A Single Gene Affects Both Ecological Divergence and Mate Choice in Drosophila Henry Chung et al. Science 343, 1148 (2014); DOI: 10.1126/science.1249998
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of March 11, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6175/1148.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2014/02/12/science.1249998.DC1.html This article cites 27 articles, 6 of which can be accessed free: http://www.sciencemag.org/content/343/6175/1148.full.html#ref-list-1 This article appears in the following subject collections: Evolution http://www.sciencemag.org/cgi/collection/evolution
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REPORTS peak QTL marker, respectively (Fig. 2C) (21). Furthermore, the tail-selected F3 individuals contained none of the shared TE insertions identified in the epiRIL population (Fig. 2D and table S6). We thus conclude that the epiRIL QTL are most likely caused by the heritable (ddm1-2 induced) loss of DNA methylation in the QTL intervals. Next, we searched for putative causal DMRs in the RL and FT QTL intervals. We analyzed the methylomes (~165–base pair resolution) of the 123 epiRILs and their founders (19) and required candidate DMRs to be in approximate linkage disequilibrium with the peak QTL marker and displaying clear differences in DNA methylation states and expression levels between the WT and the ddm1-2 founder lines (figs. S5 to S8). Our search revealed 325 candidate DMRs within the FT QTL intervals (chr1, 53; chr4, 16; chr5, 256), which mapped to 44 unique genes (including promoter regions), 153 annotated TE sequences, and 77 intergenic regions (Fig. 3, A and C, and tables S9, S10, and S12). For the RL QTL intervals, we detected 506 candidate DMRs (chr 1, 122; chr2, 367; chr4, 17), mapping to 71 unique genes (including promoter regions), 261 annotated TE sequences, and 122 intergenic regions (Fig. 3, B and C, and tables S9, S11, and S13). Further analysis of these candidate DMRs did not identify any obvious flowering time and root length genes (tables S10 and S11), which could be consistent with the lower amplitude of phenotypic variation observed among the epiRILs than among highly contrasted accessions (16). However, we cannot rule out that the candidate DMRs are in LD with causal DMRs that could not be called with our method. Ultimately, finemapping approaches and targeted manipulation
of selected DMRs will be required to identify causal regions. Our analysis of the epiRILs demonstrates that induced DMRs can be stably inherited independently of DNA sequence changes and function as epigenetic quantitative trait loci (QTLepi). Phenotypically, the detected QTLepi have all the necessary properties to become targets of natural or artificial selection. Taking advantage of the singlenucleotide resolution methylomes of 138 natural accessions (4) (table S14), we could show that about 30% of the heritable DMRs identified in the epiRIL population overlap with naturally occurring DMRs among these accessions (figs. S9 and S10). Therefore, these epiRIL DMRs may have been historical targets of epimutations in the wild, either through trans-induced ddm1-like mutation events or else through still unknown mechanisms. This finding indicates in turn that DMRs could also act as QTLepi in natural populations and thus constitute a measureable component of the so-called “missing heritability.” This possibility may have deep implications on how we delineate and interpret the heritable basis of complex traits. References and Notes 1. J. A. Law, S. E. Jacobsen, Nat. Rev. Genet. 11, 204–220 (2010). 2. S. Feng et al., Proc. Natl. Acad. Sci. U.S.A. 107, 8689–8694 (2010). 3. A. Zemach, I. E. McDaniel, P. Silva, D. Zilberman, Science 328, 916–919 (2010). 4. R. J. Schmitz et al., Nature 495, 193–198 (2013). 5. S. R. Eichten et al., Plant Cell 25, 2783–2797 (2013). 6. H. Heyn et al., Genome Res. 23, 1363–1372 (2013). 7. D. Weigel, V. Colot, Genome Biol. 13, 249 (2012). 8. C. Becker et al., Nature 480, 245–249 (2011). 9. R. J. Schmitz et al., Science 334, 369–373 (2011). 10. F. Johannes, V. Colot, R. C. Jansen, Nat. Rev. Genet. 9, 883–890 (2008).
A Single Gene Affects Both Ecological Divergence and Mate Choice in Drosophila Henry Chung,1 David W. Loehlin,1 Héloïse D. Dufour,1 Kathy Vaccarro,1 Jocelyn G. Millar,2 Sean B. Carroll1* Evolutionary changes in traits involved in both ecological divergence and mate choice may produce reproductive isolation and speciation. However, there are few examples of such dual traits, and the genetic and molecular bases of their evolution have not been identified. We show that methyl-branched cuticular hydrocarbons (mbCHCs) are a dual trait that affects both desiccation resistance and mate choice in Drosophila serrata. We identify a fatty acid synthase mFAS (CG3524) responsible for mbCHC production in Drosophila and find that expression of mFAS is undetectable in oenocytes (cells that produce CHCs) of a closely related, desiccation-sensitive species, D. birchii, due in part to multiple changes in cis-regulatory sequences of mFAS. We suggest that ecologically influenced changes in the production of mbCHCs have contributed to reproductive isolation between the two species. he evolution of traits with dual roles in ecological divergence and mate choice could cause populations to become reproductively isolated through local adaptation (1, 2).
