Seasonal but not sex-biased gene expression of the carotenoid ketolase, CYP2J19, in the sexually dichromatic southern red bishop (Euplectes orix).

Intense red colours in birds are often owing to ketocarotenoids (KCs). In many land birds, KCs are oxidized from dietary yellow precursors, presumably by the avian carotenoid ketolase CYP2J19, the regulation and constraints of which have important implications for condition-dependence and honest signalling of carotenoid colour displays. We investigated hepatic CYP2J19 gene expression in the seasonally and sexually dichromatic southern red bishop (Euplectes orix) in relation to season, sex, progression of the prenuptial moult, testis size, body condition, redness and circulating sex steroids. A coloration function of CYP2J19 is supported by a seasonal upregulation prior to and during the carotenoid-depositing stage of the male prenuptial moult. However, CYP2J19 expression was similarly high in females (which do not moult prenuptially), and remained high in males after moult, suggesting additional or alternative roles of hepatic CYP2J19 or its products, such as detoxification or antioxidant functions. In males, the CYP2J19 upregulation preceded and was unrelated to the rise in plasma testosterone, but was correlated with androstenedione, probably of adrenal origin and compatible with luteinizing hormone-induced and (in females) oestrogen-suppressed moult. Finally, contrary to ideas that carotenoid ketolation rate mediates honest signalling of male quality, CYP2J19 expression was not related to plumage redness or male body condition.

Variation in Genetic Mechanisms for Plumage Polymorphism in Skuas (Stercorarius).

Coloration is evolutionarily labile and so provides an excellent trait for examining the repeatability of evolution. Here, we investigate the repeatability of the evolution of polymorphic variation in ventral plumage coloration in skuas (Stercorarius: Stercorariidae). In 2 species, arctic (S. parasiticus) and pomarine skuas (S. pomarinus), plumage polymorphism was previously shown to be associated with coding changes at the melanocortin-1 receptor (MC1R) locus. Here, we show that polymorphism in a third species, the south polar skua (S. maccormicki), is not associated with coding variation at MC1R or with variation at a Z-linked second candidate locus, tyrosinase-related protein 1 (TYRP1). Hence, convergent evolution of plumage polymorphisms in skuas is only partly repeatable at the level of the genetic locus involved. Interestingly, the pattern of repeatability in skuas is aligned not with phylogeny but with the nature of the phenotypic variation. In particular, south polar skuas show a strong sex bias to coloration that is absent in the other species, and it may be that this has a unique genetic architecture.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio).

BACKGROUND: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama. RESULTS: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes. CONCLUSIONS: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Multiple origins of melanism in two species of North American tree squirrel (Sciurus).

BACKGROUND: While our understanding of the genetic basis of convergent evolution has improved there are still many uncertainties. Here we investigate the repeated evolution of dark colouration (melanism) in eastern fox squirrels (Sciurus niger; hereafter "fox squirrels") and eastern gray squirrels (S. carolinensis; hereafter "gray squirrels"). RESULTS: We show that convergent evolution of melanism has arisen by independent genetic mechanisms in two populations of the fox squirrel. In a western population, melanism is associated with a 24 bp deletion in the melanocortin-1-receptor gene (MC1RΔ24 allele), whereas in a south-eastern population, melanism is associated with a point substitution in the agouti signalling protein gene causing a Gly121Cys mutation. The MC1R∆24 allele is also associated with melanism in gray squirrels, and, remarkably, all the MC1R∆24 haplotypes are identical in the two species. Evolutionary analyses show that the MC1R∆24 haplotype is more closely related to other MC1R haplotypes in the fox squirrel than in the gray squirrel. Modelling supports the possibility of gene flow between the two species. CONCLUSIONS: The presence of the MC1R∆24 allele and melanism in gray squirrels is likely due to introgression from fox squirrels, although we cannot completely rule out alternative hypotheses including introgression from gray squirrels to fox squirrels, or an ancestral polymorphism. Convergent melanism in these two species of tree squirrels has evolved by at least two and probably three different evolutionary routes.

Evolution of CYP2J19, a gene involved in colour vision and red coloration in birds: positive selection in the face of conservation and pleiotropy.

BACKGROUND: Exaggerated signals, such as brilliant colours, are usually assumed to evolve through antagonistic coevolution between senders and receivers, but the underlying genetic mechanisms are rarely known. Here we explore a recently identified "redness gene", CYP2J19, that is highly interesting in this context since it encodes a carotenoid-modifying enzyme (a C4 ketolase involved in both colour signalling and colour discrimination in the red (long wavelength) spectral region.) RESULTS: A single full-length CYP2J19 was retrieved from 43 species out of 70 avian genomes examined, representing all major avian clades. In addition, CYP2J19 sequences from 13 species of weaverbirds (Ploceidae), seven of which have red C4-ketocarotenoid coloration were analysed. Despite the conserved retinal function and pleiotropy of CYP2J19, analyses indicate that the gene has been positively selected throughout the radiation of birds, including sites within functional domains described in related CYP (cytochrome P450) loci. Analyses of eight further CYP loci across 25 species show that positive selection is common in this gene family in birds. There was no evidence for a change in selection pressure on CYP2J19 following co-option for red coloration in the weaverbirds. CONCLUSIONS: The results presented here are consistent with an ancestral conserved function of CYP2J19 in the pigmentation of red retinal oil droplets used for colour vision, and its subsequent co-option for red integumentary coloration. The cause of positive selection on CYP2J19 is unclear, but may be partly related to compensatory mutations related to selection at the adjacent gene CYP2J40.

Colouration Genetics: Pretty Polymorphic Parrots.

The bright yellow, green and red feathers of parrots depend on unique pigments termed 'psittacofulvins'. The discovery of a gene underlying psittacofulvin colouration shows that this evolutionary innovation was achieved by co-opting an existing gene into feather development.

Expression of a carotenoid-modifying gene and evolution of red coloration in weaverbirds (Ploceidae).

Red carotenoid colours in birds are widely assumed to be sexually selected quality indicators, but this rests on a very incomplete understanding of genetic mechanisms and honesty-mediating costs. Recent progress was made by the implication of the gene CYP2J19 as an avian carotenoid ketolase, catalysing the synthesis of red C4-ketocarotenoids from yellow dietary precursors, and potentially a major mechanism behind red coloration in birds. Here, we investigate the role of CYP2J19 in the spectacular colour diversification of African weaverbirds (Ploceidae), represented by five genera and 16 species: eight red, seven yellow and one without carotenoid coloration. All species had a single copy of CYP2J19, unlike the duplication found in the zebra finch, with high expression in the retina, confirming its function in colouring red oil droplets. Expression was weak or undetected in skin and follicles of pigment-depositing feather buds, as well as in beaks and tarsi, including those of the red-billed quelea. In contrast, the hepatic (liver) expression of CYP2J19 was consistently higher (>14-fold) in seven species with C4-ketocarotenoid coloration than in species without (including one red species), an association strongly supported by a phylogenetic comparative analysis. The results suggest a critical role of the candidate ketolase, CYP2J19, in the evolution of red C4-ketocarotenoid colour variation in ploceids. As ancestral state reconstruction suggests that ketocarotenoid coloration has evolved twice in this group (once in Euplectes and once in the Quelea/Foudia clade), we argue that while CYP2J19 has retained its ancestral role in the retina, it has likely been co-opted for red coloration independently in the two lineages, via increased hepatic expression.

The genetic basis and enigmatic origin of melanic polymorphism in pomarine skuas (Stercorarius pomarinus).

A key outstanding issue in adaptive evolution is the relationship between the genetics of intraspecific polymorphism and interspecific evolution. Here, we show that the pale/dark ventral plumage polymorphism that occurs in both the pomarine skua (Stercorarius pomarinus) and Arctic skua (S. parasiticus) is the result of convergent evolution at the same locus (MC1R), involving some of the same amino acid sites. The dark melanic MC1R allele in the pomarine skua is strongly divergent from the pale MC1R alleles. Whereas the dark allele is closely related to MC1R alleles in three species of great skua (S. skua, S. maccormicki, S. lonnbergi), the pale pomarine skua MC1R alleles present a star-like pattern in an intermediate position on the haplotype network, closer to alleles of the long-tailed skua (S. longicaudus). Variation at other nuclear loci confirms a close relationship between the pomarine skua and the great skuas. The plumage polymorphism in pomarine skuas might have arisen in the common ancestor of pomarine and great skuas, only being retained in pomarine skuas. Alternatively, the pale and melanic MC1R alleles may have evolved independently in different lineages and been brought together in pomarine skuas by hybridization. In this case, introgression of a pale MC1R allele into the pomarine skua from another skua lineage is most likely. Our current data do not permit us to distinguish between these hypotheses, and assaying genome-wide variation holds much promise in this regard. Nevertheless, we have uncovered an intriguing example of a functionally important allele within one species that is shared across species.

