Rapid and parallel adaptive evolution of the visual system of Neotropical Midas cichlid
Julián Torres-Dowdall,1,2 Michele E.R. Pierotti,3 Andreas Härer, 1 Nidal Karagic,1 Joost M.
Woltering,1 Frederico Henning,1 Kathryn R. Elmer,1,4 Axel Meyer1*
1 Zoology and Evolutionary Biology, Department of Biology, University of Konstanz, Konstanz,
2 Zukunftskolleg, University of Konstanz, Konstanz, Germany
3 Naos Laboratories, Smithsonian Tropical Research Institute, Panama, Republic of Panama
4 Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary
and Life Sciences, University of Glasgow, Glasgow, United Kingdom
*Corresponding author: Email: (email@example.com)
Key words: Amphilophus, cichlid, crater lake, opsin, vision, visual sensitivity
Midas cichlid fish are a Central American species flock containing 13 described species that has
been dated to only few thousand years old, a historical timescale infrequently associated with
speciation. Their radiation involved the colonization of several clear water crater lakes from two
turbid great lakes. Therefore, Midas cichlids have been subjected to widely varying photic
conditions during their radiation. Being a primary signal relay for information from the environment
to the organism, the visual system is under continuing selective pressure and a prime organ
system for accumulating adaptive changes during speciation, particularly in the case of dramatic
shifts in photic conditions. Here, we characterize the full visual system of Midas cichlids at
organismal and genetic levels, to determine what types of adaptive changes evolved within the
short time span of their radiation. We show that Midas cichlids have a diverse visual system with
unexpectedly high intra- and interspecific variation in color vision sensitivity and lens transmittance.
Midas cichlid populations in the clear crater lakes have convergently evolved visual sensitivities
shifted towards shorter wavelengths compared to the ancestral populations from the turbid great
lakes. This divergence in sensitivity is driven by changes in chromophore usage, differential opsin
expression, opsin coexpression, and to a lesser degree by opsin coding sequence variation. The
visual system of Midas cichlids has the evolutionary capacity to rapidly integrate multiple
adaptations to changing light environments. Our data may indicate that, in early stages of
divergence, changes in opsin regulation could precede changes in opsin coding sequence
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Understanding the mechanisms underlying adaptive phenotypic divergence is one of the main
challenges of molecular evolutionary biology. The visual system of animals provides an excellent
model for approaching this issue for a number of reasons: it is highly diverse across organisms;
the molecular mechanisms underlying its diversity are relatively well known; and there is a clear
link between changes at the molecular level and their phenotypic consequences (Loew and
Lythgoe 1978; Chang et al. 1995; Yokoyama and Yokoyama 1996; Yokoyama 2000; Ebrey and
Koutalos 2001; Chinen et al. 2003; Hofmann and Carleton 2009; Carleton 2014; Enright et al.
2015; Dalton et al. 2017). Moreover, strong selection for tuning the visual system to the light
environment is expected given the crucial sensory role of vision for different activities including
foraging, predator avoidance, and mate choice. Particularly interesting are animals inhabiting
aquatic environments, especially freshwater habitats, given that these are among the most
spectrally diverse light environments due to the wavelength-specific absorption properties of water
combined with dissolved organic matter, suspended particles, and plankton scattering light at
various wavelengths (Cronin et al. 2014). Indeed, fishes have the most variation in spectral
sensitivities among all vertebrates, showing a strong correlation between visual sensitivities and
light environment (Loew and Lythgoe 1978; Levine and MacNichol 1979; Lythgoe 1984; Cummings
and Partridge 2001; Marshall et al. 2003; Bowmaker 2008; Cronin et al. 2014; Marshall et al.
Cichlid fishes are an interesting model system to the study of visual ecology and evolution
(Carleton 2009; Carleton et al. 2016), since they are one of the most species rich and colorful
lineages of vertebrates (Kocher 2004; Brawand et al. 2014; Henning and Meyer 2015). These fish
have undergone impressive phenotypic divergence, including visual sensitivity (Kocher 2004;
Salzburger 2009; Henning and Meyer 2014; Carleton et al. 2016). The visual system of African
cichlids is highly diverse, spanning most of the variation known from all fishes, and there is
compelling evidence that selection has shaped this diversity (Sugawara et al. 2002, 2005; Terai et
al. 2002; Carleton et al. 2005; Hofmann et al. 2009; Cronin et al. 2014; Carleton et al. 2016).
Vision is mediated by visual pigments, which are composed of an opsin protein and a light
absorbing retinal chromophore. These components are covalently bound, and variation in either of
them results in shifts of spectral sensitivity (Wald 1968; Yokoyama 2000). Eight opsin genes, one
rod-opsin that functions under dim-light conditions and seven cone opsin genes involved in color
vision, have been described from cichlids, which collectively have sensitivities that span from the
ultra-violet to the red part of the light spectrum (Carleton 2009; Escobar-Camacho et al. 2017). Of
these eight, five are hypothesized to have been present in the common ancestor of vertebrates:
the rod-opsin that functions under dim-light conditions (RH1) and four cone opsin genes that are
involved in color vision (SWS1, SWS2, RH2, LWS; Yokoyama and Yokoyama 1996; Terakita
2005). Two additional cone opsin gene duplications (SWS2a–SWS2b and RH2A–RH2B) increased
the opsin repertoire in acanthopterygians (Carleton and Kocher 2001; Parry et al. 2005). A
subsequent duplication of RH2A (RH2A
) occurred in cichlids (Parry et al. 2005).
Extensive research in the visual system of African cichlids has shown that multiple
mechanisms affect vision of these fish, including opsin gene expression and coexpression, opsin
coding sequence differences, chromophore usage, and ocular media transmittance (Carleton et al.
2016). Cichlid retinas are highly structured, with single cones expressing one of the short-
wavelength sensitive opsins (SWS1, SWS2b, or SWS2a) and double cones expressing one opsin
in each of the two cell members, either two mid-wavelength sensitive (RH2B, RH2A
or one mid-wavelength and the long-wavelength opsin (LWS; Carleton and Kocher 2001; Spady et
al. 2006; Carleton et al. 2008; Hofmann et al. 2009; O’Quin et al. 2010). Thus, African cichlids
commonly express a combination of three cone opsins (Carleton et al. 2016; but see Parry et al.
2005; Dalton et al. 2015, 2017), resulting in large differences in spectral sensitivity among species
expressing different subsets (Carleton and Kosher 2001; Spady et al. 2006; Carleton et al. 2008;
Carleton 2009; Hofmann et al. 2009). This tuning mechanism underlies much of the variation
observed among cichlid species from Lake Malawi (Hofmann et al. 2009). In contrast, fine-tuning
of visual sensitivity is mostly achieved by amino acid substitution in the opsin protein, mainly in
sites directed into the chromophore-binding pocket (Carleton et al. 2005). This has been shown to
be an important tuning mechanism for the dim-light sensitive RH1 (Sugawara et al. 2005) and for
SWS1 and LWS that have sensitivities at opposite extremes of the visible spectrum (Terai et al.
2002, 2006; Seehausen et al. 2008; Hofmann et al. 2009; O’Quin et al. 2010; Miyagi et al. 2012).
Visual sensitivity can also be tuned by changing chromophore type, and this mechanism is
known to underlie some of the phenotypic variation between African cichlids that inhabit turbid
versus clear waters (Sugawara et al. 2005; Terai et al. 2006; Carleton et al. 2008; Miyagi et al.
2012). Two types of chromophores can be found in fish, 11-cis retinal derived from vitamin A1 and
3,4 didehydroretinal derived from vitamin A2. Switching from A1- to A2-derived chromophores
results in sensitivities shifting towards longer wavelengths (Wald 1961; Hárosi 1994; Cronin et al.
2014). Another way to alter sensitivity is to filter light passing the cornea and lens before reaching
the retina; and African cichlids are known to vary strongly in the clearness of the lenses (Hofmann
et al. 2010; O’Quin et al. 2010).
The study of the visual system of African cichlids has furthered our understanding of the
mechanisms involved in adaptive divergence (reviewed in Carleton et al. 2016). Yet, there remain
numerous unanswered questions regarding how this diversity has evolved that might be difficult to
address without exploring younger cichlid radiations (Carleton et al. 2016). One such question
concerns the likelihood of different mechanisms driving early stages of differentiation. Is early
divergence characterized by structural changes of opsin genes or by modifications in the pattern of
opsin expression? Does one tuning mechanism or the interaction of multiple mechanisms underlie
early spectral sensitivity divergence? The Midas cichlid fishes from Nicaragua (Amphilophus cf.
citrinellus) provide an excellent system to address these questions, as they have recently
colonized new visual environments from known source populations and are ecologically divergent
in parallel along the benthic-limnetic axis within crater lakes (Elmer et al. 2014; Kautt et al. 2016a).
Nicaragua has a rich diversity of freshwater environments including the largest lakes in
Central America and a series of young (< 24,000 years) and completely isolated crater lakes that
are part of the Central American Volcanic Arc (Kutterolf et al. 2007). Midas cichlid populations of
the great lakes Managua and Nicaragua have recently (less than 2,000 generations ago) and
independently colonized multiple crater lakes (Barluenga et al. 2006; Barluenga and Meyer 2010;
Elmer et al. 2010; Elmer et al. 2014; Kautt et al. 2012, 2016a, 2016b). The newly colonized crater
lakes differ in many aspects from the great lakes, including a drastic difference in the light
environment. The great lakes are very turbid due to a high level of suspended particles whereas
crater lakes tend to have clearer waters (Cole 1976; Elmer et al. 2010). This is particularly true for
two of the oldest and deepest crater lakes, Apoyo and Xiloá. These crater lakes harbor small
Midas cichlid radiations along a benthic–limnetic axis of divergence (4 to 6 endemic species each;
Kautt et al. 2016a). Benthic and limnetic Midas cichlids might experience different light conditions.
Limnetic Midas cichlids forage in open water, a relatively homogenous light environment with a
broad spectral bandwidth (Sabbah et al. 2011). Benthic Midas cichlids forage in the littoral zone
where the light environment is likely shifted toward longer wavelength and with a narrower spectral
bandwidth (Sabbah et al. 2011). Thus, Midas cichlids are an excellent system to study the
evolution of sensitivities after the very recent colonization of, and speciation in a new light
So far, relatively little is known about the visual system of Neotropical cichlids. Early
microspectrophotometry (MSP) studies suggested that Neotropical cichlids have long wavelength
shifted spectral sensitivities (Muntz 1973; Loew and Lythgoe 1978; Levine and MacNichol 1979;
Kröger et al. 1999; Weadick et al. 2012). Opsin gene expression in Neotropical cichlids supports
these findings as these fish express a long wavelength sensitive palette of opsins (i.e., SWS2a,
RH2A and LWS, Escobar-Camacho et al. 2017). Interestingly, the most short-wavelength shifted
opsins in single cones (i.e., SWS1) and double cones (i.e., RH2B) were suggested to be lost or to
have become pseudogenized (Weadick et al. 2012; Fisher et al. 2015, Escobar-Camacho et al.
2017). Measures of lens transmittance in Neotropical cichlids show that the UV and violet parts of
the visible spectrum are often filtered out before reaching the retina (Muntz 1973). Finally, usage of
the A2-derived chromophore producing long-wavelength shifted sensitivities appears to be
common in Neotropical cichlids (Loew and Lythgoe 1978; Levine and MacNichol 1979; Weadick et
al. 2012). In combination, those results suggested that Neotropical cichlids might have a reduced
diversity in their visual system and the potential for adaptation to new light environments with
short-wavelength shifted spectra might be limited (Weadick et al. 2012).
Based on our knowledge of the evolutionary history of the Midas cichlid species complex,
we aimed to understand the phenotypic and molecular consequences of colonization of new light
environments. First, we compared light irradiances between great and crater lakes to better predict
the expected phenotypic divergence in spectral sensitivities. Second, we used
microspectrophotometry to compare visual pigment sensitivity and lens spectral transmittance
measurements between different Midas cichlid species inhabiting great and crater lakes and
between benthic and limnetic ecomorphs within crater lakes. Finally, we explored the molecular
mechanisms underlying divergence in the visual sensitivity of Midas cichlids by studying the
evolution of opsin amino acid sequences, opsin gene expression, and chromophore usage.
VARIATION IN THE VISUAL ENVIRONMENT IN NICARAGUAN LAKES
To determine the different photic environments experienced by Midas cichlids we took underwater
light measurements in a turbid great lake (Lake Managua) and two clear crater lakes (Lakes Apoyo
and Xiloá). These lakes differed in many aspects of their underwater light environment. Spectral
irradiance measurements in the turbid great lake showed that light attenuation was dramatically
higher than in the crater lakes, as expected due to their differences in turbidity (fig. 1). Therefore,
the photic environment was restricted to shallower waters in the turbid great lake, but it expanded
into deeper waters in the clear crater lakes. Moreover, light spectra differ among lakes. While long-
wavelengths were attenuated with depth similarly in crater lakes and the great lake, short-
wavelength light was better transmitted in crater lakes, resulting in a blue-shifted light spectrum
compared to that of the great lake (fig. 1). A useful measure to compare the light environments of
different lakes is λP50, the wavelength at which the total number of photons is divided in two equal
parts (McFarland and Munz 1975). Higher λP50 values suggest a light spectrum shifted towards
longer wavelengths, whereas lower λP50 values indicate short-wavelength shifted light
environments. In the turbid great lake, λP50 was 529 nm, but in the crater lakes it was shifted
towards shorter wavelengths (Apoyo λP50 = 504–511; Xiloá λP50 = 505–523). Thus, the underwater
photic environment of the crater lakes is richer both in bandwidth and intensity compared to the
great lake, providing a source of strong divergent selection on the visual system of aquatic
PHENOTYPIC DIVERSITY IN THE VISUAL SYSTEM OF MIDAS CICHLIDS
SPECTRAL SENSITIVITIES OF VISUAL PIGMENTS
To determine if the colonization of clear water crater lakes (i.e., a new photic environment) resulted
in adaptive phenotypic divergence in the visual system of Midas cichlids, we conducted
microspectrophotometry analyses (MSP) on retinas of specimens from a turbid great lake (Lake
Nicaragua) and two clear crater lakes (Lakes Apoyo and Xiloá). Additionally, to explore the
divergence between benthic and limnetic species within crater lakes, both ecomorphs were studied
from the same two crater lakes (there are no limnetic species in the great lakes; supplementary
table S1, Supplementary Material online). The peaks of maximum absorbance (λmax) as well as
estimates of A1/A2 chromophore ratios of rod and cone photoreceptors were determined. Analysis
repeated with fish reared under common light conditions provided qualitatively similar results
(supplemental fig. S1, Supplemental Material online). Thus, we infer that the patterns described
below have a genetic basis.
Retina rod photoreceptor cells are particularly tuned to dim light conditions, which in aquatic
environments are characteristic of deep and murky waters (Bowmaker 1995, 2008). In Midas
cichlids, peaks of maximum sensitivity (λmax) from 101 rod cells (number of specimens NNicaragua = 2,
number of cells: nNicaragua = 9; NApoyo = 8, nApoyo = 35; NXiloá = 11, nXiloá = 57) ranged from 495 to 525
nm (fig. 2). All these were assigned to one spectral class based on the estimated pure-A1 visual
pigment (λA1 = 497 ± 1 nm, mean ± SD; fig. 2), suggesting various A1/A2 chromophore ratios. No
clear pattern of variation of rod photoreceptor sensitivity with lake of origin was found
(supplemental fig. S2, Supplemental Material online). However, λmax values in the limnetic species
of both clear crater lakes were less variable (Bartlett's k2 = 21.065, df = 4, P < 0.001) and with
mean λmax shifted toward shorter wavelengths than sympatric benthic species (Kruskal-Wallis
22.370, df = 1, P < 0.001; fig. 2).
Single cone cells are one of the two types of photoreceptors involved in color vision, and their
sensitivity peaks at wavelengths between 350 and 460 nm (UV to blue part of the spectrum;
Bowmaker 2008). MSP analysis on 42 single cones of Midas cichlids (NNicaragua = 2, nNicaragua = 12;
NApoyo = 6, nApoyo = 18; NXiloá = 4, nXiloá = 12) identified two spectral classes based on the predicted
λA1, one most sensitive in the violet (431 ± 4 nm) and one in the blue (450 ± 4 nm; fig. 2) part of the
light spectrum. All single cones from turbid great lake specimens were assigned to the blue
spectral class. In contrast, specimens within clear crater lakes Apoyo and Xiloá had single cones
assigned to the blue as well as the violet spectral classes (fig. 2). The range of λmax values for the
blue spectral class varied among lakes (F = 6.190, df = 2,6, P = 0.035; fig. 2), as in specimens
from crater lake Apoyo the sensitivity of cones assigned to this class appeared to be shifted toward
shorter wavelengths (λmax: 443 – 457) compared to those seen in crater lake Xiloá (λmax: 448 – 467
nm) and the great lake (λmax: 449 – 465 nm). No differences for the blue or in the violet spectral
class (Apoyo λmax: 431 – 442 nm; Xiloá λmax: 425 – 439 nm; fig. 2) were observed between morphs
within crater lakes.
Double cones are the second type of photoreceptor involved in color vision, consisting of two
cones fused together (Cronin et al. 2014). These have peaks of sensitivities in the mid and long
parts of the visible light spectrum (blue-green to red; Bowmaker 1995, 2008). We obtained 610
MSP readings of double cones from Midas cichlids’ retinas identifying four spectral classes based
on the predicted λA1: a red (λA1 = 559 ± 2 nm; NNicaragua = 3, nNicaragua = 20; NApoyo = 5, nApoyo = 41;
NXiloá = 9, nXiloá = 37), a long-green (λA1 = 528 ± 2 nm; NNicaragua = 3, nNicaragua = 49; NApoyo = 10, nApoyo
= 142; NXiloá = 11, nXiloá = 136), a short-green (λA1 = 509 ± 1 nm; NNicaragua = 3, nNicaragua = 13; NApoyo =
9, nApoyo = 76; NXiloá = 11, nXiloá = 63), and a blue-green spectral class (λA1 = 476 ± 4 nm; NNicaragua =
2, nNicaragua = 4; NApoyo = 5, nApoyo = 12; NXiloá = 5, nXiloá = 17). In Midas cichlids’ red spectral class,
both the λmax (Kruskal-Wallis
2 = 22.295, df = 4, P < 0.001) and its associated variance (Bartlett's
k2 = 86.324, df = 4, P < 0.001; fig. 2) differed among species. Variance was higher in specimens
from the turbid great lake as these had extremely long-wavelength shifted cones (λmax: 558 – 623
nm) that were not observed in the clear crater lakes (fig. 2). No significant differences were
observed between ecomorphs in each crater lake.
Two spectral classes with sensitivities in the green part of the light spectrum (510 – 560
nm) were identified based on predicted λA1 values, a short-green and a long-green (fig. 2).
Interestingly, both of these were detected for most specimens examined. Within each of these two
spectral classes, specimens from the crater lakes had sensitivities shifted toward shorter
wavelengths than specimens from the turbid great lake (short-green: F = 5.800, df = 2,20, P = 0.
010; long-green: F = 6.500, df = 2,21, P = 0.006; fig. 2). No differences were detected when
comparing the limnetic and benthic species within the crater lakes.
A few double cones had visual pigments with sensitivities in the blue-green spectral class
(fig. 2). These had an extremely wide range of variation in λmax (469 – 505 nm), particularly in the
crater lakes (Bartlett's k2 = 6.283, df = 2, P = 0.043; fig. 2). Very few of these cones were observed
in turbid great lake specimens, and these had long wavelength-shifted sensitivities. Cones of this
class were more commonly seen in fish from the crater lakes, and these had both, long and
extremely short wavelength-shifted λmax values (fig. 2).
Collectively across lakes and species, the cones of adult Midas cichlids had six different
spectral classes that coincide with the expected λmax ranges of SWS2b (violet), SWS2a (blue),
RH2B (blue-green), RH2A (short- and long-green), and LWS (red). We found no evidence of
single-cones with maximum absorbance in the UV part of the spectrum (i.e. <400 nm, SWS1).
Remarkably, in addition to the short wavelength sensitive visual pigment of single cones at least
three different spectral classes were detected for double cones of single Midas cichlid specimens.
The presence of four different spectral channels confers these fish the potential for tetrachromatic
color vision, yet functional validation would be required to determine the roles of these channels in
chromatic or achromatic vision. Spectral sensitivities of the different photoreceptors were either
short wavelength shifted (blue, short-green, long-green and red spectral classes) or had reduced
variability (rod and blue-green) in the clear crater lakes compared to the turbid great lake. Little
divergence was observed among ecomorphs within the crater lakes.
A1/A2-DERIVED CHROMOPHORE USAGE IN MIDAS CICHLIDS
Absorbance spectra from micro-spectrophotometric measures of photoreceptor outer segments
showed significant variation in A1/A2 chromophore ratios across Midas cichlid species (Kruskal-
2 = 32.167, df = 4, P < 0.001; fig. 3a). Consistent with short wavelength shifted sensitivity
and less variation in sensitivities, Bonferroni corrected pairwise comparisons suggested that Midas
cichlids from the clear crater lakes use relatively less vitamin A2-derived chromophores than fish
from the turbid great lake. However, the benthic species from crater lake Xiloá showed vitamin A2-
derived chromophore usage not significantly different from those seen in specimens from the great
lake (fig. 3a; supplementary table S2, Supplementary Material online).
Ocular media, and in particular lenses, can selectively limit the wavelength of light reaching the
retina, thus affecting visual sensitivity (Losey et al. 2003). A large amount of variation was found in
lens transmittance (as T50, the wavelength of 50% transmission) for first generation laboratory
born individuals of five Midas cichlid species reared under common light conditions. However, lens
transmittance cut-offs were not continuously distributed but formed discrete groups (Hartigans' dip
test for unimodality D = 0.099, P = 0.003; fig. 4). One group was composed exclusively by Midas
cichlids from the turbid great lake Nicaragua, having lenses blocking UV light and part of the violet
light of the spectrum (T50 = 421.6, SD = 2.9, n = 10). A second group had UV-blocking but violet-
light-transmitting lenses and was composed of most of the fish from the clear crater lakes,
including both the limnetic (Apoyo T50 = 392.9, SD = 1.8, n = 4 and Xiloá T50 = 393.9, SD = 2.8, n
= 7) and benthic species from both lakes (Apoyo T50 = 389.9, SD = 0.4, n = 3 and Xiloá T50 =
380.4, SD = 3.0, n = 9). Interestingly, we found a third group including only two specimens of the
limnetic species from crater lake Apoyo that had UV-transmitting lenses (T50 = 352.3 and 353.6).
Thus, Midas cichlids from the clear crater lakes have shifted the lens transmittance toward shorter
wavelengths compared to the ancestral species from the turbid great lake.
MECHANISMS OF DIVERGENCE IN THE VISUAL SYSTEM OF MIDAS CICHLIDS
CODING SEQUENCE VARIATION OF MIDAS CICHLID OPSIN GENES
To determine the contribution of structural changes in opsin proteins to the phenotypic variation
observed in photoreceptors’ sensitivities (see SPECTRAL SENSITIVITIES OF VISUAL PIGMENTS above),
we sequenced rhodopsin and the seven cone opsins from 64 specimens of the Midas cichlid
species complex. Specimens from two species of Midas cichlids (A. citrinellus and A. labiatus)
from the two turbid great lakes (Nicaragua and Managua), and individuals from one benthic and
one limnetic species from the clear crater lakes Apoyo and Xiloá (A. astorquii and A. zaliosus in
the former, and A. xiloaensis and A. sagittae in the latter) were included. Opsin genes and their
inferred amino acid sequences were found to be highly similar across the analyzed species.
Collectively across rhodopsin and all seven cone opsins, we identified a total of 16 variable
nucleotide sites of which eight resulted in amino acid substitutions (table 1). In none of these cases
were different alleles fixed in different species, but rather the alleles were segregating in one or
more of the Midas cichlid species.
The eight non-synonymous substitutions found were not homogeneously distributed across
opsin genes. Only one substitution was found in RH2A
and LWS, two in SWS1, and four in
(none were found in RH1, SWS2a, SWS2b, and RH2B, table 1). Seven of these occurred
in transmembrane regions, but only one occurred in a site directed into the retinal-binding pocket:
A164S in LWS. We determined the frequency of alanine and serine at this position by genotyping a
larger number of A. citrinellus (great lake Nicaragua, n = 63), A. zaliosus (crater lake Apoyo, n =
24) and A. xiloaensis (crater lake Xiloá, n = 24) individuals using a PCR-RFLP approach since the
polymorphism generates cutting sites for different restriction enzymes (Ala = SatI, Ser = Fnu4HI).
This confirmed our previous result, finding that LWS segregates for these two alleles only in the
turbid great lake Nicaragua, but not in the species from the crater lakes.
Overall, given that no fixed differences across species were found, coding sequence
variation appears to have a minor impact on the divergence of Midas cichlids’ visual system. The
only possible exception is LWS, where the A164S substitution could explain some variation seen in
the great lake but not in the crater lakes. For other variable sites, mutagenesis experiments will be
needed to determine their contribution to divergence in visual sensitivity.
CONE OPSIN EXPRESSION IN MIDAS CICHLIDS
Using quantitative real-time PCR (qRT-PCR), we quantified opsin expression in retinas of 25 wild-
caught individuals of Midas cichlids, including specimens from the turbid great lake Managua and
of a limnetic and a benthic species from clear crater lakes Xiloá and Apoyo. The proportion of the
total cone opsin gene expression (Tall) comprised by each of the seven cone opsins (Ti; Carleton
and Kocher 2001; Fuller et al. 2004) is reported (fig. 5).
Significant differences were found in the expression of wild-caught fish from different lakes
(AMRPP=0.43, P=0.001). In the species from the turbid great lake LWS constituted more than 60%
of the total cone opsin expressed whereas RH2A
represented almost 24% of total opsin
expression. SWS2a was the only single cone opsin expressed (~15% of total expression; fig. 5a).
This pattern of opsin expression reflects the results of the MSP analyses showing that Midas
cichlids in the turbid great lake have visual sensitivities shifted toward longer wavelengths.
Opsin expression of clear crater lake Midas cichlids differed from that in the great lake in
two aspects. First, in the crater lakes fish expressed proportionally less LWS and more RH2A
than those in the great lake (fig. 5b-c). Second, in crater lake Apoyo some individuals expressed
the blue-green sensitive RH2B and the violet sensitive SWS2b gene (fig. 5c). Individuals
expressing SWS2b expressed only traces of SWS2a and vice versa, suggesting a trade-off
between single cone opsins (supplementary fig. S3, Supplementary Material online). No variation
was evident between species within each crater lake.
In summary, opsin expression differences suggest shift in sensitivity towards shorter
wavelengths in fish from the clear crater lakes compared to a turbid great lake. This is achieved by
changes in the relative proportion of LWS and RH2A
expressed, and by the novel expression of
SWS2b and RH2B. These patterns of opsin expression were maintained in fish reared under
common light conditions (supplementary fig. S4, Supplementary Material online), suggesting a
genetic basis for the divergence between species.
OPSIN COEXPRESSION IN MIDAS CICHLIDS
To better understand the phenotypic consequences of differential opsin gene expression, we
performed triple fluorescent in situ hybridization (FISH) in laboratory reared Midas cichlids from a
great lake (A. citrinellus, Lake Nicaragua) and a clear crater lake representative species (A.
astorquii, Lake Apoyo), with a focus in double cones (8265 double cone members counted). The
retina of Midas cichlids from the turbid great lake was dominated by double cones expressing LWS
(>75% of double cones consistently across the retina), including multiple twin cones (fig. 6a-e).
Most of the rest of double cone members expressed RH2A
(17% - 24%; fig 6e). Two specimens
coexpressed LWS and RH2A
in the dorsal part of the retina and one of these also coexpressed
RH2B and LWS (fig 6e).
Retinas of A. astorquii differed in many aspects from those in A. citrinellus, and overall were
more variable (fig. 6j). Contrary to A. citrinellus, in A. astorquii most double cones had one member
expressing LWS and the second member expressing RH2A
(fig. 6f-j). Also, across all individuals
and retinal regions there was an average of 6% of cones coexpressing LWS and RH2A
cases representing up to 20% of the cones). Three specimens (all of them females) expressed
RH2B, either by itself or in combination with RH2A
or LWS (fig. 6f and j), which could explain
some of the variation seem in the blue-green spectral class (fig. 2).
SOURCES OF A1/A2-DERIVED CHROMOPHORE VARIATION IN MIDAS CICHLIDS
The enzyme Cyp27c1 mediates the conversion of vitamin A1 into vitamin A2 in the retinal pigment
epithelium and the level of vitamin A2 is strongly correlated with the expression of cyp27c1
(Enright et al. 2015). cyp27c1 expression in retinas of Midas cichlids is in agreement with the A2
proportions estimated in the MSP experiment. Those species showing higher levels of A2-derived
chromophore usage (fig. 3a) also had higher levels of cyp27c1 expression (fig. 3b; supplementary
fig. S5a, Supplementary Material online). In addition to significant differences in cyp27c1
expression level (Kruskal-Wallis c2 = 17.513, df = 4, P = 0.002), Midas cichlid species also differed
in their variance in expression (Bartlett's k2 = 11.337, df = 4, P = 0.023), with Midas cichlids from
the turbid great lake being significantly more variable than all other analyzed species (fig. 3b).
Bonferroni corrected pairwise comparisons suggested that cyp27c1 expression in the limnetic
species from both crater lakes was significantly lower than those seen in Midas cichlids from the
great lake (supplementary table S2, Supplementary Material online). All other pairwise
comparisons were not significant after Bonferroni correction. Similar results in laboratory-reared
specimens suggest a genetic basis for the observed pattern of variation (supplementary fig. S5b,
Supplementary Material online). When comparing cyp27c1 coding sequence among species
showing high (i.e. A. citrinellus from great lake Nicaragua) and low (i.e., A. sagittae from crater lake
Xiloá and A. astorquii from crater lake Apoyo) levels of expression of this gene, we found almost
no variation. The exception was A. astorquii, in which two alleles (V540 and E540) were found.
Our results suggest rapid and parallel adaptive evolution of Midas cichlid vision in response to the
colonization of a new light environment that occurred by taking advantage of different molecular
mechanisms (fig. 7). Midas cichlids have colonized crater lakes Apoyo and Xiloá from the great
lakes Nicaragua and Managua, respectively, less than 2000 generations ago (Kautt et al. 2016a).
This event resulted in Midas cichlids experiencing a novel light environment in the crater lakes.
The most important differences found between the ancestral and derived environments are that in
the crater lakes light attenuation is lower, the light spectrum is broader and the overall visual
environment is shifted toward shorter wavelengths compared to the great lake (fig. 1). Given the
differences in the visual environments occupied by Midas cichlid species, we predicted phenotypic
divergence in visual sensitivity between fish from the great lakes and the crater lakes. We found
Midas cichlids to have a highly diverse visual system, both within and across species, with
particularly high levels of intraspecific variation in turbid great lake Midas cichlids. Importantly,
Midas cichlids from both crater lakes were found to have an overall shift in their visual sensitivities
toward shorter wavelengths when compared to the source populations from the great lakes in
agreement to the change observed in the photic environment (supplementary fig. S6,
Supplementary Material online). This shift could not be explained by a single mechanism, but
involved an integrated change that includes changes in lens transmittance, differential opsin gene
expression, opsin coexpression, and the use of various A1/A2 chromophore mixes. Because most
of the observed differences between species are maintained in laboratory-reared specimens
(supplementary figs. S1, S4 and S5, Supplementary Material online), these traits appear to have a
THE VISUAL SYSTEM OF MIDAS CICHLIDS FROM THE GREAT LAKES
Midas cichlids from the turbid great lake Nicaragua (Amphilophus citrinellus) have lenses blocking
UV and partially violet light. Additionally, the MSP experiment showed that Midas cichlids from this
great lake have peaks of maximum sensitivity in the blue, the green and the red parts of the light
spectrum (fig. 2) that correspond with the observed expression of SWS2a, RH2A and LWS seen in
fish from the great lake Managua (fig. 5). Interestingly, the retinas of fish from the turbid lake
Nicaragua are dominated by double cones expressing LWS in both members (fig. 6). This
dominance of the long sensitive cones might be an adaptation to the dim-light conditions
experienced in the turbid great lake (fig. 1), as the long sensitive cones could be used for
achromatic vision (Chiao et al. 2000; Cronin et al. 2014). The low genetic differentiation between
Midas cichlids from the two great lakes (Fst = 0.05; Kautt et al. 2016a) and the congruence of the
measures taken from specimens of both lakes (i.e., opsin expression and opsin sequences from
Lake Managua; and MSP, opsin expression, coexpression, opsin sequence and lens transmittance
from Lake Nicaragua) suggest that these two populations share a common phenotype.
The lens transmittance and photopigment sensitivities of Midas cichlids from the great lake
Nicaragua are in agreement with what is known for Neotropical cichlids. So far, there have been
few attempts to characterize lens transmittance and visual sensitivities in Neotropical cichlids (e.g.,
Muntz 1973; Loew and Lythgoe 1978; Levine and MacNichol 1979; Kröger et al. 1999; Weadick et
al. 2012, Escobar-Camacho et al. 2017). Yet, a clear picture emerges suggesting that in the
neotropics, cichlids tend to have lenses blocking UV and partially violet light, and blue sensitive
single cones, green and red sensitive double cones representing a long wavelength sensitive
palette of opsins (sensu Carleton et al. 2016). Thus, Midas cichlids from the great lakes have a
visual system similar to that seen in South American cichlids, but given the age of these lakes (i.e.,
Early Pleistocene; Kutterolf et al. 2007) this species likely had enough evolutionary time to fine
tune its visual system to the particular light conditions of the lakes. However, Midas cichlids depart
from the general pattern in two interesting ways: by showing a large degree of intraspecific
variation in visual sensitivity and by having at least four functionally visual pigments in cone cells.
INTRASPECIFIC VARIATION IN VISUAL SENSITIVITY IN MIDAS CICHLID FROM THE TURBID GREAT LAKES
Freshwater animals inhabiting turbid environments can adaptively shift their visual sensitivities
toward longer wavelengths without changing the opsin protein by using chromophores derived
from vitamin A2 rather than from vitamin A1 in their photopigments (Wald 1961; Hárosi 1994;
Cronin et al. 2014). Midas cichlids from the turbid great lakes use this mechanism to adjust their
visual sensitivity, although there was a great degree of variation among specimens (see fig. 3a). A
similar pattern was previously reported by Levine and MacNichol (1979), who analyzed ten Midas
cichlid individuals finding two discrete groups, one with mean lmax at 454, 532, and 562 nm and the
second at 463, 543, and 607 nm (for the single cones and the two members of double cones,
respectively). Although the origin of fish used by Levine and MacNichol (1979) is unclear, we
confirmed this variation among individuals from great lake Nicaragua (fig. 2). Since a similar
variation in the blue Acara (Aequidens pulcher) was found (Kröger et al. 1999), it suggests that this
high variability in A1/A2 might be a common pattern in Neotropical cichlids.
