The molecular genetics of clinal variation: a case study of ebony and thoracic trident pigmentation in Drosophila melanogaster from eastern Australia

Widespread pigmentation diversity coupled with a well‐defined genetic system of melanin synthesis and patterning in Drosophila provides an excellent opportunity to study phenotypes undergoing evolutionary change. Pigmentation variation is highly correlated with different ecological variables and is thought to reflect adaptations to different environments. Several studies have linked candidate genes from Drosophila melanogaster to intra‐population variation and interspecific morphological divergence, but less clearly to variation among populations forming pigmentation clines. We characterized a new thoracic trident pigmentation cline in D. melanogaster populations from eastern Australia, and applied a candidate gene approach to explain the majority of the geographically structured phenotypic variation. More melanized populations from higher latitudes tended to express less ebony than their tropical counterparts, and an independent artificial selection experiment confirmed this association. By partitioning temperature dependent effects, we showed that the genetic differences underlying clinal patterns for trident variation at 25 °C do not explain the patterns observed at 16 °C. Changes in thoracic trident pigmentation could be a common evolutionary response to climatically mediated environmental pressures. On the Australian east coast most of the changes appear to be associated with regulatory divergence of the ebony gene but this depends on temperature.     

Redundancy analysis,genome-wide association studies and the pigmentation of brown trout (Salmo trutta L.)

The association of molecular variants with phenotypic variation is a main issue in biology, often tackled with genome-wide association studies (GWAS). GWAS are challenging, with increasing, but still limited, use in evolutionary biology. We used redundancy analysis (RDA) as a complimentary ordination approach to single- and multitrait GWAS to explore the molecular basis of pigmentation variation in brown trout (Salmo trutta) belonging to wild populations impacted by hatchery fish. Based on 75,684 single nucleotide polymorphic (SNP) markers, RDA, single- and multitrait GWAS allowed the extraction of 337 independent colour patterning loci (CPLs) associated with trout pigmentation traits, such as the number of red and black spots on flanks. Collectively, these CPLs (i) mapped onto 35 out of 40 brown trout linkage groups indicating a polygenic genomic architecture of pigmentation, (ii) were found to be associated with 218 candidate genes, including 197 genes formerly mentioned in the literature associated to skin pigmentation, skin patterning, differentiation or structure notably in a close relative, the rainbow trout (Onchorhynchus mykiss), and (iii) related to functions relevant to pigmentation variation (e.g., calcium- and ion-binding, cell adhesion). Annotated CPLs include genes with well-known pigmentation effects (e.g., PMEL, SLC45A2, SOX10), but also markers associated with genes formerly found expressed in rainbow or brown trout skins. RDA was also shown to be useful to investigate management issues, especially the dynamics of trout pigmentation submitted to several generations of hatchery introgression.     

Genetic architecture,biochemical underpinnings and ecological impact of floral UV patterning

Floral attraction traits can significantly affect pollinator visitation patterns, but adaptive evolution of these traits may be constrained by correlations with other traits. In some cases, molecular pathways contributing to floral attraction are well characterized, offering the opportunity to explore loci potentially underlying variation among individuals. Here, we quantify the range of variation in floral UV patterning (i.e. UV ‘bulls‐eye nectar guides) among crop and wild accessions of Brassica rapa. We then use experimental crosses to examine the genetic architecture, candidate loci and biochemical underpinnings of this patterning as well as phenotypic manipulations to test the ecological impact. We find qualitative variation in UV patterning between wild (commonly lacking UV patterns) and crop (commonly exhibiting UV patterns) accessions. Similar to the majority of crops, recombinant inbred lines (RILs) derived from an oilseed crop × WI fast‐plant^® cross exhibit UV patterns, the size of which varies extensively among genotypes. In RILs, we further observe strong statistical‐genetic and QTL correlations within petal morphological traits and within measurements of petal UV patterning; however, correlations between morphology and UV patterning are weak or nonsignificant, suggesting that UV patterning is regulated and may evolve independently of overall petal size. HPLC analyses reveal a high concentration of sinapoyl glucose in UV‐absorbing petal regions, which, in concert with physical locations of UV‐trait QTLs, suggest a regulatory and structural gene as candidates underlying observed quantitative variation. Finally, insects prefer flowers with UV bulls‐eye patterns over those that lack patterns, validating the importance of UV patterning in pollen‐limited populations of B. rapa.     

Identifying genes underlying skin pigmentation differences among human populations

Skin pigmentation is a human phenotype that varies greatly among human populations and it has long been speculated that this variation is adaptive. We therefore expect the genes that contribute to these large differences in phenotype to show large allele frequency differences among populations and to possibly harbor signatures of positive selection. To identify the loci that likely contribute to among-population human skin pigmentation differences, we measured allele frequency differentiation among Europeans, Chinese and Africans for 24 human pigmentation genes from 2 publicly available, large scale SNP data sets. Several skin pigmentation genes show unusually large allele frequency differences among these populations. To determine whether these allele frequency differences might be due to selection, we employed a within-population test based on long-range haplotype structure and identified several outliers that have not been previously identified as putatively adaptive. Most notably, we identify the DCT gene as a candidate for recent positive selection in the Chinese. Moreover, our analyses suggest that it is likely that different genes are responsible for the lighter skin pigmentation found in different non-African populations. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The evolution of teleost pigmentation and the fish‐specific genome duplication

Teleost fishes have evolved a unique complexity and diversity of pigmentation and colour patterning that is unmatched among vertebrates. Teleost colouration is mediated by five different major types of neural‐crest derived pigment cells, while tetrapods have a smaller repertoire of such chromatophores. The genetic basis of teleost colouration has been mainly uncovered by the cloning of pigmentation genes in mutants of zebrafish Danio rerio and medaka Oryzias latipes. Many of these teleost pigmentation genes were already known as key players in mammalian pigmentation, suggesting partial conservation of the corresponding developmental programme among vertebrates. Strikingly, teleost fishes have additional copies of many pigmentation genes compared with tetrapods, mainly as a result of a whole‐genome duplication that occurred 320–350 million years ago at the base of the teleost lineage, the so‐called fish‐specific genome duplication. Furthermore, teleosts have retained several duplicated pigmentation genes from earlier rounds of genome duplication in the vertebrate lineage, which were lost in other vertebrate groups. It was hypothesized that divergent evolution of such duplicated genes may have played an important role in pigmentation diversity and complexity in teleost fishes, which therefore not only provide important insights into the evolution of the vertebrate pigmentary system but also allow us to study the significance of genome duplications for vertebrate biodiversity.     

Morphological organization of abdominal colour patterns in pyrrhocorid bugs (Hemiptera-Heteroptera: Pentatomomorpha)

Postembryonic development of abdominal colour patterns, both epidermal pigmentation and cuticular melanization, of two model species, Pynhocoris apterus and Dysdercus cingulatus (Heteroptera: Pyrrhocoridae) is analysed with the aim of revealing morphological regularities involved in colour-pattern organization. This analysis is supplemented with a comparative study of diversity of colour patterns among 90 species of the Pyrrhocoridae. Comparison of both these approaches suggests that epidermal and cuticular patterns are ontogenetically independent of each other; that the ventral cuticular melanization is paired and respects boundaries delineated by epidermal pigmentation; that the dorsal cuticular melanization is unpaired and does not respect epidermal-colour boundaries; that the adult cuticular melanization develops almost independently of the larval one; and that the anterior and posterior regions of different segments are developmentally (and also evolutionarily) more tightly correlated than anterior and posterior parts of the same segment. These regularities are then compared with data concerning intrasegmental patterning of Drosophila and other insects.     

Wing pigmentation in Calopteryx damselflies: a role in thermoregulation?

Body melanization may show adaptive variation related to thermoregulation ability, and it is to be expected that the degree of melanization will change among populations or closely related species across environmental gradients of solar radiation and/or environmental temperature. Some melanized secondary sexual traits may also play a role in sexual selection, leading to interpopulation variation, which would not be predicted by thermoregulation pressures alone. We studied the relationships between the interpopulation variation in wing pigmentation level (i.e. melanized secondary sexual trait) of two closely related species of Calopteryx damselfly, and both solar radiation and maximum environmental temperature estimates. Wing pigmentation differs between these species, is gender specific and is used in species' discrimination. Only Calopteryx virgo meridionalis males showed a significant negative partial correlation between wing pigmentation degree and temperature. However, C. virgo meridionalis females showed a positive significant partial correlation between wing pigmentation degree and solar radiation. Wing pigmentation in Calopteryx xanthostoma males was not related to solar radiation or temperature. Thus, thermoregulation pressures poorly explained the observed variations in wing pigmentation between populations, although they might have an adaptive significance at the species' level. As wing pigmentation showed important latitudinal variation, several other selection pressures which might act on melanized traits are briefly discussed. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 36–44.     

COARSE DARK PATTERNING FUNCTIONALLY CONSTRAINS ADAPTIVE SHIFTS FROM APOSEMATISM TO CRYPSIS IN STRAWBERRY POISON FROGS

Ecological specialization often requires tight coevolution of several traits, which may constrain future evolutionary pathways and make species more prone to extinction. Aposematism and crypsis represent two specialized adaptations to avoid predation. We tested whether the combined effects of color and pattern on prey conspicuousness functionally constrain or facilitate shifts between these two adaptations. We combined data from 17 natural populations of strawberry poison frogs, Oophaga pumilio with an experimental approach using digitalized images of frogs and chickens as predators. We show that bright coloration often co‐occurs with coarse patterning among the natural populations. Dull green frogs with coarse patterning are rare in nature but in the experiment they were as easily detected as bright red frogs suggesting that this trait combination represents a transient evolutionary state toward aposematism. Hence, a gain of either bright color or coarse patterning leads to conspicuousness, but a transition back to crypsis would be functionally constrained in populations with both bright color and coarse patterning by requiring simultaneous changes in two traits. Thus, populations (or species) signaling aposematism by conspicuous color should be less likely to face an evolutionary dead end and more likely to radiate than populations with both conspicuous color and coarse patterning.     

Genetic basis of continuous variation in the levels and modular inheritance of pigmentation in cichlid fishes

Variation in pigmentation type and levels is a hallmark of myriad evolutionary radiations, and biologists have long been fascinated by the factors that promote and maintain variation in coloration across populations. Here, we provide insights into the genetic basis of complex and continuous patterns of colour variation in cichlid fishes, which offer a vast diversity of pigmentation patterns that have evolved in response to both natural and sexual selection. Specifically, we crossed two divergent cichlid species to generate an F₂ mapping population that exhibited extensive variation in pigmentation levels and patterns. Our experimental design is robust in that it combines traditional quantitative trait locus (QTL) analysis with population genomics, which has allowed us to move efficiently from QTL interval to candidate gene. In total, we detected 41 QTL and 13 epistatic interactions that underlie melanocyte‐ and xanthophore‐based coloration across the fins and flanks of these fishes. We also identified 2 QTL and 1 interaction for variation in the magnitude of integration among these colour traits. This finding in particular is notable as there are marked differences both within and between species with respect to the complexity of pigmentation patterns. While certain individuals are characterized by more uniform ‘integrated’ colour patterns, others exhibit many more degrees of freedom with respect to the distribution of colour ‘modules’ across the fins and flank. Our data reveal, for the first time, a genetic basis for this difference. Finally, we implicate pax3a as a mediator of continuous variation in the levels of xanthophore‐based colour along the cichlid flank.     

Primers for the amplification of nuclear introns in Heliconius butterflies

I described the development of degenerate polymerase chain reaction (PCR) primers for intron‐containing regions of nine candidate wing patterning and pigmentation genes in Heliconius butterflies. Primers were developed by comparing sequence data from Drosophila melanogaster, Precis coenia, and a variety of other insects so they are likely to be applicable widely among the butterfly family Nymphalidae and perhaps Lepidoptera in general. The amplified regions are highly variable and should be useful for inferring relationships among closely related species and estimating the phylogeographical and population genetic structure of individual species.     

Genetic diversity in Australian ancient asexual <Emphasis Type="Italic">Vestalenula</Emphasis> (Ostracoda,Darwinulidae): little variability down under

Darwinulid ostracods are putative ancient asexuals, and are thus assumed to be unable to purge deleterious mutations from their genomes. Some darwinulids species can be found both above (epigeic) and below ground (hypogeic). We hypothesize that surface populations carry more mutations than their below-ground counterparts, which are buffered from mutagens such as UV-B. Given the age of the investigated area, the Pilbara in Western Australia, we also expect geographic patterning of observed haplotypes. We have used DNA sequence data from the nuclear ITS and the mitochondrial COI region to investigate a (limited) data set on two Australian species, the endemic Vestalenula matildae and V. marmonieri from the Pilbara region. We do not find differences in genetic variability between specimens from subterranean habitats as compared to those from habitats above ground. There was also no congruence between hydrological basins and distribution patterns of the haplotypes identified. Although our data indicate that the two species may have split from each other ca. 70 myr ago, this has not resulted in any clear phylogeographic patterns among the analysed specimens across the regions of the Pilbara.     

Abdominal pigmentation in Drosophila melanogaster females from natural Indian populations

Females of Drosophila melanogaster collected from five geographically distant populations in India were analysed for the intensity of pigmentation in the 5th, 6th and 7th segments of the abdomen. In all three segments, this intensity was found to vary among individuals of any given population, and, furthermore, different populations differ with respect to this phenotypic trait. Statistical analysis revealed significant intra- and interpopulational variation. A clinical pattern was detected: females from populations closer to the equator tended to have lighter cuticle, in which case differences between the three segments could not be detected and all three segments responded both independently and jointly to latitudinal variation, as indicated by a statistically significant positive correlation between latitude and pigmentation score. This is the first report on abdominal pigmentation analysis in natural populations of D. melanogaster that provides evidence that phenotypic flexibility reflects temperature differences, as a result of which abdominal pigmentation shows geographic differentiation.     

Quantitative morphometrical analysis of a North African population of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: sexual dimorphism,and comparison with European populations

Genetic variability of quantitative traits was investigated in aMoroccan population of Drosophila melanogaster, with an isofemale line design. Results were compared with data previously obtained from French populations. Although the environmental and thermal conditions are very different in France and Morocco, only two significant differences were observed: a shorter wing and a lighter abdomen pigmentation in Morocco. It is, therefore, concluded that Moroccan D. melanogaster are quite typical temperate flies, belonging to the Palaearctic region, and very different from the ancestral Afrotropical populations. Almost all traits were genetically variable, as shown by significant intraclass correlations among lines. Genetic correlations were highly significant among three size-related traits, while much lower between size and bristle numbers. Fluctuating asymmetry was greater for abdominal bristles than for sternopleural bristles. Sex dimorphism, analysed as a female/male ratio, was identical in French and Moroccan populations. Examination of the thorax length/thorax width ratio showed that the thorax is more elongated in females. Sexual dimorphism of wing length was significantly more correlated to thorax width than to thorax length. The results illustrate the value of measuring numerous quantitative traits on the same flies for characterizing the genetic architecture of a natural population. In several cases, and especially for genetic correlations, some interesting suggestions could be made, which should be confirmed, or invalidated, by more extensive investigations.     

Evolution of wing pigmentation in Drosophila: Diversity,physiological regulation,and cis-regulatory evolution

Fruit flies (Drosophila and its close relatives, or “drosophilids”) are a group that includes an important model organism, Drosophila melanogaster, and also very diverse species distributed worldwide. Many of these species have black or brown pigmentation patterns on their wings, and have been used as material for evo-devo research. Pigmentation patterns are thought to have evolved rapidly compared with body plans or body shapes; hence they are advantageous model systems for studying evolutionary gains of traits and parallel evolution. Various groups of drosophilids, including genus Idiomyia (Hawaiian Drosophila), have a variety of pigmentations, ranging from simple black pigmentations around crossveins to a single antero-distal spot and a more complex mottled pattern. Pigmentation patterns are sometimes obviously used for sexual displays; however, in some cases they may have other functions. The process of wing formation in Drosophila, the general mechanism of pigmentation formation, and the transport of substances necessary for pigmentation, including melanin precursors, through wing veins are summarized here. Lastly, the evolution of the expression of genes regulating pigmentation patterns, the role of cis-regulatory regions, and the conditions required for the evolutionary emergence of pigmentation patterns are discussed. Future prospects for research on the evolution of wing pigmentation pattern formation in drosophilids are presented, particularly from the point of view of how they compare with other studies of the evolution of new traits.     

Quantitative dissection of color patterning in the foliar ornamental coleus

Coleus (Coleus scutellarioides) is a popular ornamental plant that exhibits a diverse array of foliar color patterns. New cultivars are currently hand selected by both amateur and experienced plant breeders. In this study, we reimagine breeding for color patterning using a quantitative color analysis framework. Despite impressive advances in high-throughput data collection and processing, complex color patterns remain challenging to extract from image datasets. Using a phenotyping approach called “ColourQuant,” we extract and analyze pigmentation patterns from one of the largest coleus breeding populations in the world. Working with this massive dataset, we can analyze quantitative relationships between maternal plants and their progeny, identify features that underlie breeder-selections, and collect and compare public input on trait preferences. This study is one of the most comprehensive explorations into complex color patterning in plant biology and provides insights and tools for exploring the color pallet of the plant kingdom.

