).
Differential gene
expression
Retinal transcriptome assemblies and subsequent DGE mapping of all
samples/species found 26,774 expressed transcripts, of which 11,227
passed the pre-processing filtering, and 7,769 transcripts were
converted to Ensembl gene IDs (41.9% of total transcripts). PCA
revealed that independent of species, larvae formed one cluster and
juvenile and adult stages formed a second cluster along PC1, which
accounted for 36.8% of the variance (Fig. 2A). Along PC2 (13.9% of the
variance), three major clusters could be distinguished: one comprising
the two Chrominae species (Chro. atripectoralis , D.
aruanus ), a second one including all the Pomacentrus spp., Chry.
flavipinnis , N. melas and D. perspicillatus , and a third
for the anemonefish A. akindynos .
An in-depth analysis of P. amboinensis separated the retinal gene
expression profiles by life stage (n = 3 specimens/life stage; Fig. 2B).
PC1 explained 84.7% of the variance, while PC2 explained 4.5%. A total
of 23,190 transcripts were expressed in the retinas of the nine P.
amboinensis samples, of which 20,952 passed the pre-processing
filtering. Of these, 13,038 genes were converted to Ensembl IDs (56.2%
of total transcripts). The top 1000 most variable genes clustered into
four major groups based on their expression pattern across samples using
k-means (Fig. 2C). GO enrichment analysis of overrepresented biological
processes showed that three of the clusters were mostly comprised of
genes upregulated in larvae, which were involved in developmental
processes (muscle structure development [GO:0061061], tissue
development [GO:0009888]). The fourth cluster comprised genes
involved in visual processes (visual perception [GO:0007601],
response to light stimulus [GO:0009416]) that were primarily
upregulated in juvenile and adult P. amboinensis (Fig. 2C).
Details about the pairwise DGE analyses for the three life stages
(larval vs. juvenile, larval vs. adult, juvenile vs. adult) in P.
amboinensis , C. atripectoralis and A. akindynos are
provided in Fig. 6 and Tables S1-9. In the larval vs. juvenile
comparison, 4,325; 2,998, and 6,049 genes were upregulated in the larvae
of each species, respectively. In contrast, 4,075; 1,830; and 4,175 were
upregulated in the juveniles. In the larval vs. adult comparison, 4,591;
3,081; and 5,322 genes were upregulated in the larvae, and 4,121; 1,688;
and 3,636 were upregulated in the adults. In the juvenile vs. adult
comparison, 868, 811, and 630 genes were upregulated in the juveniles.
In contrast, 435, 682, and 1,468 were upregulated in the adults. A GO
enrichment analysis of the top 15 up- and down-regulated genes for each
comparison revealed that most of the genes that were upregulated in the
larval stage, when compared to later stages, were involved in
developmental processes, predominantly of the lens (Fig. 6, Tables
S1-9). Specifically, developmental genes upregulated in larvae included
keratin 5 (krt5 ), involved in epidermal cell differentiation
(GO:0009913); a crystallin, gamma M3 (crygm3 ), involved in lens
development in camera-type eyes [GO:0002088]; and a periostin,
osteoblast-specific factor b (postnb ), involved in extracellular
matrix organisation (GO:0030198). Conversely, genes upregulated in
juveniles and adults, when compared to larval expression were primarily
involved in visual processes and intracellular pathways including
beta-carotene oxygenase 2b (bco2b ), involved in retinal metabolic
processes (GO:0042574); red-sensitive opsin-1 (opn1lw1 ), involved
in visual perception (GO:0007601); and, the G protein-coupled receptor
kinase (grk1b ), involved in the cone photoresponse recovery
(GO:0036368).
In the comparison between juveniles and adults, genes upregulated in the
juvenile stage were involved in developmental processes and cellular
division, such as the centromere protein W (CENPW ), involved in
the central nervous system development (GO:0007417); and the protein
atonal homolog 7 (atoh7 ), involved in retinal development in
camera-type eyes (GO:0060041). Genes upregulated in the adults were
primarily involved in visual processes including relaxin family peptide
receptor 3 (rxfp3r ), involved in the G protein-coupled receptor
signalling pathway (GO:0007186); cellular retinol-binding protein type
II (rbp2a ), involved in the vitamin A metabolic process
(GO:0006776); and purpurin (rbp4l ), which is involved in retinol
transport (GO:0034633).
