Evolution of abdominal pigmentation differences across species in the Drosophila dunni subgroup

The Drosophila dunni subgroup displays a nearly perfect latitudinal cline in abdominal pigmentation that likely resulted from selective forces acting in the habitat of each species during speciation. Here we characterize the nature of this clinal variation by developing a quantitative measure to assess variation in abdominal pigmentation within and between the D. dunni subgroup species. Using discriminant analysis, we confirm the existence of a cline and find that our quantitative measure of pigmentation distinguishes each of the species with singular efficacy. We then combine our quantitative phenotypic analysis of pigmentation with the phylogeny of the D. dunni subgroup species and map the species relationships into the three-dimensional morphological space defined by our pigmentation measures. In this manner, we can visualize how the species have traversed the morphological pigmentation space during the course of speciation. Our analysis reveals that natural selection has caused overall intensity of pigmentation among the northernmost species of the cline to converge. Along with this convergence in phenotype has been a relaxation in expression of sexual dimorphism in these species, indicating a possible shift in the relative intensity of natural and sexual selection. Our analysis indicates an accelerated rate of change in pigmentation for the darkest species in addition to this species evolving a novel abdominal pigmentation trait.     

Two genomic regions together cause dark abdominal pigmentation in Drosophila tenebrosa

Pigmentation is a rapidly evolving trait that is under both natural and sexual selection in many organisms. In the quinaria group of Drosophila, nearly all of the 30 species have an abdomen that is light in color with distinct markings; D. tenebrosa is the exception in that it has a completely melanic abdomen with no visible markings. In this study, we use a combination of quantitative genetic and candidate gene approaches to investigate the genetic basis of abdominal pigmentation in D. tenebrosa. We find that abdominal pigmentation is invariant across wild-caught lines of D. tenebrosa and is not sexually dimorphic. Quantitative genetic mapping utilizing crosses between D. tenebrosa and the light-colored D. suboccidentalis indicates that two genomic regions together underlie abdominal pigmentation, including the X-chromosome and an autosome (Muller Element C/E). Further support for their central importance in pigmentation is that experimental introgression of one phenotype into the other species, in either direction, results in introgression of these two genomic regions. Finally, the expression of the X-linked gene yellow in the pupae exactly foreshadows the adult melanization pattern in the abdomen of both species, suggesting that changes in the regulation of yellow are important for the phenotypic divergence of D. tenebrosa from the rest of the quinaria group. These results contribute to a body of work that demonstrates how changes in expression of highly conserved genes can cause substantial phenotypic differences even between closely related species.     

Genetic basis of sex-specific color pattern variation in Drosophila malerkotliana

Pigmentation is a rapidly evolving trait that can play important roles in mimicry, sexual selection, thermoregulation, and other adaptive processes in many groups of animals. In Drosophila, pigmentation can differ dramatically among closely related taxa, presenting a good opportunity to dissect the genetic changes underlying species divergence. In this report, we investigate the genetic basis of color pattern variation between two allopatric subspecies of Drosophila malerkotliana, a widespread member of the ananassae species subgroup. In D. malerkotliana malerkotliana, the last three abdominal segments are darkly pigmented in males but not in females, while in D. malerkotliana pallens both sexes lack dark pigmentation. Composite interval mapping in F(2) hybrid progeny shows that this difference is largely controlled by three quantitative trait loci (QTL) located on the 2L chromosome arm, which is homologous to the 3R of D. melanogaster (Muller element E). Using highly recombinant introgression strains produced by repeated backcrossing and phenotypic selection, we show that these QTL do not correspond to any of the candidate genes known to be involved in pigment patterning and synthesis in Drosophila. These results, in combination with similar analyses in other Drosophila species, indicate that different genetic and molecular changes are responsible for the evolution of similar phenotypic traits in different lineages. This feature makes Drosophila color patterns a powerful model for investigating how the genetic basis of trait evolution is influenced by the intrinsic organization of regulatory pathways controlling the development of these traits.     

Tinker where the tinkering's good

Do general principles govern the genetic causes of phenotypic evolution? One promising idea is that mutations in cis-regulatory regions play a predominant role in phenotypic evolution because they can alter gene activity without causing pleiotropic effects. Recent evidence that revealed the genetic basis of pigmentation pattern evolution in Drosophila santomea supports this notion. Multiple mutations that disrupt an abdominal enhancer of the pleiotropic gene tan partly explain the reduced pigmentation observed in this species.     

Evolution of yellow gene regulation and pigmentation in Drosophila

BACKGROUND: Changes in developmental gene expression are central to phenotypic evolution, but the genetic mechanisms underlying these changes are not well understood. Interspecific differences in gene expression can arise from evolutionary changes in cis-regulatory DNA and/or in the expression of trans-acting regulatory proteins, but few case studies have distinguished between these mechanisms. Here, we compare the regulation of the yellow gene, which is required for melanization, among distantly related Drosophila species with different pigment patterns and determine the phenotypic effects of divergent Yellow expression. RESULTS: Yellow expression has diverged among D. melanogaster, D. subobscura, and D. virilis and, in all cases, correlates with the distribution of black melanin. Species-specific Yellow expression patterns were retained in D. melanogaster transformants carrying the D. subobscura and D. virilis yellow genes, indicating that sequence evolution within the yellow gene underlies the divergence of Yellow expression. Evolutionary changes in the activity of orthologous cis-regulatory elements are responsible for differences in abdominal Yellow expression; however, cis-regulatory element evolution is not the sole cause of divergent Yellow expression patterns. Transformation of the D. melanogaster yellow gene into D. virilis altered its expression pattern, indicating that trans-acting factors that regulate the D. melanogaster yellow gene have also diverged between these two species. Finally, we found that the phenotypic effects of evolutionary changes in Yellow expression depend on epistatic interactions with other genes. CONCLUSIONS: Evolutionary changes in Yellow expression correlate with divergent melanin patterns and are a result of evolution in both cis- and trans-regulation. These changes were likely necessary for the divergence of pigmentation, but evolutionary changes in other genes were also required.     

Quantitative trait loci responsible for variation in sexually dimorphic traits in Drosophila melanogaster

To understand the mechanisms of morphological evolution and species divergence, it is essential to elucidate the genetic basis of variation in natural populations. Sexually dimorphic characters, which evolve rapidly both within and among species, present attractive models for addressing these questions. In this report, we map quantitative trait loci (QTL) responsible for variation in sexually dimorphic traits (abdominal pigmentation and the number of ventral abdominal bristles and sex comb teeth) in a natural population of Drosophila melanogaster. To capture the pattern of genetic variation present in the wild, a panel of recombinant inbred lines was created from two heterozygous flies taken directly from nature. High-resolution mapping was made possible by cytological markers at the average density of one per 2 cM. We have used a new Bayesian algorithm that allows QTL mapping based on all markers simultaneously. With this approach, we were able to detect small-effect QTL that were not evident in single-marker analyses. Our results show that at least for some sexually dimorphic traits, a small number of QTL account for the majority of genetic variation. The three strongest QTL account for >60% of variation in the number of ventral abdominal bristles. Strikingly, a single QTL accounts for almost 60% of variation in female abdominal pigmentation. This QTL maps to the chromosomal region that Robertson et al. have found to affect female abdominal pigmentation in other populations of D. melanogaster. Using quantitative complementation tests, we demonstrate that this QTL is allelic to the bric a brac gene, whose expression has previously been shown to correlate with interspecific differences in pigmentation. Multiple bab alleles that confer distinct phenotypes appear to segregate in natural populations at appreciable frequencies, suggesting that intraspecific and interspecific variation in abdominal pigmentation may share a similar genetic basis.     