Quantitative analysis of color patterning in a large coleus breeding population reveals color features that are associated with aesthetic value.     

Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Protoporphyrin‐based eggshell pigmentation predicts hatching success and offspring sex ratio in the barn swallow

Inter‐ and intraspecific variation in eggshell colouration has long fascinated evolutionary biologists. Among species, such variation may accomplish different functions, the most obvious of which is camouflage and background matching. Within species, it has been proposed that inter‐female variation in eggshell pigmentation patterns can reflect egg, maternal or paternal traits and hence may provide cues to conspecifics about egg, maternal or paternal phenotypic quality. However, the relationship between protoporphyrin‐based eggshell pigmentation and egg or maternal/paternal traits appears to be highly variable among species. We investigated patterns of intraspecific variation in Eurasian barn swallow Hirundo r. rustica protoporphyrin‐based eggshell pigmentation, and analysed its association with egg and clutch characteristics, maternal/paternal phenotypic traits and parental feeding effort. Eggshell pigmentation pattern significantly varied between breeding colonies, was significantly repeatable in first clutches laid by the same females in different years (intraclass correlation coefficient ranging between 0.56 and 0.63), but it was not significantly associated with egg traits, such as position in the laying sequence, egg mass, yolk testosterone concentration and antioxidant capacity. It was weakly or non‐significantly associated with female and male traits (sexual ornaments), but females laying darker (higher pigment intensity) first clutches had higher hatching success, suggesting that eggshell pigment intensity may predict fitness. Male nestling feeding effort was not predicted by eggshell pigmentation. In addition, females with darker breast plumage colouration (a melanin‐based trait related to fitness) laid highly protoporphyrin‐covered eggs, suggesting the presence of a previously unappreciated link between protoporphyrin biosynthesis and plumage melanisation. Moreover, the proportion of male offspring increased in clutches originating from highly protoporphyrin‐covered eggs, suggesting that parents could acquire visual cues about their future brood sex composition before egg hatching. Our results support the idea that intraspecific signalling via eggshell pigmentation is a species‐specific rather than a general feature of avian taxa.     

Validation of photo‐identification as a mark–recapture method in the spotted eagle ray Aetobatus narinari

The spotted eagle ray Aetobatus narinari is characterized by pigmentation patterns that are retained for up to 3·5 years. These pigmentations can be used to identify individuals through photo‐identification. Only one study has validated this technique, but no study has estimated the percentage of correct identification of the rays using this technique. In order to carry out demographic research, a reliable photographic identification technique is needed. To achieve this validation for A. narinari, a double‐mark system was established over 11 months and photographs of the dorsal surface of 191 rays were taken. Three body parts with distinctive natural patterns were analysed (dorsal surface of the cephalic region, dorsal surface of the pectoral fins and dorsal surface of the pelvic fins) in order to determine the body part that could be used to give the highest percentage of correct identification. The dorsal surface of the pectoral fins of A. narinari provides the most accurate photo‐identification to distinguish individuals (88·2%).     

Population genetic structure of a rare unionid (<Emphasis Type="Italic">Lampsilis cariosa</Emphasis>) in a recently glaciated landscape

The yellow lampmussel (Lampsilis cariosa) is a rare unionid species in need of conservation, as it is declining throughout most of its Atlantic slope range in North America. Because freshwater mussels rely on a fish host for dispersal of their larvae, barriers to the movement of hosts, such as habitat fragmentation by dams, may indirectly affect population genetic structure. We used microsatellite loci to assess genetic variation for L. cariosa within and among three river drainages in the northern part of its range, which emerged from glaciation only ∼ ∼8–10 kya. Despite this relatively recent emergence, significant differences were observed among populations both within and among drainages, possibly because low effective population sizes meant that populations of these mussels achieved drift-migration equilibrium rapidly following glaciation. L. cariosa individuals could be assigned to their own drainages with 89.3% accuracy. Among-population differences were modest, however, in comparison to differences observed in another study of rare mussels south of the recently glaciated region. L. cariosa populations exhibited significant isolation by distance, but there was no additional variation explained by the number, size, or age of intervening dams. An understanding of mussel population genetic structure provides information, useful for conservation planning, on patterns of isolation and connectivity among populations.