Discussion
In this study we uncovered that all Pomacentrus species express
two UV-sensitive sws1 opsin genes at later developmental stages.
The expression profile of these copies correlates with the appearance of
complex UV patterns with variable stripes, lines and dotted motifs at
the juvenile stage of Pomacentrus spp. In P. amboinensis ,
these complex patterns differ in spectral reflectance between pattern
elements and might be used for individual recognition. We also found
that larval fishes differ significantly in retinal gene expression and
colouration from later stages, while differences between juveniles and
adults were less pronounced. In the following we discuss each of our
findings in detail.
Ontogeny of damselfish UV colouration and visual
adaptations
Using a comparative approach in eleven damselfish species, we show that
complex UV facial and body patterns are more widespread than previously
thought. Specifically, complex UV patterns were found in allPomacentrus species and D. perspicillatus (Table S10, Fig.
S10, S12-16). Previously, only the adults of the sister species ofP. amboinensis and P. moluccensis have been shown to have
complex facial UV patterns that they use for individual recognition
(Parker et al., 2017; Siebeck, 2014, 2004; Siebeck et al., 2010). UV
patterns were present from the juvenile stage onwards inPomacentrus spp. (also see Gagliano et al., 2015 for previous
work on P. amboinensis ) and from the sub-adult stage onwards inD. perspicillatus . It is possible that complex UV patterns
emerged a second time independently in D. perspicillatus .
Alternatively, these patterns might have appeared in the Pomacentrinae
ancestor and been lost multiple times after that (Fig. 4). Supporting
the former hypothesis is that the patterns would have had to be lost at
least four times independently (Pomacentrinae 1 – 4; McCord et al.,
2021) in the Pomacentrinae history. The latter hypothesis is supported
by the expression of two sws1 copies correlating with complex UV
patterns in Pomacentrus spp. Because the sws1 duplication
occurred in the Pomacentrinae ancestor, it is possible that the patterns
emerged around the same time. This was then followed by a loss in
complex patterns in species that retained only one sws1 copy
(e.g., Chry. Flavipinnis ) or a simplification of patterns into
stripes in others (e.g., A. akindynos and juvenile N.
melas ), with D. perspicillatus maintaining the ancestral trait
but shifting its appearance to later in development (Fig. 4).
The expression of two or more sws1 paralogues has so far only
been reported from butterflies, snakes, mantis shrimp, a species of gar,
and some adult damselfishes (mostly anemonefishes) (Bok et al., 2018;
Briscoe et al., 2010; Cronin et al., 1994; Hauzman et al., 2021;
Mitchell et al., 2021; Stieb et al., 2024; Sukeena et al., 2016). Here,
we show a co-expression of the paralogs in Pomacentrus spp. In
butterflies, the expression of two sws1 copies allows for colour
vision in the ultraviolet, with females able to discriminate between
narrow UV spectra of 380 nm versus 390 nm (Finkbeiner and Briscoe,
2021). Similarly, mantis shrimp have been shown to distinguish between
different UV colours (Bok et al., 2014). The expression of twosws1 opsins that potentially confer different spectral
sensitivities in the UV (~15 nm apart) and the occurrence
of differently coloured UV patterns in the fish (Fig. 1D) strongly
suggest that Pomacentrus species are also able to distinguish
between UV colours. However, in-situ hybridisation, single-cell RNA
sequencing and microspectrophotometry are needed to show that the two
damselfish sws1 copies are in separate cone photoreceptors with
different spectral sensitivities and their distribution across the
retina. Finally, behavioural assays, like the ones in A.
ocellaris (Mitchell et al., 2024), are necessary to prove UV-colour
discrimination in these fishes.
Sws1 evolution and spectral tuning
The phylogenetic reconstruction showed that the damselfish visual opsins
mined from the retinal transcriptomes belong to the major five
vertebrate clades (Yokoyama, 2000) (Fig. S1). A more detailed
evolutionary reconstruction of the sws1 genes revealed that the
damselfish ancestor most likely possessed a single sws1 copy
(Fig. 4). These results are congruent with a more extensive phylogenetic
comparison of damselfish sws1 opsins conducted in Stieb et al.,
2024. Also, by adding the transcriptomes of D. perspicillatus andC. flavipinnis (both Cheiloprionini), we could place the
duplication of sws1 at the base of the Pomacentrinae subfamily.
Previously, the duplication was thought to have occurred only after the
split of the Cheiloprionini from the rest of the Pomacentrinae (Stieb et
al., 2024). Since only sws1a was recovered from the two
Cheiloprionini species analysed here, and a single sws1 was also
recovered in the two Chrysiptera species from Stieb et al., 2024,
it is likely that the tribe has lost sws1β ancestrally (Fig. 4).N. melas was the most ancestrally derived Pomacentrinae species
from our dataset, with two copies (Fig. 4).
Interestingly, the N. melas sws1 paralogs showed extensive
patterns of gene conversion, with distinct breaking sites revealed by
GARD analysis (Fig. S2) and sws1β being pulled towards thesws1a clade in the phylogeny (Fig. 3). This was also evident from
the amino acid comparison at sites 114 and 118 (relative to BRH). Most
damselfish sws1α orthologues had A114 and A118 (shorter-shifted
phenotype), and sws1β had S114 and S118 (longer-shifted
phenotype). However, the N. melas copies had the opposite amino
acids at these two sites, with sws1α S114, S118 and sws1β A114, A118. GARD also revealed that gene conversion generally affected
the sws1 paralogs of Pomacentrinae, with higher sequence
similarities detected in the first and last sections of the coding
regions (Fig. S2). N. melas differed from the rest of the
Pomacentrinae species in that the middle part, containing sites 114 and
118, was also affected. Future work should sample more broadly within
the Hemiglyphidodontini (the tribe containing Hemiglyphidodon,
Amblyglyphidodon, Altrichthys, Acanthochromis andNeoglyphidodon; Tang et al., 2021; Whitley, 1929) to reveal
whether the pattern observed here is specific to N. melas or
common to the tribe.
Opsin gene expression
We found that damselfishes use a variety of visual opsin gene
repertories with ontogenetic differences in gene expression. Consistent
with previous research (Luehrmann et al., 2018; Stieb et al., 2024,
2023, 2019, 2017, 2016), each species expressed rh1 and at least
three cone opsin genes (sws1 , rh2a and rh2b ), with
some species also expressing sws2b , lws and/or multiplesws1 , rh2a and lws paralogs (Table 1). Some of this
variation might be explained by short-term reversible changes in opsin
gene expression, which are common in teleosts (reviewed in Carleton et
al., 2020; Musilova et al., 2021) and have previously been documented in
the adults of P. amboinensis and P. moluccensis (Luehrmann
et al., 2018). Moreover, damselfishes are known to tune their visual
pigments over evolutionary timeframes through changes in opsin gene
sequences (Stieb et al., 2017). These adaptations are thought to be
driven by species-specific ecologies. For example, some damselfishes may
show intraspecific differences in sws1 (UV-sensitivity)
expression with depth (Stieb et al., 2016), and, as mentioned above,
they might also show differences in lws (red-sensitivity)
expression and spectral tuning that correlate with feeding mode and fish
colouration (Stieb et al., 2023). As we show here, we also find
consistent developmental plasticity in opsin gene expression between
larval, juvenile and adult stages of all species (Table 1, Fig 4).