Abdominal pigmentation variation in drosophila polymorpha: geographic variation in the trait, and underlying phylogeography

Drosophila polymorpha is a widespread species that exhibits abdominal pigmentation variation throughout its range. To gain insight into this variation we combined phenotypic and genotypic data to test a series of nested hypotheses. First, we tested the null hypothesis that geographic variation in pigmentation is due to neutral factors. We used nested clade analysis to examine the distribution of haplotypes from a nuclear and a mitochrondrial locus. Restricted gene flow via isolation by distance, the primary inference of this phylogeographic analysis, was then used to generate and test the hypothesis of increasing average abdominal pigmentation difference with increasing geographic distance. We found no correlation between geographic distance and phenotypic distance. We then tested the hypothesis that pigmentation is affected by environmental differences among localities. We found a significant effect of habitat type on the average abdominal pigmentation phenotype of different localities. Finally, we tested the hypothesis that pigmentation in D. polymorpha is associated with desiccation resistance. We found that dark individuals of both sexes survived significantly longer in a desiccating environment than light individuals. These patterns combined lead us to hypothesize that abdominal pigmentation variation in D. polymorpha is important in mediating the organism's interactions with local ecological factors.     

Genetics of a difference in pigmentation between Drosophila yakuba and Drosophila santomea

Abstract.— Drosophila yakuba is a species widespread in Africa, whereas D. santomea, its newly discovered sister species, is endemic to the volcanic island of São Tomé in the Gulf of Guinea. Drosophila santomea probably formed after colonization of the island by its common ancestor with D. yakuba. The two species differ strikingly in pigmentation: D. santomea, unlike the other eight species in the D. melanogaster subgroup, almost completely lacks dark abdominal pigmentation. D. yakuba shows the sexually dimorphic pigmentation typical of the group: both sexes have melanic patterns on the abdomen, but males are much darker than females. A genetic analysis of this species difference using morphological markers shows that the X chromosome accounts for nearly 90% of the species difference in the area of abdomen that is pigmented and that at least three genes (one on each major chromosome) are involved in each sex. The order of chromosome effects on pigmentation area are the same in males and females, suggesting that loss of pigmentation in D. santomea may have involved the same genes in both sexes. Further genetic analysis of the interspecific difference between males in pigmentation area and intensity using molecular markers shows that at least five genes are responsible, with no single locus having an overwhelming effect on the trait. The species difference is thus oligogenic or polygenic. Different chromosomal regions from each of the two species influenced pigmentation in the same direction, suggesting that the species difference (at least in males) is due to natural or sexual selection and not genetic drift. Measurements of sexual isolation between the species in both light and dark conditions show no difference, suggesting that the pigmentation difference is not an important cue for interspecific mate discrimination. Using DNA sequence differences in nine noncoding regions, we estimate that D. santomea and D. yakuba diverged about 400,000 years ago, a time similar to the divergences between two other well‐studied pair of species in the subgroup, both of which also involved island colonization.     

The reaction norm for abdominal pigmentation and its curve in Drosophila mediopunctata depend on the mean phenotypic value

The idea of a general independence between the phenotypic plasticity and the mean value of a trait is, presently, a consensus. Here, we use the reaction norm of abdominal pigmentation (number of dark spots) of Drosophila mediopunctata in response to temperature, to test this idea. We raised eight strains, bearing two different chromosomal inversions and with varying mean phenotypic values, under 11 temperatures in a thermal gradient to test for predictions concerning mean phenotypic values, chromosomal inversions, and reaction norms. Our results revealed a strong effect of different phenotypic groups and no effect of different karyotypes on reaction norms. Moreover, we found a significant negative correlation between mean phenotypic value and the curvature of the reaction norms, revealing a high dependency of the reaction norm shape on mean phenotypic value. These results clearly reject the idea of genetic independence between mean value and phenotypic plasticity, and may indicate a pattern of correlation, which may include results from other traits and species, with an importance that has not been fully appreciated.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Phylogenetic analysis of the Cardini group of Drosophila with respect to changes in pigmentation

Phenotypic variability is the engine that drives future diversification with the expectation that polymorphic ancestors give rise to descendants harboring a subset of the ancestral variation. Here we examine evolutionary transitions from polymorphism to monomorphism in a visually striking New World radiation of fruit flies, the Drosophila cardini group. This group is distributed across the Americas and the Caribbean islands and exhibits a wide spectrum of abdominal pigmentation variation. Specifically, the D. dunni subgroup consists of Caribbean island endemics, each of which is monomorphic for its pigmentation pattern, with an interspecific cline of pigmentation across the islands. The D. cardini subgroup consists of American continental species with wide-ranging distributions and intraspecifically variable abdominal pigmentation. We determined the phylogeny of 18 species and subspecies using three nuclear and three mitochondrial regions analyzed with maximum parsimony, maximum likelihood, and Bayesian methods. The topology produced from a combined dataset exhibited high support values at all nodes, and differed from earlier phylogenetic hypotheses based on polytene chromosome inversion patterns and isozyme data. We find that the D. dunni subgroup species, with the exception of D. belladunni, are derived from a single source not of direct South American origin and their dispersal across the islands of the Caribbean does not follow a simple stepping-stone model. Morphological changes in pigmentation across the island species are incongruent with the colonization history of the group indicating that natural selection may have played a role in the determination of this character. Finally, we demonstrate that monomorphic species have arisen independently from polymorphic ancestors two to three times.     

Many ways to make darker flies: Intra‐ and interspecific variation in Drosophila body pigmentation components

Body pigmentation is an evolutionarily diversified and ecologically relevant trait with substantial variation within and between species, and important roles in animal survival and reproduction. Insect pigmentation, in particular, provides some of the most compelling examples of adaptive evolution, including its ecological significance and genetic bases. Pigmentation includes multiple aspects of color and color pattern that may vary more or less independently, and can be under different selective pressures. We decompose Drosophila thorax and abdominal pigmentation, a valuable eco‐evo‐devo model, into distinct measurable traits related to color and color pattern. We investigate intra‐ and interspecific variation for those traits and assess its different sources. For each body part, we measured overall darkness, as well as four other pigmentation properties distinguishing between background color and color of the darker pattern elements that decorate each body part. By focusing on two standard D. melanogaster laboratory populations, we show that pigmentation components vary and covary in distinct manners depending on sex, genetic background, and temperature during development. Studying three natural populations of D. melanogaster along a latitudinal cline and five other Drosophila species, we then show that evolution of lighter or darker bodies can be achieved by changing distinct component traits. Our results paint a much more complex picture of body pigmentation variation than previous studies could uncover, including patterns of sexual dimorphism, thermal plasticity, and interspecific diversity. These findings underscore the value of detailed quantitative phenotyping and analysis of different sources of variation for a better understanding of phenotypic variation and diversification, and the ecological pressures and genetic mechanisms underlying them.     

QTL analysis reveals different and independent modes of inheritance for diagnostic achene characters in Microseris (Asteraceae)

There are few reliable diagnostic morphological characters for species of the asteracean genus Microseris, and quantitative differences in the shapes of the achenes and the paleaceous pappus parts play a decisive role in species recognition. The genetic basis of species and strain differences in various characters has been studied previously, but little is known about quantitative characters of the achenes.

We performed a quantitative trait locus (QTL) analysis in the F2 of an interspecific cross between Microseris douglasii and M. bigelovii for achene length, achene diameter, achene shape, palea length, awn length, and achene pigmentation.

Independent inheritance of the main heritable achene characters – achene length, palea length and achene pigmentation – was revealed by the detection of distinct and specific QTLs for these characters. For palea length five QTLs with about equal phenotypic effects were mapped on four different linkage groups. Achene length and achene shape (achene length / achene diameter) were determined by two different genetic systems with one major gene and two modifiers. The detection of QTLs with a polarity of the effects opposite to that in the parental strains for achene length and achene shape reveals genetic variation for a potential increase in species differences. For the highly heritable trait, achene pigmentation, the bimodal F2 distribution suggested single-factor inheritance for absence versus presence of spots, with dominance for the spotted condition. However, only relatively weak QTL effects on that trait could be detected. Additional molecular markers (RAPDs, AFLPs) have to be tested for cosegregation with that major gene. The results are discussed in the context of different theories for the evolution of morphological characters.     