Evidence of a Conserved Molecular Response to Selection for Increased Brain Size in Primates.

The adaptive significance of human brain evolution has been frequently studied through comparisons with other primates. However, the evolution of increased brain size is not restricted to the human lineage but is a general characteristic of primate evolution. Whether or not these independent episodes of increased brain size share a common genetic basis is unclear. We sequenced and de novo assembled the transcriptome from the neocortical tissue of the most highly encephalized nonhuman primate, the tufted capuchin monkey (Cebus apella). Using this novel data set, we conducted a genome-wide analysis of orthologous brain-expressed protein coding genes to identify evidence of conserved gene-phenotype associations and species-specific adaptations during three independent episodes of brain size increase. We identify a greater number of genes associated with either total brain mass or relative brain size across these six species than show species-specific accelerated rates of evolution in individual large-brained lineages. We test the robustness of these associations in an expanded data set of 13 species, through permutation tests and by analyzing how genome-wide patterns of substitution co-vary with brain size. Many of the genes targeted by selection during brain expansion have glutamatergic functions or roles in cell cycle dynamics. We also identify accelerated evolution in a number of individual capuchin genes whose human orthologs are associated with human neuropsychiatric disorders. These findings demonstrate the value of phenotypically informed genome analyses, and suggest at least some aspects of human brain evolution have occurred through conserved gene-phenotype associations. Understanding these commonalities is essential for distinguishing human-specific selection events from general trends in brain evolution.

Novel opsin gene variation in large-bodied, diurnal lemurs.

Some primate populations include both trichromatic and dichromatic (red-green colour blind) individuals due to allelic variation at the X-linked opsin locus. This polymorphic trichromacy is well described in day-active New World monkeys. Less is known about colour vision in Malagasy lemurs, but, unlike New World monkeys, only some day-active lemurs are polymorphic, while others are dichromatic. The evolutionary pressures underlying these differences in lemurs are unknown, but aspects of species ecology, including variation in activity pattern, are hypothesized to play a role. Limited data on X-linked opsin variation in lemurs make such hypotheses difficult to evaluate. We provide the first detailed examination of X-linked opsin variation across a lemur clade (Indriidae). We sequenced the X-linked opsin in the most strictly diurnal and largest extant lemur, Indri indri, and nine species of smaller, generally diurnal indriids (Propithecus). Although nocturnal Avahi (sister taxon to Propithecus) lacks a polymorphism, at least eight species of diurnal indriids have two or more X-linked opsin alleles. Four rainforest-living taxa-I. indri and the three largest Propithecus species-have alleles not previously documented in lemurs. Moreover, we identified at least three opsin alleles in Indri with peak spectral sensitivities similar to some New World monkeys.

The differential expression of MC1R regulators in dorsal and ventral quail plumages during embryogenesis: Implications for plumage pattern formation.

Melanin pigmentation patterns are ubiquitous in animals and function in crypsis, physical protection, thermoregulation and signalling. In vertebrates, pigmentation patterns formed over large body regions as well as within appendages (hair/feathers) may be due to the differential distribution of pigment producing cells (melanocytes) and/or regulation of the melanin synthesis pathway. We took advantage of the pigmentation patterns of Japanese quail embryos (pale ventrum and patterned feathers dorsally) to explore the role of genes and their transcripts in regulating the function of the melanocortin-1-receptor (MC1R) via 1. activation: pro-opiomelanocortin (POMC), endoproteases prohormone convertase 1 (PC1) and 2 (PC2), and 2. inhibition-agouti signaling and agouti-related protein (ASIP and AGRP, respectively). Melanocytes are present in all feather follicles at both 8 and 12 days post-fertilisation (E8/E12), so differential deposition of melanocytes is not responsible for pigmentation patterns in embryonic quail. POMC transcripts expressed were a subset of those found in chicken and POMC expression within feather follicles was strong. PC1 was not expressed in feather follicles. PC2 was strongly expressed in all feather follicles at E12. ASIP transcript expression was variable and we report four novel ASIP transcripts. ASIP is strongly expressed in ventral feather follicles, but not dorsally. AGRP expression within feather follicles was weak. These results demonstrate that the pale-bellied quail phenotype probably involves inhibition of MC1R, as found previously. However, quail may require MC1R activation for eumelanogenesis in dorsal feathers which may have important implications for an understanding of colour pattern formation in vertebrates.

Characterisation of urinary WFDC12 in small nocturnal basal primates, mouse lemurs (Microcebus spp.).

