Maria D. S. Nunes

A Perspective on Micro-Evo-Devo: Progress and Potential

The term “micro-evo-devo” refers to the combined study of the genetic and developmental bases of natural variation in populations and the evolutionary forces that have shaped this variation. It thus represents a synthesis of the fields of evolutionary developmental biology and population genetics. As has been pointed out by several others, this synthesis can provide insights into the evolution of organismal form and function that have not been possible within these individual disciplines separately. Despite a number of important successes in micro-evo-devo, however, it appears that evo devo and population genetics remain largely separate spheres of research, limiting their ability to address evolutionary questions. This also risks pushing contemporary evo devo to the fringes of evolutionary biology because it does not describe the causative molecular changes underlying evolution or the evolutionary forces involved. Here we reemphasize the theoretical and practical importance of micro-evo-devo as a strategy for understanding phenotypic evolution, review the key recent insights that it has provided, and present a perspective on both the potential and the remaining challenges of this exciting interdisciplinary field.

Multiple hybridization events between Drosophila simulans and Drosophila mauritiana are supported by mtDNA introgression

The study of speciation has advanced considerably in the last decades because of the increased application of molecular tools. In particular, the quantification of gene flow between recently diverged species could be addressed. Drosophila simulans and Drosophila mauritiana diverged, probably allopatrically, from a common ancestor approximately 250 000 years ago. However, these species share one mitochondrial DNA (mtDNA) haplotype indicative of a recent episode of introgression. To study the extent of gene flow between these species, we took advantage of a large sample of D. mauritiana and employed a range of different markers, i.e. nuclear and mitochondrial sequences, and microsatellites. This allowed us to detect two new mtDNA haplotypes (MAU3 and MAU4). These haplotypes diverged quite recently from haplotypes of the siII group present in cosmopolitan populations of D. simulans. The mean divergence time of the most diverged haplotype (MAU4) is approximately 127 000 years, which is more than 100 000 years before the assumed speciation time. Interestingly, we also found some evidence for gene flow at the nuclear level because an excess of putatively neutral loci shows significantly reduced differentiation between D. simulans and D. mauritiana. Our results suggest that these species are exchanging genes more frequently than previously thought.

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele‐specific real‐time quantitative PCR (RT‐qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter‐selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.

Contrasting patterns of natural variation in global Drosophila melanogaster populations

Despite the popularity of Drosophila melanogaster in functional and evolutionary genetics, the global pattern of natural variation has not yet been comprehensively described in this species. For the first time, we report a combined survey using neutral microsatellites and mitochondrial sequence variation jointly. Thirty‐five populations originating from five continents were compared. In agreement with previous microsatellite studies, sub‐Saharan African populations were the most variable ones. Consistent with previous reports of a single ‘out of Africa’ habitat expansion, we found that non‐African populations contained a subset of the African alleles. The pattern of variation detected for the mitochondrial sequences differed substantially. The most divergent haplotypes were detected in the Mediterranean region while Africa harboured most haplotypes, which were all closely related. In the light of the well‐established African origin of D. melanogaster, our results cast severe doubts about the suitability of mtDNA for biogeographic inference in this model organism.

Nonrandom Wolbachia Infection Status of Drosophila melanogaster Strains with Different mtDNA Haplotypes

Wolbachia are maternally inherited bacteria, which typically spread in the host population by inducing cytoplasmic incompatibility (CI). In Drosophila melanogaster, Wolbachia is quite common but CI is variable, with most of the studies reporting low levels of CI. Surveying mitochondrial DNA (mtDNA) variation and infection status in a worldwide D. melanogaster collection, we found that the Wolbachia infection was not randomly distributed among flies with different mtDNA haplotypes. This preferential infection of some mtDNA haplotypes could be caused by a recent spread of mtDNA haplotypes associated with the infection. The comparison of contemporary D. melanogaster samples with lines collected more than 50 years ago shows that indeed one haplotype with a high incidence of Wolbachia infection has increased in frequency. Consistent with this observation, we found that the acquisition of a Wolbachia infection in a population from Crete was accompanied with an almost complete mtDNA replacement, with the Wolbachia-associated haplotype becoming abundant. Although it is difficult to identify the evolutionary forces causing the global increase of wMel, the parallel sweep of Wolbachia and an mtDNA haplotype suggests a fitness advantage of the Wolbachia infection.