In damselfishes, such as A. akindynos (Stieb et al., 2019),rh2a and rh2b are expressed in opposite double cone
members, forming a pair of visual pigments that is best tuned to the
underwater light environment (blue-green) (Jerlov, 1976; Yokoyama and
Jia, 2020). A slight change in rh2b gene expression with
development was found for most species, which shift from a higher
expression of rh2b in the settlement larval stage to a more even
expression between rh2b and rh2a at later stages (Table
1). This could be explained by the open ocean light environment being
blue-shifted compared to the broad light spectrum found on shallow reefs
(all juveniles and adults, except for C. flavipinnis , were caught
is similar to the intraspecific depth differences discovered previously
in the adults of some damselfishes. Individuals caught at shallower
depths had a higher expression of rh2a than rh2b , while a
more even expression between genes was seen in deeper-caught specimens
(Stieb et al., 2016). Changing opsin gene expression this way likely
tunes the double cone spectral sensitivities to the most abundant
wavelengths of light available in each life stage. However, as is the
case in Killifish (Lucania goodei ) (Fuller et al., 2004), this
might result from environmental plasticity rather than a pre-determined
part of development.
Two species (P. amboinensis and P. nagasakiensis )
expressed a second rh2a paralog (rh2a-2 ). Interestingly,
some, but not all, anemonefish species have also been found to have a
second rh2a gene in their genomes (Mitchell et al., 2021;
Musilova et al., 2019). The two damselfish species expressingrh2a-2 diverged ~20 Mya, while more closely related
species, such as P. chrysurus (divergence from P.
nagasakiensis ~2.5 Mya; McCord et al., 2021), expressed
only one rh2a copy. It is possible that the second copy was not
expressed in most of the species analysed here. However, because of the
abundant depth at which we sequenced the transcriptomes, we can usually
detect traces of all visual opsins present in a species’ genome. Also,
many more percomorph fishes have two rh2a gene copies (Lin et
al., 2017; Musilova and Cortesi, 2023). This suggests that rh2a has duplicated early in percomorph evolution and that the orthologs were
lost multiple times in different species. Unfortunately, extensive and
frequent gene conversion between rh2 genes has made it nearly
impossible to conclusively disentangle their evolutionary history
(Musilova and Cortesi, 2023).
Sws1 was expressed in all stages and species investigated.
Interestingly, species in the Pomacentrus clade, plus the larval
stage of N. melas , were found to express two copies ofsws1 , which could be assigned to short- and long-wavelength
shifted groups (see Fig. 3 for classification and Fig. 5 for
expression). Multiple sws1 genes are rare in fishes (Musilova et
al., 2019) and in coral reef species have so far only been found in the
damselfishes (Mitchell et al., 2021; Musilova et al., 2019; Stieb et
al., 2024). The expression of sws1 is often associated with UV
communication and could also be an adaptation for feeding on
UV-absorbing or reflecting zooplankton (Flamarique, 2016; Job and
Bellwood, 2007; Novales-Flamarique and Hawryshyn, 1994; Siebeck, 2014,
2004; Siebeck et al., 2010; Stieb et al., 2017). Expression ofsws1 for foraging could be substantiated by damselfishes being
highly opportunistic feeders; while pomacentrid species are often
grouped into three major dietary classes (planktivore, herbivore and
omnivore), they can readily shift feeding mode to exploit nutrient-rich
food (Frédérich et al., 2006). Thus, retaining sws1 expression
even in prevalently benthic feeding species could benefit opportunistic
predation on zooplankton and other small organisms. Feeding ecology,
however, does not explain the expression of a second sws1 gene in
the Pomacentrus spp.
Retinal gene expression throughout
ontogeny
PCA revealed that in damselfishes, the retinal gene expression in larvae
is distinctly different to later stages (Fig. 2A), suggesting that
juvenile damselfishes are already displaying adult expression profiles.
PCA and k-means clustering in P. amboinensis supported these
findings by also showing a separation of gene expression between the
larval and later stages (Fig. 2B, 2C).
Using DGE between developmental stages in three damselfish species (Fig.
6), the top 15 upregulated genes in larvae compared to juveniles and
adults, independent of species, were primarily involved in developmental
processes of the eye and especially in lens formation (e.g.,krt5 , crygm3 , postnb ) (Tables S1, S2, S4, S5, S8,
and S9). This is similar to the retinal gene expression in settling
surgeonfish larvae (Fogg et al., 2022b), and supports the metamorphosis
seen in other organs of coral reef fishes during settlement on the reef
(Hu et al., 2019; Lim and Mukai, 2014). Notably, various crystallin
paralogues were upregulated in the larvae. These encode water-soluble
proteins responsible for the transparency and the gradient of
refractiveness of the vertebrate lens (Palczewski et al., 2000). The
latter is a crucial aspect of teleost fish multifocal lenses, which
enable focusing different wavelengths of light on the same plane (Gagnon
et al., 2012; Karpestam et al., 2007; Kröger et al., 1999).
Compared to the larval stage, the top 15 upregulated genes in the
juvenile and adult stages were primarily involved in visual processing
and homeostasis of the retina (e.g., grk1b , nfil3-5 ) (Fig.
6). Specifically, an orthologue of the zebrafish beta-carotene oxygenase
2b (bco2b ) was upregulated in juveniles and adults of P.
amboinensis . In teleost fishes, beta-carotene oxygenases have evolved
an extended repertoire through repeated gene duplications, andbco2 paralogues are thought to participate in retinoic acid (RA)
biosynthesis (Helgeland et al., 2014; Kiefer et al., 2001). Retinoic
acid is involved in cone photoreceptor survival in the mouse retina
(Amamoto et al., 2022). The upregulation of bco2 in the juvenile
and adult stages highlights the need for a continuous supply of RA for
the long-term survival of their cones, which might be less critical in
the still rapidly developing retina at the larval stage.
Unsurprisingly, the juvenile versus adult comparison also revealed an
upregulation of developmental genes in the juvenile stage. For example,
the atonal homolog 7 (atoh7 ), which is crucial for developing
ganglion cells (Brown et al., 2001), was upregulated in juveniles
compared to adults. Genes upregulated in adults compared to juveniles
were primarily involved in visual processing and phototransduction,
including the long-wavelength-sensitive opsin-1 (opn1lw1 orlws ). An increased proportional expression of lws was also
present in the adults of several other damselfish species, includingD. perspicillatus , N. melas , A. akindynos , andP. chrysurus (Table 1). This increase might be linked to changes
in their feeding ecologies to a more herbivorous diet, as chlorophyll
reflects strongly in the far-red, or due to the emergence of orange/red
colours in later stages, as discussed in detail in Stieb et al., 2024.
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Acknowledgements
We would like to thank the Lizard Island Research Station staff for
their support during fieldwork and acknowledge the Dingaal, Ngurrumungu
and Thanhil peoples as traditional owners of the lands and waters of the
Lizard Island region. We would also like to thank Professor Mark
McCormick and his team for generously lending us their light traps and
assisting with some animal collections and Dr Sam B. Powell for
technical support with the UV camera system. We would like to
acknowledge the Australian Museum and the Lizard Island Reef Research
Foundation for supporting the fieldwork component of this research with
a doctoral fellowship awarded to V.T. This work was supported by an
Australian Research Council (ARC) Discovery Project (DP180102363) to
J.M. and F.C., the AFOSR/AOARD to J.M., a UQ Amplify and ARC DECRA
Fellowships (DE200100620) to F.C., and the Zoltan Florian Marine Biology
Fellowship from the Lizard Island Reef Research Foundation (LIRRF)
awarded to V.T.
Data availability
Raw RNA sequencing data was uploaded to SRA (XXXX) and opsin genes
sequences were uploaded to NCBI (XXXX) and will be made available upon
acceptance. Transcriptome assemblies, pictures and phylogenetic trees
are available on Dryad (XXXX).
Author contributions
V.T. conceived the study and designed the experiments together with
F.C., N.J.M. and K.L.C. V.T. performed molecular experiments and
analysed gene expression data. V.T., F.C. and J.M. collected ultraviolet
pictures and spectral reflectance from specimens. V.T. and F.C. wrote
the initial manuscript and all authors contributed to the final
submitted version.
Competing interests
The authors declare no competing or financial interests.
Table 1 Opsin gene expression through development in eleven
damselfish species. Shown are the proportional expression within single
and double cones, and the rod-opsin expression versus total opsin
expression.