Current Biology 23, 1–6, March 18, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.02.018 Report Evolution of mir-92a Underlies Natural Morphological Variation in Drosophila melanogaster Saad Arif, 1,2,7 Sophie Murat, 1,2,7 Isabel Almudi, 1 Maria D.S. Nunes, 1 Diane Bortolamiol-Becet, 6 Naomi S. McGregor, 1 James M.S. Currie, 1 Harri Hughes, 1 Matthew Ronshaugen, 3 E ´ lio Sucena, 4,5 Eric C. Lai, 6 Christian Schlo ¨ tterer, 2 and Alistair P. McGregor 1,2, * 1 Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, UK 2 Institut fu ¨ r Populationsgenetik, Veterina ¨ rmedizinische Universita ¨ t Wien, Veterina ¨ rplatz 1, 1210 Vienna, Austria 3 Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK 4 Instituto Gulbenkian de Cie ˆ ncia, Apartado 14, 2781-901 Oeiras, Portugal 5 Departamento de Biologia Animal, Faculdade de Cie ˆ ncias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal 6 Sloan-Kettering Institute, 1017C Rockefeller Research Labs, 1275 York Avenue, Box 252, New York, NY 10065, USA 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 expres- sion. 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 mm 2 . We investigated the genetic basis of intra- specific 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 evolu- tion of naked valley size in D. melanogaster through repres- sion 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. Results and Discussion Intraspecific Variation in the Naked Valley The naked valley exhibits considerable intraspecific variation in D. melanogaster, ranging from a trichome-free patch as small as 1,000 mm 2 to a naked region of up to approximately 30,000 mm 2 (Figure 1; see also Figure S1 available online). Moreover, small and large naked valley phenotypes segregate within natural D. melanogaster populations (Figures 1 and S1). In contrast, among D. simulans (Figure S1), D. mauritiana, and D. sechellia populations, as well as D. yakuba, we have only observed naked valley areas at the higher end of the size range (13,000 to 30,000 mm 2 ). Therefore, small naked valleys (SNVs) appear to be a derived morphological feature within D. melanogaster, whereas larger naked valleys (LNVs) are likely to be ancestral with respect to the D. melanogaster species subgroup. Mapping the Genetic Basis of Naked Valley Variation in D. melanogaster It was previously shown that the Hox gene Ultrabithorax (Ubx) contributes to the difference in naked valley size between a D. melanogaster strain with a small naked valley and D. simulans [6]. Therefore, to determine whether Ubx is also responsible for intraspecific naked valley variation in D. melanogaster, we performed quantitative trait locus (QTL) mapping of naked valley size on chromosome 3 among back- cross progeny from crosses between strains st, ss, e (LNV) and Oregon-R (SNV). We found a single QTL at 88.2 cM on chromo- some 3 that explains up to 91% of the difference in naked valley size between the two parental strains (Figure S2A; Table S1), and, using a male F1 backcrossing strategy, we deter- mined that the remaining effect (approximately 10%) is caused by chromosome 2 (p < 0.017, Bonferroni corrected pairwise comparison of means). Chromosomes X and 4 have no signif- icant effect. Our mapping thus excludes both Ubx, which is at 58.8 cM on chromosome 3 (Figure 2), and the X-linked gene shavenbaby (svb), which is known to underlie variation in larval trichome patterns [10–12]. To verify that variation in Ubx is not responsible for differ- ences in the naked valley in D. melanogaster, we carried out two further experiments. First, we repeated our chromosome 3 mapping strategy with two different D. melanogaster strains, RAL514 and ebony (e), white ocelli (wo), rough (ro), which have SNVs and LNVs, respectively. QTL mapping using these three recessive markers confirmed the position of a single, large- effect QTL on chromosome 3 at 79.7 to 89.7 cM (2 LOD interval), between wo and ro (Figure S2A; Table S1). Second, we generated flies with recombinant third chromosomes: homozygous for the Ubx allele from a LNV background (Ubx L ) and homozygous for the QTL region from a SNV back- ground (QTL S ), and vice versa (Ubx S and QTL L )(Figure S2B). The size of the naked valley of these flies was determined by the background from which the QTL region originated (Fig- ure S2), and no significant effect could be attributed to Ubx: flies homozygous for Ubx L and QTL S had a small naked valley, whereas flies homozygous for Ubx S and QTL L had a large naked valley. Furthermore, the effect on naked valley area of homozygosity for QTL L or QTL S was consistent with the QTL mapping results (Figure S2). Our mapping results therefore showed that neither Ubx nor svb contributes to naked valley variation in D. melanogaster. To fine map the causative locus or loci in the QTL region, we took advantage of the large effect of the QTL and employed the 7 These authors contributed equally to this work *Correspondence: amcgregor@brookes.ac.uk Please cite this article in press as: Arif et al., Evolution of mir-92a Underlies Natural Morphological Variation in Drosophila mela- nogaster, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.02.018