It has been recently shown that the enzyme Cyp27c1 is responsible for the conversion of
vitamin A1 into vitamin A2 in the retinal pigment epithelium (Enright et al. 2015). In zebrafish
(Danio rerio) the ratio of A1- to A2-derived chromophore covaries with the expression level of
cyp27c1 and knocking down this gene result in an inability of individuals to shift sensitivities toward
longer wavelengths by means of differential chromophore usage (Enright et al. 2015). Midas
cichlids from the turbid great lake show high intraspecific variation in the expression levels of
cyp27c1 (fig. 3b), providing a likely molecular mechanism for the observed variation in A1/A2
Coding sequence variation could also explain some of the intraspecific variation seen in
turbid great lake Midas cichlid visual sensitivity. A164S in LWS was the only variable site directed
into the retinal binding pocket identified in this study (table 1). These allelic variants of LWS have
been found in several other organisms (e.g. Terai et al. 2006; Hofmann et al. 2009; Sandkam et al.
2015) and also as divergence among LWS paralogs (e.g. Asenjo et al. 1994; Ward et al. 2008;
Phillips et al. 2016). The replacement of alanine with serine at this site is known to result in λmax
shift towards longer wavelengths (+ 7nm; Asenjo et al. 1994). Measurements of absorption spectra
on reconstituted LWS proteins of African cichlids showed that this substitution produced the
expected λmax shifts only if combined with an A2-derived chromophore (Terai et al. 2006).
Interestingly, the 164A-164S allelic variants solely occur in the great lakes, where fish varied in
chromophore usage. The combination of 164S and A2-derived retinal in red-sensitive pigments is
proposed to be an adaptation to visual environments with a red-shifted light spectrum (Terai et al.
2006), as those experienced by fish in the great lakes. Yet, 164S is not fixed in Lake Nicaragua but
it is segregating in the population. It is possible that photic environment variation across the lake
favors the maintenance of the polymorphism (e.g., Terai et al. 2006).
FOUR FUNCTIONAL PHOTOPIGMENTS IN MIDAS CICHLID CONE CELLS
Functional analyses with MSP suggested that Midas cichlids have four different photopigments in
their cone cells. Most examined individuals had double cones corresponding to three different
spectral classes (a red, a long green, and a short green; fig. 2), that, in combination with the
spectral class of single cones confer them the potential for tetrachromatic color vision.
Remarkably, the fourth spectral class identified in Midas cichlid retinas appears to be the result of
the coexpression of RH2A
and LWS on the same double cone member, rather than the
expression of a different opsin gene. Combining the MSP, qPCR and FISH experiments, we
inferred that the red spectral class with λmax from 560 to 623 nm corresponds to photoreceptors
using LWS as the protein component of their visual pigments, and the short-green spectral class
ranging from 517 to 539 nm corresponds to photoreceptors using RH2Ab. The observed range of
sensitivities within these two spectral groups is explained by variations in A1 to A2 chromophore
proportions in visual pigments having predicted pure-A1 sensitivities at ~560 and ~510 nm,
respectively. Yet, there are several double cone members with λmax values between these two
groups that could not be assigned to either spectral class by just adjusting A1/A2 proportions.
These cones have to be classified into a new spectral class, the long-green, and FISH staining
suggests that this spectral class is the product of RH2A
and LWS coexpression in double cone
That the long-green spectral class is the product of coexpression begs the question, why
Midas cichlids do not use RH2A
based visual pigments as African cichlids do? This is intriguing
given that the predicted protein coded by RH2A
appears be functional. Although speculative, it is
possible that gene conversion between RH2A paralogs (Escobar-Camacho et al. 2017) plays a
role, either because both paralogs are functionally very similar or because the regulatory
machinery has been affected by gene conversion. In addition, visual sensitivity curves deriving
from coexpression will be significantly different (wider) compared to their pure RH2A
effectively changing the sensitivity bandwidth of this color channel and, by varying coexpression
proportions, maintaining a flexible mechanism for spectral tuning. The function of coexpression in
Midas cichlids is unclear, but it may be related to increased contrast detection (Dalton et al. 2017).
Departures from trichromacy have been previously proposed for African cichlids based on
measurements of maximum sensitivity by MSP (e.g., Parry et al. 2005; Dalton et al. 2014) or
electroretinography (e.g., Sabbah et al. 2010), and by determining gene expression using qPCR
(e.g., Hofmann et al. 2009) and in situ hybridization (e.g., Dalton et al. 2014). Recently, Dalton et
al. (2017) showed that in the African cichlid Metriaclima zebra extensive regions of the retina could
have very high levels of coexpression, with an incidence of more than 90%. This would imply that
cones expressing only one opsin got almost completely replaced by cones showing coexpression.
Midas cichlids differ from this in that cones coexpressing two opsin are distributed in low number
across the retina, not replacing cones with only one opsin expressed, but coexisting with those.
Thus, this extensive coexpression pattern appears to be novel to Midas cichlids. It is not clear if
this is common in other Neotropical cichlids. It would be interesting to explore this issue in the
neotropical pike cichlid (C. frenata) given that it was reported to have a very long-shifted green
sensitive double cone member (~547 nm; Weadick et al. 2012).
ADAPTIVE CHANGES IN THE VISUAL SENSITIVITY OF CRATER LAKES MIDAS CICHLIDS
Midas cichlids from the crater lakes have a visual system that departs in several aspects from that
seen in fish from the great lakes, resulting in an overall shift in sensitivity toward shorter
wavelengths (fig. 7). The mechanisms underlying this shift include more transmissive ocular
media, and changes in the chromophore and the protein component of visual pigments. Although
this was apparent in species from both crater lakes, the biggest differences were observed in the
species from crater lake Apoyo. This was expected given that this crater lake is the oldest (Elmer
et al. 2010), had been occupied by Midas cichlids the longest (Kautt et al. 2016a), and it differs the
most in terms of photic environment from the great lakes (fig.1).
OCULAR MEDIA TRANSMITTANCE IN MIDAS CICHLIDS FROM THE CRATER LAKES
Eye lenses have become clearer in the crater lakes showing no overlap with the transmitting
values seen in fish from the great lakes. This includes two extreme cases of UV-transmitting
lenses in A. zaliosus, the limnetic species from crater lake Apoyo (fig. 4). Vertebrate lenses are
formed by concentric layers of translucent proteins called crystallins, belonging to three large
protein families (Fernald 2006). Crystallin proteins differ in their refractive indexes, so changes in
crystallin usage across populations or developmental stages can result in variation in lens
transmittance (Sabbah et al. 2012; Wages et al. 2013; Mahendiran et al. 2014). Additionally,
Neotropical cichlids tend to deposit pigments in their lenses that work as filters for short-
wavelength light (Muntz 1973). Whereas having UV- and violet-blocking lenses might help reduce
the loss of contrast detection due to the scattering of short-wavelength light; bearing clearer lenses
in blue-shifted light environments could be adaptive, since it would allow fish to better utilize the
whole available light spectrum (Muntz 1982). This is supported by a positive correlation between
lens transmission and single cone’s sensitivity in African cichlids (Hofmann et al. 2010). Thus,
more short-wavelength transmitting lenses might be an adaptation to the light environment of clear
water crater lakes. Given that these differences are observed in laboratory-born specimens reared
under common conditions, we suggest that the use of different crystallin proteins or the deposition
of pigments in lenses resulting in the observed cut-offs does not strictly depend on diet or light
conditions, but also has a genetic component.
CONE OPSIN EXPRESSION IN MIDAS CICHLIDS FROM THE CRATER LAKES
There is evidence for genetically based differential opsin gene expression between Midas cichlids
from the ancestral population of the great lakes and the derived populations from crater lakes that
appears to be adaptive to the visual environment they experience (see fig. 1 and 5; supplementary
fig. S6, Supplementary Material online). Moreover, this variation in opsin expression is consistent
with the phenotypic variation determined by MSP (see fig. 2 and fig. 5). One way in which crater
lake Midas cichlids differ from great lake fish is in the proportional expression of different opsin.