Mouse lemurs are basal primates that rely on chemo- and acoustic signalling for social interactions in their dispersed social systems. We examined the urinary protein content of two mouse lemurs species, within and outside the breeding season, to assess candidates used in species discrimination, reproductive or competitive communication. Urine from Microcebus murinus and Microcebus lehilahytsara contain a predominant 10 kDa protein, expressed in both species by some, but not all, males during the breeding season, but at very low levels by females. Mass spectrometry of the intact proteins confirmed the protein mass and revealed a 30 Da mass difference between proteins from the two species. Tandem mass spectrometry after digestion with three proteases and sequencing de novo defined the complete protein sequence and located an Ala/Thr difference between the two species that explained the 30 Da mass difference. The protein (mature form: 87 amino acids) is an atypical member of the whey acidic protein family (WFDC12). Seasonal excretion of this protein, species difference and male-specific expression during the breeding season suggest that it may have a function in intra- and/or intersexual chemical signalling in the context of reproduction, and could be a cue for sexual selection and species recognition.

Population genetics of mouse lemur vomeronasal receptors: current versus past selection and demographic inference.

BACKGROUND: A major effort is underway to use population genetic approaches to identify loci involved in adaptation. One issue that has so far received limited attention is whether loci that show a phylogenetic signal of positive selection in the past also show evidence of ongoing positive selection at the population level. We address this issue using vomeronasal receptors (VRs), a diverse gene family in mammals involved in intraspecific communication and predator detection. In mouse lemurs, we previously demonstrated that both subfamilies of VRs (V1Rs and V2Rs) show a strong signal of directional selection in interspecific analyses. We predicted that ongoing sexual selection and/or co-evolution with predators may lead to current directional or balancing selection on VRs. Here, we re-sequence 17 VRs and perform a suite of selection and demographic analyses in sympatric populations of two species of mouse lemurs (Microcebus murinus and M. ravelobensis) in northwestern Madagascar. RESULTS: M. ravelobensis had consistently higher genetic diversity at VRs than M. murinus. In general, we find little evidence for positive selection, with most loci evolving under purifying selection and one locus even showing evidence of functional loss in M. ravelobensis. However, a few loci in M. ravelobensis show potential evidence of positive selection. Using mismatch distributions and expansion models, we infer a more recent colonisation of the habitat by M. murinus than by M. ravelobensis, which most likely speciated in this region earlier on. CONCLUSIONS: These findings suggest that the analysis of VR variation is useful in inferring demographic and phylogeographic history of mouse lemurs. In conclusion, this study reveals a substantial heterogeneity over time in selection on VR loci, suggesting that VR evolution is episodic.

Population genomics fits the bill: genetics of adaptive beak variation in Darwin's finches.

Darwin's finches are an iconic case of adaptive radiation. The size and shape of their beaks are key adaptive traits related to trophic niche that vary among species and evolve rapidly when the food supply changes. Building on recent studies, a paper in this issue of Molecular Ecology (Chaves et al. ) investigates the genomic basis of beak size variation in sympatric populations of three species of ground finch (Geospiza) by performing a Genome-wide association study using RAD-seq data. The authors find that variation in a small number of markers can explain a substantial proportion of variation in beak size. Some of these markers are in genomic regions that have previously been implicated in beak size variation in Darwin's finches, whereas other markers have not, suggesting both conservation and divergence in the genetic basis of morphological evolution. Overall, the study confirms that loci of large effect are involved in beak size variation, which helps to explain the high heritability and rapid response to selection of this trait. The independent identification of regions containing HMGA2 and DLK1 loci in a GWAS makes them prime targets for functional studies. The study also shows that under the right conditions, RAD-seq can be a viable alternative to genome sequencing for GWAS in wild vertebrate populations.

Evolutionary pathways to convergence in plumage patterns.