Quantitative trait loci affecting the difference in pigmentation between Drosophila yakuba and D. santomea

Using quantitative trait locus (QTL) mapping, we studied the genetic basis of the difference in pigmentation between two sister species of Drosophila: Drosophila yakuba, which, like other members of the D. melanogaster subgroup, shows heavy black pigmentation on the abdomen of males and females, and D. santomea, an endemic to the African island of S?o Tomé, which has virtually no pigmentation. Here we mapped four QTL with large effects on this interspecific difference in pigmentation: two on the X chromosome and one each on the second and third chromosomes. The same four QTL were detected in male hybrids in the backcrosses to both D. santomea and D. yakuba and in the female D. yakuba backcross hybrids. All four QTL exhibited strong epistatic interactions in male backcross hybrids, but only one pair of QTL interacted in females from the backcross to D. yabuka. All QTL from each species affected pigmentation in the same direction, consistent with adaptive evolution driven by directional natural selection. The regions delimited by the QTL included many positional candidate loci in the pigmentation pathway, including genes affecting catecholamine biosynthesis, melanization of the cuticle, and many additional pleiotropic effects.     

Pleiotropic effects of regulatory variation in tan result in correlation of two pigmentation traits in Drosophila melanogaster

Traits with a common genetic basis frequently display correlated phenotypic responses to selection or environmental conditions. In Drosophila melanogaster, pigmentation of the abdomen and a trident‐shaped region on the thorax are genetically correlated. Here, we used a pooled replicated genomewide association approach (Pool‐GWAS) to identify the genetic basis of variation in thoracic trident pigmentation in two Drosophila melanogaster populations. We confirmed the previously reported large effect of ebony and the association of the cosmopolitan inversion In(3R)Payne. For the first time, we identified tan as another major locus contributing to variation in trident pigmentation. Intriguingly, the regulatory variants of tan that were most strongly associated with female abdominal pigmentation also showed a strong association with trident pigmentation. We validated this common genetic basis in transgenic assays and found qualitatively similar effects on trident and abdominal pigmentation. Further work is required to determine whether this genetic correlation is favoured by natural selection or reflects a neutral by‐product of a shared regulatory architecture.     

The ontogeny of color: developmental origins of divergent pigmentation in Drosophila americana and D. novamexicana

Pigmentation is a model trait for evolutionary and developmental analysis that is particularly amenable to molecular investigation in the genus Drosophila. To better understand how this phenotype evolves, we examined divergent pigmentation and gene expression over developmental time in the dark‐bodied D. americana and its light‐bodied sister species D. novamexicana. Prior genetic analysis implicated two enzyme‐encoding genes, tan and ebony, in pigmentation divergence between these species, but questions remain about the underlying molecular mechanisms. Here, we describe stages of pupal development in both species and use this staging to determine when pigmentation develops and diverges between D. americana and D. novamexicana. For the developmental stages encompassing pigment divergence, we compare mRNA expression of tan and ebony over time and between species. Finally, we use allele‐specific expression assays to determine whether interspecific differences in mRNA abundance have a cis‐regulatory basis and find evidence of cis‐regulatory divergence for both tan and ebony. cis‐regulatory divergence affecting tan had a small effect on mRNA abundance and was limited to a few developmental stages, yet previous data suggests that this divergence is likely to be biologically meaningful. Our study suggests that small and developmentally transient expression changes may contribute to phenotypic diversification more often than commonly appreciated. Recognizing the potential phenotypic impact of such changes is important for a scientific community increasingly focused on dissecting quantitative variation, but detecting these types of changes will be a major challenge to elucidating the molecular basis of complex traits.     

Changes in micro RNA expression in a wild tuber-bearing Solanum species induced by 5-Azacytidine treatment

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species.