Whereas LWS represents > 60% of the total expression in fish from the great lakes, it is
consistently below 50% in the crater lakes. The opposite pattern is seen when comparing the
expression of RH2A
. In Midas cichlids, this differential expression is translated into a higher
proportion of green sensitive cones (both, RH2Ab-based and LWS-RH2Ab coexpression-based
cones; fig. 6). It is apparent that one of the mechanisms used by Midas cichlids to improve vision in
the shorter wavelength shifted light environment of the crater lakes is to increase the number of
green-sensitive cones at the cost of fewer red-sensitive ones. Similar patterns of change in the
proportional expression of cone opsins have been found in other cichlids suggesting it as a
common mechanism of visual tuning (e.g., Carleton and Kocher 2001).
Also, Midas cichlids from the clear crater lakes have expanded their sensitivities toward the
shorter part of the spectrum. Violet sensitive single cones have not been reported before for
Neotropical cichlids, although they are commonly seen in African cichlids and correspond to a
change from SWS2a to SWS2b as the protein component of photopigments (Carleton and Kocher
2001; Hofmann et al. 2009; O’Quin et al. 2010). Midas cichlids from the great lakes express
exclusively SWS2a, RH2A
, and LWS. In the crater lakes, more distinctly in crater lake Apoyo
some specimens expressed the violet sensitive SWS2b instead of SWS2a in single cones, and
expressed the green-blue sensitive RH2B in combination with other double cone opsins (fig. 6).
The expression of SWS2b and RH2B was coupled at the individual level (supplementary fig. S3,
Supplementary Material online), suggesting a general change in the pattern of expression.
To summarize, differential opsin expression is an important molecular mechanism in
adaptive phenotypic divergence of Midas cichlids visual system. By changing the relative
proportion of the different opsins expressed and by expressing other opsins (e.g., SWS2b and
RH2B), crater lake Midas cichlids have diverged from the ancestral population in the great lake in
the direction predicted based on the light environment differences (supplementary fig. S6,
Supplementary Material online).
CHROMOPHORE USAGE IN MIDAS CICHLIDS FROM THE CRATER LAKES
In the spectral classes common to Midas cichlids from the great lakes and the crater lakes, we
observed divergence in mean lmax and the associated variance (fig. 2). This is most evidently in
red-sensitive receptors where lmax estimates were limited to the yellow in fish from the crater lakes,
but expanding into the red part of the spectrum in the great lake specimens. In other spectral
classes the differences are subtler, but there is still a clear trend for crater lake Midas cichlids to
have lmax shifted toward shorter wavelengths. This shift could be the result of structural changes in
the opsin protein (Yokoyama et al. 2008) or due to differential chromophore usage (Wald 1961;
Hárosi 1994). Given that only one amino acid substitution was identified in sites directed into the
binding pocket across all Midas cichlid opsins (see table 1), different usage of A1- and A2-derived
chromophores is the most plausible mechanism behind the observed variation in photoreceptor
sensitivity. This conclusion is supported by the significant decrease in A2-derived chromophore
usage seen in clear crater lake Midas cichlids compared to fish from the turbid great lakes. The
down-regulation of cyp27c1 expression in Midas cichlids from the crater lakes is the most likely
mechanism underlying the changes in chromophore usage (Enright et al. 2015; supplementary fig.
S5a, Supplementary Material online). Moreover, this variation is interpreted to have a genetic basis
given that the differences in cyp27c1 expression between species were maintained under
laboratory conditions (supplementary fig. S5b, Supplementary Material online).
An interesting exception to this general pattern was the benthic species from crater lake
Xiloá (A. xiloaensis) that showed high proportions of vitamin A2-derived chromophore usage and
high levels of cyp27c1 expression similar to the ancestral phenotype seen in great lake Midas
cichlids. This could be adaptive in Xiloá, as this lake departs less in the photic condition from great
lake Managua than crater lake Apoyo does. However, the down-regulation of LWS in A. xiloaensis
strongly departs from the ancestral phenotype. Thus, this species might be using a different
strategy to tune sensitivity to the new environment, but further studies are necessary to clarify this
issue. Nonetheless, we did not observe this in laboratory-reared individuals of A. xiloaensis,
suggesting that this phenotype might be plastic (supplementary fig. S4 and S5, Supplementary
Material online). This highlights the multitude of mechanism that this extremely closely related set
of species is capable of using during repeated adaptation to the crater lake environments.
MECHANISMS OF ADAPTATION TO DIVERGENT VISUAL ENVIRONMENTS IN MIDAS CICHLIDS
There is much debate about the relative importance of changes in coding sequence and of gene
expression as the molecular mechanisms underlying phenotypic diversification (Hoekstra and
Coyne 2007; Carroll 2008; Stern and Orgogozo 2008; Elmer and Meyer 2011; Rosemblum et al.
2014). Evidence supporting the importance of amino acid substitutions for phenotypic evolution
has steadily accumulated for many decades, establishing it as an important mechanism of
diversification (Hoekstra and Coyne 2007; Stern and Orgogozo 2008). On the other hand, the
importance of regulatory processes for phenotypic divergence has become strongly supported
more recently, as new molecular techniques resulted in the accumulation of new evidence (Wray
2007; Carroll 2008; Stern and Orgogozo 2008; Kratochwil and Meyer 2015). Changes in
expression of cone opsins and cyp27c1, the gene responsible for changes in chromophore usage,
seem to contribute the most to the observed variation in visual sensitivity. In contrast, structural
changes might play only a limited role in vision tuning of Midas cichlids. Surely, amino acid
substitutions are not unimportant for the phenotypic evolution of vision, as there is compelling
evidence for its role in divergence in sensitivity, both, among paralogs (e.g., Yokoyama 2000) and
among homologs when comparing different populations or species (e.g. Terai et al. 2002, 2006;
Sugawara et al. 2005; Migayi et al. 2012; Torres-Dowdall et al. 2015). Yet, in Midas cichlids
structural changes might become more relevant in later stages of diversification as genetic
variation in coding sequence would be expected to take time to appear by de novo mutations in
young and initially small populations.
We presented evidence that the visual system of Midas cichlids has rapidly and adaptively
evolved since the colonization of crater lakes, a few thousand generations ago (Kautt et al. 2016a;
fig. 7). The observed changes in visual sensitivity are the result of a combination of different
mechanisms including changes in the ocular media and in both, the opsin protein and the light
absorbing chromophore components of photopigments. Previous research has shown that all
these mechanisms can independently tune visual sensitivity in African cichlids (reviewed in
Carleton 2009; Carleton et al. 2016). Here, we showed that all these underlying mechanisms
respond extremely rapidly and in an integrated way to adapt these fishes to changed light
conditions that their ancestors experienced due to the colonization of the clear water crater lakes.
Despite the divergence in visual sensitivity of crater lake Midas cichlids compared to the great lake
ancestral populations, we did not find striking differences in sensitivity within the small radiations in
each crater lake. Yet, in the limnetic species from Apoyo we observed a trend to have sensitivities
shifted toward shorter wavelengths compared to the benthic species that suggests that differences
might be accumulating.
The Midas cichlid species complex is only one of the many fish species that colonized
Nicaraguan crater lakes from the source populations in the great lakes Managua and Nicaragua
(Elmer et al. 2010; Kautt et al. 2016a). Yet, it is clearly the most abundant species in these lakes
(Dittmann et al. 2012) and the only lineage that has radiated in the crater lakes, resulting in a
species complex composed of at least 13 species (Barluenga et al. 2006, 2010; Elmer et al. 2010;
Recknagel et al. 2013; Kautt et al. 2016a). The reasons why this species has become dominant in
terms of biomass and has diversified but other species that colonized the crater lakes have not,
remain largely unclear (Franchini et al. 2017). Uncovering the molecular mechanisms contributing
to the adaptation of Midas cichlids to the novel conditions experienced in the crater lakes, such as
a short-wavelength shifted light environment, is fundamental to progress in our understanding of
Material and Methods
UNDERWATER LIGHT MEASUREMENTS
Underwater light measurements were taken at one site in Lake Managua, 4 sites in Lake Xiloá,
and 7 sites in Lake Apoyo, characterized by different bottom structure (rocky outcrops, boulders
covered in algal material, Chara beds, sandy bottoms). Underwater spectral irradiance was
measured with an Ocean Optics USB2000 connected to a 15m UV-VIS optical fiber fitted with a
cosine corrector, just under the surface and at 2m depth, orienting the probe upwards (for
downwelling light) and towards four orthogonal directions horizontally (sidewelling light). The four
horizontal measurements were averaged to derive a single measurement of side-welling light at
depth. Downwelling irradiance is presented in the main text; sidewelling irradiance is presented in
supplementary fig. S7 (Supplementary Material online). We calculated the total quantal flux for
each irradiance integrating each spectral measurement in the range (350-700nm) relevant to
cichlid vision. Following McFarland and Munz (1975), we derived λP50, i.e. the wavelength that
halves the total number of photons in the selected range of visible spectrum and that identifies the
spectral region with the highest abundance of quanta.
RETINAL MICROSPECTROPHOTOMETRY MEASUREMENTS
We conducted microspectrophotometry (MSP) in wild-caught Midas cichlids from great lake
Nicaragua (n = 5), crater lake Apoyo (n = 10), and crater lake Xiloá (n = 12; species identities,
number of rods and cones analyzed per species, mean peak of maximum absorption, and A1%
are noted in supplementary table S1; Supplementary Material online) and in laboratory reared
Midas cichlids from great lake Nicaragua (n = 2), crater lake Apoyo (n = 4), and crater lake Xiloá (n
= 8). Analyses followed standard methods (Loew 1994; Losey et al. 2003; Fuller et al. 2003).
Before conducting MSP, fish were maintained under dark conditions for a minimum of four hours
and then euthanized with an overdose of MS-222 followed by cervical dislocation. The eyes were
rapidly enucleated under dim red light, and the retinas removed and maintained in phosphate-
buffered saline (pH 7.2) with 6% sucrose. Small pieces of the retina were placed on a cover slide,
fragmented to isolate individual photoreceptors, and sealed with a second cover slide and Corning
High Vacuum grease. We used a single-beam, computer-controlled MSP, with a 100-W quartz
iodine lamp that allowed for accurate absorption measurements down to 340 nm (Loew 1994;
Losey et al. 2003). Peak of maximum absorption (λmax) of photoreceptors was obtained by fitting
A1- or A2 templates to the smoothed, normalized absorbance spectra (Lipetz and Cronin 1988;
Govardovskji et al. 2000). We used the criteria for data inclusion into the analysis of λmax described
in Loew (1994) and Losey et al. (2003).
We conducted statistical comparisons at two levels. First, to test for the effect of
colonization of clear water crater lakes on the visual system of Midas cichlids, we considered lake
of origin as explanatory variable, ignoring species or ecomorphs within crater lakes. Second, to
test for the effect of microhabitat (i.e. limnetic versus benthic) we only used data from the crater
lakes, where both ecomorphs are found, and included lake of origin and ecomorph as explanatory
variables in the statistical model. In both cases, we first conducted a Bartlett’s k2 test of
homoscedasticity within each spectral class to determine if there were differences in variance
among groups. This was interpreted as a test for variation in A1- to A2-derived chromophore
usage as we found little structural variation in opsin proteins that could explain variation within
spectral class (see Results: Coding Sequence Variation of Midas Cichlid Opsin Genes above). If
the Bartlett’s k2 test did not reject homoscedasticity, we conducted a linear mixed model using λmax
values for individual photoreceptors within each spectral class as response variable, lake of origin
as explanatory variable, and specimen as a random variable. When testing for the effect of
microhabitat, ecomorph and its interaction with lake of origin were also included as explanatory
variables. If the Bartlett’s k2 test suggested heteroscedasticity, we used a non-parametric Kruskal-
Wallis test. All analyses were conducted in R (R Core Team 2014). Significant results are reported
in the main text, non-significant tests are reported in supplemental table S3 (Supplemental Material
OCULAR MEDIA TRANSMISSION
We measured ocular media transmission in laboratory-reared individuals of A. citrinellus from great
lake Nicaragua (n = 10), the limnetic A. zaliosus (n = 6) and the benthic A. astorquii (n = 3) from
crater lake Apoyo, and the limnetic A. sagittae (n = 7) and the benthic A. amarillo (n = 9) from
crater lake Xiloá. All fish were euthanized using an overdose of MS-222 and subsequent cervical
dislocation. The eyes were enucleated, carefully hemisected, and the corneas and lenses were
placed on a black paper with a small hole. A pulsed xenon lamp (PX-2, Ocean Optics) was
directed through the hole and transmission was measured with an USB2000+UV-VIS-ES
spectrometer (Ocean Optics). For each specimen, three measures of transmission were obtained
from each of the two eye ocular media. As previously reported for cichlids (Hofmann et al. 2010;
O’Quin et al. 2010), we found that the lenses are the limiting ocular media, so we subsequently
measured only lens transmission. We calculated lens transmission (T50) following Hofmann et al.
(2010), measuring the wavelength of maximum slope (i.e., inflection point in the sigmoid curve)
within the range of 300 to 700 nm. This method was shown to be less sensitive to departures from
perfect sigmoid shape than methods that determine T50 as the halfway point between the
minimum transmission and that of maximum transmission, and both are highly correlated
(Hofmann et al. 2010). Using this last method did not produce a qualitative difference in our
OPSIN CODING REGIONS AMPLIFICATION AND SEQUENCING
Genomic DNA was isolated using standard phenol–chloroform extractions from a total of 64
specimens of Midas cichlids, including representatives of two species from each of the great lakes
Managua and Nicaragua, and two species from each of the crater lakes Apoyo and Xiloá (table 1).
Genomic sequences of all opsin genes were obtained by polymerase chain reaction (PCR) using
standard protocols. Primers were designed in PRIMER 3 (Rozen and Skaletsky 2000) using the A.
citrinellus draft genome as a template (Elmer et al. 2014; primer list and PCR conditions in
supplementary table S4; Supplementary Material online). Samples were sequenced bi-directionally
and using internal primers on a 3130xl Genetic Analyzer. Sequence editing and assembly was
performed using SeqMan II (DNAstar).
ANALYSES OF OPSIN AND CYP27C1 GENE EXPRESSION
We measured cone opsin and cyp27c1 expression in wild-caught (WC) and laboratory-reared (LR)
individuals of A. citrinellus (nWC = 6 from Lake Managua; nLR = 8 from Lake Nicaragua), the limnetic
A. zaliosus (nWC = 6; nLR = 4) and the benthic A. astorquii (nWC = 6; nLR = 4) from crater lake Apoyo,
and the limnetic A. sagittae (nWC = 4; nLR = 4) and the benthic A. xiloaensis (nWC = 4; nLR = 4) from
crater lake Xiloá. All fish were sacrificed using an overdose of MS-222 and subsequent cervical
dislocation. The eyes were rapidly enucleated and the retinas removed and stored in RNAlater
(Sigma-Aldrich, USA) until RNA extraction. RNA was extracted using a commercial kit (RNeasy
Mini Kit, Qiagen) and RNA concentrations were measured using the Colibri Microvolume
Spectrometer, (Titertek Berthold, Germany). Total RNA was reverse transcribed with the first-
strand cDNA synthesis kit (GoScriptTM Reverse Transcription System, Promega, Madison,
Gene expression levels were quantified using Quantitative Real-Time PCR (qPCR). Real-
Time reactions were run in a CFX96TM Real-Time System (Bio-Rad Laboratories, Hercules,
California) using specifically designed primers (supplementary table S4; Supplementary Material
online). Amplification efficiencies were determined for each primer pair. Standard PCR and Sanger
sequencing of PCR products were performed for each opsin gene to check for specificity of
amplification. Expression levels of genes were quantified with three technical replicates and mean
Ct values were used for further analyses. Quantitative Real-Time PCR was performed under
standard conditions following the manufacturer’s protocol (GoTaq qPCR Master Mix, Promega,
Madison, Wisconsin). Proportional opsin expression was determined for each specimen by
calculating the proportion of each opsin (Ti) relative to the total opsin expression (Tall) after Fuller et
al. (2004) using the following equation:
%&' &&' ( )"*+,-**
&' &&' ( )"*+,-**
Ei represents the primer efficiency for primer i and Cti is the critical cycle number for gene i (the
proportional expression values of the seven cone opsins add up to 1 for each specimen). cyp27c1
expression was normalized using the geometric mean of two selected housekeeping genes (ldh2
and imp2) using the following equation:
./"% 0 )"
Non-parametric Multi-Response Permutation Procedures (MRPP) tests (Mielke et al. 1981)
were used to compare cone opsin expression among species and between wild-caught and
laboratory-reared specimens. Pairwise comparisons between wild-caught and laboratory-reared
specimens within each species were also conducted and significant differences were found only
for the benthic species of crater lake Xiloá (A. xiloaensis; supplementary table S5; Supplementary
Material online). Kruskal Wallis tests were used to compare expression of cyp27c1 among species
and between wild-caught and laboratory-reared specimens. As with opsin gene expression, using
pairwise comparisons we only found differences in cyp27c1 due to rearing condition for A.
xiloaensis (supplementary table S2; Supplementary Material online).
ANALYSES OF OPSIN GENE COEXPRESSION
We performed triple FISH (fluorescent in situ hybridization) in five laboratory reared individuals per
species of a Midas cichlid from a turbid great lake (A. citrinellus) and one from a clear crater lake
(A. astorquii). All samples were probed for all three cone opsin genes. Probes for RH2B, RH2A
and LWS were cloned into the pGEMT or pGEMTE vector systems (Promega #A3600 and
#A3610) using primers: RH2B-FW ATGGCATGGGATGGAGGACTTG; RH2B-RV
GAAACAGAGGAGACTTCTGTC; RH2A-FW TGGGTTGGGAAGGAGGAATTG; RH2A-RV
ACAGAGGACACCTCTGTCTTG; LWS-FW ATGGCAGAAGAGTGGGGAAA; LWS-RV
The fluorescent in situ hybridization was performed as described (Woltering et al. 2009)
with modifications enabling triple fluorescent instead of single colorimetric detection. Briefly, eyes
were rapidly enucleated and retinas fixed in 4% PFA in PBS overnight and stored in methanol at -
20 °C until further use. Duration of tissue bleaching in 1.5% H2O2 in methanol and Proteinase K
treatment were decreased to three minutes each. Probes with three different detection labels were
synthesized using DIG-labeling mix (Roche #11277073910), Fluorescein labeling mix (Roche
#116855619910), custom made DNP labeling mix 10x [DNP-11-UTP (Perkin Elmer
#NEL555001EA) 3.5mM combined with UTP 6.5mM, CTP 10mM, GTP 10mmM, ATP 10mM
(ThermoFischer #R0481)]. Antibody incubation was performed overnight at 4°C using anti-
Fluorescein-POD (Roche #11426346910), anti-DIG-POD (Roche #11207733910) and anti-DNP-
HRP (Perkin Elmer #FP1129). To amplify fluorescent signal, we used tyramide signal amplification
(TSA) for each of the different labels; TSA plus-Fluorescein (Perkin Elmer #NEL753001KT), TSA
plus-Cyanine 3 (Perkin Elmer #NEL753001KT), and TSA plus-Cyanine 5 (Perkin Elmer
#NEL745001KT). Antibody incubation and corresponding signal amplification were performed
sequentially. Prior to incubation with the next antibody, POD activity of the previous one was
deactivated in 100 mM glycine solution (pH 2.0) for 15 minutes followed by 15 washes for 10
minutes each in TBS-T and once overnight. Before mounting, retinas were cleared in 70 % glycerol
overnight at 4 °C.
Expression levels were quantified in four quadrants of the retina divided as dorsal-nasal,
dorsal-temporal, ventral-nasal and ventral-temporal. Per retinal region, five sampling areas were
randomly chosen and in each all the cones in a frame of 55 x 55 µm were examined for RH2B,
RH2A and LWS expression and for coexpression genes within one member of a double cone. This
assured that more than 200 double cone members were characterized in each region for each fish.
We are thankful to the members of the Meyer lab, particularly Sina Rometsch for helping with
samples, Ralf Schneider for helping with ocular media analyses, and Gonzalo Machado-Schiaffino
and Andreas Kautt for fruitful discussions. We especially thank Ellis Loew for allowing us to use his
microspectrophotometer and for advice on data analysis. We appreciate the assistance of Kenneth
McKaye during the collection of specimens for microspectrophotometry. MARENA granted permits
for fieldwork and collections (DGPN/DB-IC-004-2013). Laboratory reared fish were euthanized
under University of Konstanz permit (T13/13TFA). This work was supported by the European
Research Council through ERC-advanced (grant number 293700-GenAdap to A.M), the Deutsche
Forschungsgemeinschaft (grant number 914/2-1 to J.T.D.), the EU FP7 Marie Curie Zukunftskolleg
Incoming Fellowship Programme, University of Konstanz (grant number 291784 to J.T.D.), and the
Young Scholar Fund of the University of Konstanz (grant number FP 794/15 to J.T.D.).
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Table 1. Non-synonymous nucleotide substitution observed in the Midas cichlid species complex. Amino acid replacement and location for each
non-synonymous substitution are indicated at the bottom of the table.
Midas cichlid species
amino acid substitution
1 Nucleotide positions, the transmembrane helices (TM 1-5) and the extracellular interhelical loop (E-2) are defined and numbered based on the
bovine crystal structure of rhodopsin (Palczewski et al. 2000).
2 In all cases we observed different alleles segregating in the corresponding population. A IUPAC/IUB single-letter amino acid code (Leonard 2003)
is used to denote the nucleotides segregating at each position in the corresponding species (r: either a or g; s: either c or g; m: either a or c; k:
either g or t). A dot (·) represents implies no departure from the consensus.
Figure 1. Difference in the photic environment of a great lake and the crater lakes.
Normalized downwelling irradiance is narrower at two meters deep in the turbid great lakes in
comparison to the clear crater lakes. Hence, a higher proportion of light in the blue and red
part of the spectrum penetrates in the crater lakes compared to the great lakes. The insert
shows the absolute downwelling irradiance at 2 meters in each lake, showing the differences
among lakes in light extinction with depth.
350 400 450 500 550 600 650 700
Normalized downwelling irradiance at 2m
400 500 600 700
(µmol m s )
Figure 2. Individual level peaks of maximum absorbance (lmax ± SE) of visual pigments
determined by microspectrophotometry from wild-caught Midas cichlids from a turbid great
lake and to clear crater lakes. Unfilled symbols correspond to specimens of the benthic
ecomorph within each lake, whereas filled symbols correspond to limnetic specimens. Visual
pigments were assigned to different spectral classes (indicated by the vertical lines of
different colors) based on their estimated pure A1 peak of maximum absorbance (lA1). The
ranges of estimated lA1 are shown as shaded areas of the same color. From left to right,
these spectral classes correspond to the violet, blue, blue-green, rod, green (short), green
(long), and red previously identified in African cichlids (Carleton et al. 2009). The grey
shading separates samples from the different lakes.
420 440 460 480 500 520 540 560 580 600
λ max (nm)
Clear CL Apoyo Clear CL Xiloá Turbid great lake
Figure 3. Distribution of estimated proportion of vitamin A2-derived chromophore usage in
Midas cichlids from the turbid great lake Nicaragua and two clear water crater lakes (a).
Estimates of A2-derived usage for the long-green spectral class were not included due to the
confounded effect of opsin coexpression. cyp27c1 expression relative to the geometric mean
of two housekeeping genes (ldh2 and inp2) in the same crater lakes and the turbid great lake
Managua (b). Horizontal lines are the mean for each group. Bonferroni corrected pair-wised
comparisons are reported in supplementary table S2, Supplementary Materials online.
Figure 4. Lens transmittance grouped into different categories. Example of these are shown
in (a). Histogram depicting the frequency of lens transmittance cut-offs (T50) of lab-reared
Midas cichlids (b). Over-imposed is a density kernel showing the bimodal distribution of T50.
Clear crater lake ApoyoClear crater lake XiloáTurbid great lake
b. cyp27c1 expression
0 100 0 100 0 100 0 100
(n = 58)
(n = 128) (n = 56) (n = 38)
(n = 144)
limnetic speciesbenthic specieslimnetic species
Clear crater lake ApoyoClear crater lake Xiloá
Turbid great lake
a. Estimated percentage of vitamin A2-derived chromophore usage
300 350 400 450 500 550
CL Xiloá benthic
CL Xiloá limnetic
CL Apoyo benthic
CL Apoyo limnetic
340 360 380 400 420 440
CL Apoyo (n= 7)
CL Xiloá (n= 16)
GL Nicaragua (n= 10)
0.000 0.005 0.010 0.015 0.020
Hartigans' D = 0.099
P = 0.003
a. Examples of transmission curves b.
2 4 6 8
Figure 5. Proportion of the total opsin expression comprised by each of the different opsin
genes in wild-caught Midas cichlids from a turbid great lake (a), and two clear crater lakes (b,
c). Means are shown as horizontal bars. Black circles represent expression in specimens of
the limnetic ecomorph, white circles denote expression in specimens of the benthic species.
Figure 6. Triple fluorescent in situ hybridization staining of the retinas of two Midas cichlids
species, one from a turbid great lake (a-e) and one from a clear crater lake (f-j) across four
quadrants of the retina. Coexpression is common in specimens from both lakes, but the
frequency is higher in specimens from the clear crater lake (f-j). Details in (g) show examples
of coexpression of LWS and RH2Aβ (upper box) and RH2B and RH2Aβ (lower box).
SWS1 SWS 2b SWS2a RH2B RH2AβRH2AαLWSSWS1 SWS 2b SWS2a RH2B RH2AβRH2AαLWS
SWS1 SWS 2b SWS2a RH2B RH2AβRH2AαLWS
a. Turbid great lake b. Clear Crater Lake Xiloá c. Clear Crater Lake Apoyo
Figure 7. The visual system of Midas cichlids evolved in parallel in the two clear crater lakes
Apoyo and Xiloá after their colonization from the turbid great lakes Nicaragua and Managua,
respectively. Phenotypic changes in opsin expression, chromophore usage and lens
transmittance are mapped on a phylogenetic reconstruction modified from Kautt et al.
(2016a). In fish from both crater lakes, lenses became more transmissive and expression of
RH2A increased and that of LWS decreased. In all crater lake species, except for A.
xiloaensis, chromophore usage changed from mainly A2 to mainly A1.