BACKGROUND: Avian plumage is ideal for investigating phenotypic convergence because of repeated evolution of the same within-feather patterns. In birds, there are three major types of regular patterns within feathers: scales, bars and spots. Existing models of within-feather pattern development suggest that scales have the simplest developmental mechanism, bars require more stringent regulation than scales, and spots have the strictest developmental parameters. We hypothesized that increasing stringency in the mechanism of pattern formation predicts the evolutionary trajectory of patterns, and hence scales should evolve first, followed by bars and finally spots. Here, using Bayesian phylogenetic modeling we reconstructed pattern evolution in the most spectacularly patterned avian clades - aquatic waterfowl (Anseriformes) and terrestrial gamebirds (Galliformes). RESULTS: Our analyses suggest that the ancestral state of plumage is an absence of patterns, but with some variability. Independent analyses of seven feather patches reveal that spots evolve after bars and scales. However, both scales and bars evolve frequently from an absence of patterns, contradicting our predictions. Over the whole body, many constraints are conserved from the level of patches, for example the largest number of steps from the ancestral state was required for spots to evolve. CONCLUSIONS: Overall there was remarkable similarity in the inferred evolutionary trajectories of plumage pattern evolution in Galliformes and Anseriformes, suggesting that developmental constraint is similar in these two orders, despite large ecological differences. These evolutionary transitions are largely congruent with a reaction-diffusion based model of pattern formation, but the evolution of bars from an unpatterned ancestor is more common than expected. Our study highlights the promise of testing models of development using comparative methods.

Brain evolution and development: adaptation, allometry and constraint.

Phenotypic traits are products of two processes: evolution and development. But how do these processes combine to produce integrated phenotypes? Comparative studies identify consistent patterns of covariation, or allometries, between brain and body size, and between brain components, indicating the presence of significant constraints limiting independent evolution of separate parts. These constraints are poorly understood, but in principle could be either developmental or functional. The developmental constraints hypothesis suggests that individual components (brain and body size, or individual brain components) tend to evolve together because natural selection operates on relatively simple developmental mechanisms that affect the growth of all parts in a concerted manner. The functional constraints hypothesis suggests that correlated change reflects the action of selection on distributed functional systems connecting the different sub-components, predicting more complex patterns of mosaic change at the level of the functional systems and more complex genetic and developmental mechanisms. These hypotheses are not mutually exclusive but make different predictions. We review recent genetic and neurodevelopmental evidence, concluding that functional rather than developmental constraints are the main cause of the observed patterns.

Seeing red to being red: conserved genetic mechanism for red cone oil droplets and co-option for red coloration in birds and turtles.

Avian ketocarotenoid pigments occur in both the red retinal oil droplets that contribute to colour vision and bright red coloration used in signalling. Turtles are the only other tetrapods with red retinal oil droplets, and some also display red carotenoid-based coloration. Recently, the CYP2J19 gene was strongly implicated in ketocarotenoid synthesis in birds. Here, we investigate CYP2J19 evolution in relation to colour vision and red coloration in reptiles using genomic and expression data. We show that turtles, but not crocodiles or lepidosaurs, possess a CYP2J19 orthologue, which arose via gene duplication before turtles and archosaurs split, and which is strongly and specifically expressed in the ketocarotenoid-containing retina and red integument. We infer that CYP2J19 initially functioned in colour vision in archelosaurs and conclude that red ketocarotenoid-based coloration evolved independently in birds and turtles via gene regulatory changes of CYP2J19 Our results suggest that red oil droplets contributed to colour vision in dinosaurs and pterosaurs.

Potential arms race in the coevolution of primates and angiosperms: brazzein sweet proteins and gorilla taste receptors.

OBJECTIVES: We explored whether variation in the sweet taste receptor protein T1R3 in primates could contribute to differences in sweet taste repertoire among species, potentially reflecting coevolution with local plants. Specifically, we examined which primates are likely to be sweet "tasters" of brazzein, a protein found in the fruit of the African plant Pentadiplandra brazzeana that tastes intensely sweet to humans, but provides little energy. Sweet proteins like brazzein are thought to mimic the taste of sugars to entice seed dispersers. We examined the evolution of T1R3 and assessed whether primates are likely "deceived" by such biochemical mimicry. METHODS: Using published and new sequence data for TAS1R3, we characterized 57 primates and other mammals at the two amino acid sites necessary to taste brazzein to determine which species are tasters. We further used dN/dS-based methods to look for statistical evidence of accelerated evolution in this protein across primate lineages. RESULTS: The taster genotype is shared across most catarrhines, suggesting that most African primates can be "tricked" into eating and dispersing P. brazzeana's seeds for little caloric gain. Western gorillas (Gorilla gorilla), however, exhibit derived mutations at the two brazzein-critical positions, and although fruit is a substantial portion of the western gorilla diet, they have not been observed to eat P. brazzeana. Our analyses of protein evolution found no signature of positive selection on TAS1R3 along the gorilla lineage. DISCUSSION: We propose that the gorilla-specific mutations at the TAS1R3 locus encoding T1R3 could be a counter-adaptation to the false sweet signal of brazzein.

Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.