Evolution of mir-92a Underlies Natural Morphological Variation in Drosophila melanogaster

Summary Identifying the genetic mechanisms underlying phenotypic change is essential to understanding how gene regulatory networks and ultimately the genotype-to-phenotype map evolve. It is recognized that microRNAs (miRNAs) have the potential to facilitate evolutionary change [1–3]; however, there are no known examples of natural morphological variation caused by evolutionary changes in miRNA expression. Therefore, the contribution of miRNAs to evolutionary change remains unknown [1, 4]. Drosophila melanogaster subgroup species display a portion of trichome-free cuticle on the femur of the second leg called the “naked valley.” It was previously shown that Ultrabithorax (Ubx) is involved in naked valley variation between D. melanogaster and D. simulans [5, 6]. However, naked valley size also varies among populations of D. melanogaster, ranging from 1,000 up to 30,000 μm2. We investigated the genetic basis of intraspecific differences in the naked valley in D. melanogaster and found that neither Ubx nor shavenbaby (svb) [7, 8] contributes to this morphological difference. Instead, we show that changes in mir-92a expression underlie the evolution of naked valley size in D. melanogaster through repression of shavenoid (sha) [9]. Therefore, our results reveal a novel mechanism for morphological evolution and suggest that modulation of the expression of miRNAs potentially plays a prominent role in generating organismal diversity.

Evolution of Eye Morphology and Rhodopsin Expression in the Drosophila melanogaster Species Subgroup

A striking diversity of compound eye size and shape has evolved among insects. The number of ommatidia and their size are major determinants of the visual sensitivity and acuity of the compound eye. Each ommatidium is composed of eight photoreceptor cells that facilitate the discrimination of different colours via the expression of various light sensitive Rhodopsin proteins. It follows that variation in eye size, shape, and opsin composition is likely to directly influence vision. We analyzed variation in these three traits in D. melanogaster, D. simulans and D. mauritiana. We show that D. mauritiana generally has larger eyes than its sibling species, which is due to a combination of larger ommatidia and more ommatidia. In addition, intra- and inter-specific differences in eye size among D. simulans and D. melanogaster strains are mainly caused by variation in ommatidia number. By applying a geometric morphometrics approach to assess whether the formation of larger eyes influences other parts of the head capsule, we found that an increase in eye size is associated with a reduction in the adjacent face cuticle. Our shape analysis also demonstrates that D. mauritiana eyes are specifically enlarged in the dorsal region. Intriguingly, this dorsal enlargement is associated with enhanced expression of rhodopsin 3 in D. mauritiana. In summary, our data suggests that the morphology and functional properties of the compound eyes vary considerably within and among these closely related Drosophila species and may be part of coordinated morphological changes affecting the head capsule.

Genetic Architecture and Functional Characterization of Genes Underlying the Rapid Diversification of Male External Genitalia Between Drosophila simulans and Drosophila mauritiana

Male sexual characters are often among the first traits to diverge between closely related species and identifying the genetic basis of such changes can contribute to our understanding of their evolutionary history. However, little is known about the genetic architecture or the specific genes underlying the evolution of male genitalia. The morphology of the claspers, posterior lobes, and anal plates exhibit striking differences between Drosophila mauritiana and D. simulans. Using QTL and introgression-based high-resolution mapping, we identified several small regions on chromosome arms 3L and 3R that contribute to differences in these traits. However, we found that the loci underlying the evolution of clasper differences between these two species are independent from those that contribute to posterior lobe and anal plate divergence. Furthermore, while most of the loci affect each trait in the same direction and act additively, we also found evidence for epistasis between loci for clasper bristle number. In addition, we conducted an RNAi screen in D. melanogaster to investigate if positional and expression candidate genes located on chromosome 3L, are also involved in genital development. We found that six of these genes, including components of Wnt signaling and male-specific lethal 3 (msl3), regulate the development of genital traits consistent with the effects of the introgressed regions where they are located and that thus represent promising candidate genes for the evolution these traits.

Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana

Eye and head morphology vary considerably among insects and even between closely related species of Drosophila. Species of the D. melanogaster subgroup, and other Drosophila species, exhibit a negative correlation between eye size and face width (FW); for example, D. mauritiana generally has bigger eyes composed of larger ommatidia and conversely a narrower face than its sibling species. To better understand the evolution of eye and head morphology, we investigated the genetic and developmental basis of differences in eye size and FW between male D. mauritiana and D. simulans. QTL mapping of eye size and FW showed that the major loci responsible for the interspecific variation in these traits are localized to different genomic regions. Introgression of the largest effect QTL underlying the difference in eye size resulted in flies with larger eyes but no significant difference in FW. Moreover, introgression of a QTL region on the third chromosome that contributes to the FW difference between these species affected FW, but not eye size. We also observed that this difference in FW is detectable earlier in the development of the eye‐antennal disc than the difference in the size of the retinal field. Our results suggest that different loci that act at different developmental stages underlie changes in eye size and FW. Therefore, while there is a negative correlation between these traits in Drosophila, we show genetically that they also have the potential to evolve independently and this may help to explain the evolution of these traits in other insects.

Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary ‘hotspots’ are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the ‘naked valley’ on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. We compared the expression and function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution.