Human activities frequently alter environmental conditions and affect the use of sexually selected traits like color in animals. However, the effects of environmental stressors are unlikely to be uniform across populations that experience different environments or between sexes. We aimed to understand the underlying genetic, environmental, and gene-by-environment contributions to color expression in males and females of a sexually dimorphic fish. Pseudocrenilabrus multicolor is a haplochromine cichlid found in environments that vary dramatically, particularly with respect to oxygen and turbidity levels. We reared fish from one swamp (hypoxic, clear) and one river (normoxic, turbid) population in a split-brood design (hypoxic/normoxic × clear/turbid) and then quantified color and carotenoid concentrations. As expected in this sexually dimorphic species, females were far less colorful than males. In males, hypoxia and turbidity were drivers of traits associated with color, suggesting that color was modified under energetically or visually unfavorable conditions. Males in the hypoxic treatment from both populations were not as bright as males reared under normoxic conditions, which corresponds to results observed in wild fish. Males reared in turbid conditions were also marginally less bright along the ventral surface than males reared in clear water. Rearing under turbid conditions reduced carotenoid concentrations in male skin, but carotenoids were not correlated with spectral characteristics of male color. We did not find effects of population on color traits, suggesting that differences in color between wild populations are due to plastic rather than fixed genetic effects. Overall, we provide evidence that hypoxia and turbidity affect signaling traits, although the consequences for mating success remain to be determined.

Birds possess the most complex photoreceptor system among vertebrates, with one rod and six cone types, including four single cones (violet, blue, green, and red) and two constituent cells of the double cone (DC-P and DC-A). The evolutionary relationships of avian photoreceptors to those of other vertebrate taxa have not been systematically explored. Here, we perform single-cell RNA sequencing (scRNA-seq) on retinas of newly hatched chickens to trace cell-type homologies across species. Analysis of differentially expressed transcription factors (TFs) suggests that avian rods and single cone types correspond to cognate cell types in fish and placental mammals, whereas double cones have a distinct origin. We propose that DC-P arose from an ancestral red cone, as revealed by expression of the red cone cell fate determinants thyroid hormone receptor β (THRB) and SAMD7, whereas DC-A may have arisen from an ancestral blue cone, as suggested by expression of the blue cone TFs FOXQ2 and SKOR1. These expression signatures are shared by DC-P and DC-A of the green anole lizard (Anolis carolinensis), suggesting conservation throughout Sauropsida. Consistent with our hypothesis, CRISPR-mediated knockout of THRB causes loss of red cones and DC-P, but not DC-A, and the appearance of supernumerary rods and green cones, suggestive of direct transfating. Furthermore, cis-regulatory analysis suggests that separate enhancers control red cone opsin expression in DC-P and DC-A, consistent with distinct evolutionary origins. Taken together, our studies trace the evolutionary relationships of avian photoreceptors and suggest separate origins of DC-P and DC-A from ancestral red and blue cones, respectively.

Carotenoid-based coloration is an essential feature of avian diversity and has important roles in communication and mate choice. The red feathers of birds from phylogenetically diverse orders and families are pigmented with C4-ketocarotenoids produced via the successive action of Cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L) on yellow dietary precursors. Yet, the biochemistry of these enzymes remains incompletely understood. Here we present a series of experiments characterizing the substrates, intermediates, and products of CYP2J19 and BDH1L expressed in heterologous cell culture. We confirm that CYP2J19 preferentially hydroxylates the 4 and 4' positions of β-ring substrates, but can also hydroxylate the 3 and 3 positions of C4-ketocarotenoids. We confirm that BDH1L catalyzes the conversion of zeaxanthin to canary xanthophyll B (ε,ε'-carotene-3,3'-dione) a major pigment in plumage of many yellow bird species. These results suggest that the actions of CYP2J19 and/or BDH1L can explain the presence of many metabolically transformed carotenoids in avian tissues.

The carotenoid-based colours of birds are a celebrated example of biological diversity and an important system for the study of evolution. Recently, a two-step mechanism, with the enzymes cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), was described for the biosynthesis of red ketocarotenoids from yellow dietary carotenoids in the retina and plumage of birds. A common assumption has been that all birds with ketocarotenoid-based plumage coloration used this CYP2J19/BDH1L mechanism to produce red feathers. We tested this assumption in house finches (Haemorhous mexicanus) by examining the catalytic function of the house finch homologues of these enzymes and tracking their expression in birds growing new feathers. We found that CYP2J19 and BDH1L did not catalyse the production of 3-hydroxy-echinenone (3-OH-echinenone), the primary red plumage pigment of house finches, when provided with common dietary carotenoid substrates. Moreover, gene expression analyses revealed little to no expression of CYP2J19 in liver tissue or growing feather follicles, the putative sites of pigment metabolism in moulting house finches. Finally, although the hepatic mitochondria of house finches have high concentrations of 3-OH-echinenone, observations using fluorescent markers suggest that both CYP2J19 and BDH1L localise to the endomembrane system rather than the mitochondria. We propose that house finches and other birds that deposit 3-OH-echinenone as their primary red plumage pigment use an alternative enzymatic pathway to produce their characteristic red ketocarotenoid-based coloration.

In many species of birds, red carotenoid coloration serves as an honest signal of individual quality, but the mechanisms that link carotenoid coloration to animal performance remain poorly understood. Most birds that display red carotenoid coloration of feathers, bills, or legs ingest yellow carotenoids and metabolically convert the yellow pigments to red. Here, we review two lines of investigation that have rapidly advanced understanding of the production of red carotenoid coloration in birds, potentially providing an explanation for how red coloration serves as a signal of quality: the identification of the genes that enable birds to be red and the confirmation of links between production of red pigments and core cellular function. CYP2J19 and BDH1L were identified as key enzymes that catalyze the conversion of yellow carotenoids to red carotenoids both in the retinas of birds for enhanced color vision and in the feathers and bills of birds for ornamentation. This CYP2J19 and BDH1L pathway was shown to be the mechanism for production of red coloration in diverse species of birds and turtles. In other studies, it was shown that male House Finches (Haemorhous mexicanus) have high concentrations of red carotenoids within liver mitochondria and that redness is positively associated with mitochondrial function. These observations suggested that the CYP2J19 and BDH1L pathway might be tightly associated with mitochondrial function. However, it was subsequently discovered that male House Finches do not use the CYP2J19 and BDH1L pathway to produce red pigments and that both CYP2J19 and BDH1L localize in the endoplasmic reticulum, not the mitochondria. Thus, we have the most detailed understanding of links between cellular function and redness in a bird species for which the enzymes to convert yellow to red pigments remain unknown, while we have the best understanding of the enzymatic pathways to red in species for which links to cellular function are largely unstudied. Deducing whether and how signals of quality arise from these distinct mechanisms of ornamental coloration is a current challenge for scientists interested in the evolution of honest signaling.

The light environment underwater can vary dramatically over space and time, challenging the visual systems of aquatic organisms. To meet these challenges, many species shift their spectral sensitivities through changes in visual pigment chromophore composition and opsin expression. The red shiner (Cyprinella lutrensis) is a North American cyprinid minnow species that inhabits waters ranging widely in turbidity and temperature. We hypothesized that the visual system of the red shiner is plastic with chromophore composition and opsin expression varying in response to the environment. To test this hypothesis, we collected red shiners throughout the year from three Oklahoma creeks that vary in turbidity. We characterized the light environment by spectroradiometry, measured chromophore composition of the eyes with high performance liquid chromatography, characterized the mechanisms of chromophore metabolism, and examined ocular gene expression by RNA sequencing and de novo transcriptome assembly. We observed significantly higher proportions of the long wavelength-shifted A2 chromophore in the eyes of fish from the turbid site and in samples collected in winter, suggesting that there may be a temperaturedependent trade-off between chromophore-based spectral tuning and chromophore-related noise. Opsin expression varied between turbid and clear creeks, but did not align with light environment as expected, and the magnitude of these differences was limited compared with the differences in chromophore composition. We confirmed that red shiner CYP27C1 catalyzes the conversion of A1 to A2, but the ocular expression of CYP27C1 was not well correlated with A2 levels in the eye, suggesting conversion may be occurring outside the eye.

In many species of animals, red carotenoid-based coloration is produced by metabolizing yellow dietary pigments, and this red ornamentation can be an honest signal of individual quality. However, the physiological basis for associations between organism function and the metabolism of red ornamental carotenoids from yellow dietary carotenoids remains uncertain. A recent hypothesis posits that carotenoid metabolism depends on mitochondrial performance, with diminished red coloration resulting from altered mitochondrial aerobic respiration. To test for an association between mitochondrial respiration and red carotenoids, we held wild-caught, molting male house finches in either small bird cages or large flight cages to create environmental challenges during the period when red ornamental coloration is produced. We predicted that small cages would present a less favorable environment than large flight cages and that captivity itself would decrease both mitochondrial performance and the abundance of red carotenoids compared with free-living birds. We found that captive-held birds circulated fewer red carotenoids, showed increased mitochondrial respiratory rates, and had lower complex II respiratory control ratios - a metric associated with mitochondrial efficiency - compared with free-living birds, though we did not detect a difference in the effects of small cages versus large cages. Among captive individuals, the birds that circulated the highest concentrations of red carotenoids had the highest mitochondrial respiratory control ratio for complex II substrate. These data support the hypothesis that the metabolism of red carotenoid pigments is linked to mitochondrial aerobic respiration in the house finch, but the mechanisms for this association remain to be established.

Carotenoid pigments underlie most of the red, orange, and yellow visual signals used in mate choice in vertebrates. However, many of the underlying processes surrounding the production of carotenoid-based traits remain unclear due to the complex nature of carotenoid uptake, metabolism, and deposition across tissues. Here, we leverage the ability to experimentally induce the production of a carotenoid-based red plumage patch in the red-backed fairywren (Malurus melanocephalus), a songbird in which red plumage is an important male sexual signal. We experimentally elevated testosterone in unornamented males lacking red plumage to induce the production of ornamentation and compared gene expression in both the liver and feather follicles between unornamented control males, testosterone-implanted males, and naturally ornamented males. We show that testosterone upregulates the expression of CYP2J19, a gene known to be involved in ketocarotenoid metabolism, and a putative carotenoid processing gene (ELOVL6) in the liver, and also regulates the expression of putative carotenoid transporter genes in red feather follicles on the back, including ABCG1. In black feathers, carotenoid-related genes are downregulated and melanin genes upregulated, but we find that carotenoids are still present in the feathers. This may be due to the activity of the carotenoid-cleaving enzyme BCO2 in black feathers. Our study provides a first working model of a pathway for carotenoid-based trait production in free-living birds, implicates testosterone as a key regulator of carotenoid-associated gene expression, and suggests hormones may coordinate the many processes that underlie the production of these traits across multiple tissues.

An enormous body of research is focused on finding ways to commercialize carotenoids produced by the unicellular green alga, Haematococcus, often without the benefit of a sound phylogenetic assessment. Evidence of cryptic diversity in the genus means that comparing results of pigment studies may be confounded by the absence of a phylogenetic framework. Moreover, previous work has identified unnamed strains that are likely candidates for species status. We reconstructed the phylogeny of an expanded sampling of Haematococcus isolates utilizing data from nuclear ribosomal markers (18S rRNA gene, 26S rRNA gene, internal transcribed spacer [ITS]-1, 5.8S rRNA gene, and ITS-2) and the rbcL gene. In addition, we gathered morphological, ultrastructural and pigment data from key isolates of Haematococcus. Our expanded data and taxon sampling support the concept of a new species, H. privus, found exclusively in North America. Despite overlap in numerous morphological traits, results indicate that ratios of protoplast length to width and akinete diameter may be useful for discriminating Haematococcus lineages. High growth rate and robust astaxanthin yield indicate that H. rubicundus (SAG 34-1c) is worthy of additional scrutiny as a pigment source. With the description of H. privus, the evidence supports the existence of at least five, species-level lineages in the genus. Our phylogenetic assessment provides the tools to frame future pigment investigations of Haematococcus in an updated evolutionary context. In addition, our investigation highlighted open questions regarding polyploidy and sexuality in Haematococcus which demonstrate that much remains to be discovered about this green flagellate.