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However, the direct contribution of ecological divergence to speciation is uncertain, in part because relatively few systems have been investigated experimentally, and the genes affecting
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
F. Johannes et al., PLOS Genet. 5, e1000530 (2009). Z. Lippman et al., Nature 430, 471–476 (2004). S. Tsukahara et al., Nature 461, 423–426 (2009). T. Kakutani, K. Munakata, E. J. Richards, H. Hirochika, Genetics 151, 831–838 (1999). F. K. Teixeira et al., Science 323, 1600–1604 (2009). V. Latzel, Y. Zhang, K. Karlsson Moritz, M. Fischer, O. Bossdorf, Ann. Bot. 110, 1423–1428 (2012). F. Roux et al., Genetics 188, 1015–1017 (2011). F. Johannes, M. Colomé-Tatché, Genetics 188, 215–227 (2011). M. Colomé-Tatché et al., Proc. Natl. Acad. Sci. U.S.A. 109, 16240–16245 (2012). E. S. Lander, D. Botstein, Genetics 121, 185–199 (1989). Materials and methods and supplementary text are available as supporting material on Science Online.
Acknowledgments: We thank O. Bossdorf, C. Richards, T. Day, and K. Verhoeven for their input on an earlier version of this report. This work was supported by grants from the Netherlands Organization for Scientific Research (to F.J., R.C.J., R.W., and M.C.-T.); a University of Groningen Rosalind Franklin Fellowship to M.C.-T.; the Agence Nationale de la Recherche (ANR-09-BLAN-0237 EPIMOBILE to V.C. and P.W.; ANR-06GPLA-010 TAG, Investissements d’Avenir ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-0001-02 PSL* Research University to V.C.); and the European Union (EpiGeneSys FP7 Network of Excellence number 257082, to V.C.). S.C. and M.E. were supported by Ph.D. studentships from the Ministère de l’Enseignement Supérieur et de la Recherche, with additional support from the Fondation pour la Recherche Médicale (to S.C.). Sequence reads for the 52 epiRILs and parental lines are deposited at the European Bioinformatics Institute under accession number ERP004507.
Supplementary Materials www.sciencemag.org/content/343/6175/1145/suppl/DC1 Materials and Methods Supplementary Text Figs. S1 to S11 Tables S1 to S14 References (22–28) 5 November 2013; accepted 22 January 2014 Published online 6 February 2014; 10.1126/science.1248127
dual traits have not been identified. Insect cuticular hydrocarbons (CHCs) are potential dual traits (3). CHCs seal the cuticle, protecting against desiccating environments (4), and can also act as pheromones that influence mate choice and mating success (5). Whereas specific CHC molecules act as pheromones (6, 7), no specific CHC or class of CHCs has been demonstrated to be involved directly in desiccation resistance. One class of CHCs, methyl-branched CHCs (mbCHCs), which have melting points above ambient temperature, are hypothesized to help maintain a barrier against evaporative water loss (8). D. serrata, a habitat generalist found outside of and on the fringes of the rainforest on the east coast of Australia, is relatively desiccation resistant and produces relatively large amounts of mbCHCs (29% of all CHCs) (Fig. 1A). In contrast, its close relative D. birchii, a habitat specialist found exclusively in the humid rainforest, is ex1 Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA. 2Department of Entomology, University of California, Riverside, CA 92521, USA.
*Corresponding author. E-mail: [email protected]
www.sciencemag.org
REPORTS tremely sensitive to desiccation and produces very low amounts of mbCHCs (9–11) (3% of all CHCs) (Fig. 1 and fig. S1A). D. serrata and D. birchii show prezygotic isolation, which appears to be mediated by chemical signals (12). Notably, mbCHC levels are one factor correlated with male mating success in D. serrata (13, 14). If mbCHCs contribute to both mating success and desiccation resistance, then mbCHCs would constitute a dual trait, and the loss of mbCHCs in D. birchii could have contributed to its reproductive isolation from D. serrata. To explore this scenario, we identified the gene(s) responsible for the differences in mbCHCs between D. serrata and D. birchii. Because the levels of both major mbCHCs (2Me-C26 and 2MeC28) are lower in D. birchii than in D. serrata (10) (fig. S1), we reasoned that the underlying genetic differences between these two species must have occurred early in the fatty acid synthase (FAS) pathway responsible for mbCHC synthesis (Fig. 1B). Biochemical studies have identified two forms of FASs in insects, one for producing unbranched CHCs such as alkanes, monoenes, and dienes, and the other for producing mbCHCs (15). The D. melanogaster genome contains three putative FASs: CG3523, CG3524, and CG17374 (16). In situ hybridization of RNA probes for all three FASs in adult abdomens revealed that CG3523 is expressed only in the adult fat body, whereas CG17374 and CG3524 are both expressed in adult
B
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D. melanogaster oenocytes (Fig. 1C), where CHCs are produced (17). RNA interference (RNAi)–mediated knockdown of CG3524 and CG17374 in D. melanogaster by a ubiquitously expressed driver (tubulin-GAL4) was lethal. An oenocyte-specific driver (oenoGAL4) (17) inducing CG17374 RNAi was also lethal, whereas oenoGAL4-driven RNAi knockdown of CG3524 produced viable adult flies in which mbCHC production was strongly and specifically reduced (Fig. 1D). This suggests that CG3524 is a mbCHC-specific FAS (mFAS). RNAi-mediated knockdown of CG17374 in oenocytes after adult flies eclosed yielded viable adults with unchanged CHC profiles, whereas knockdown of CG3524 in adults reduced mbCHCs, thus confirming the specific role of CG3524 as mFAS. We then isolated the orthologous gene in the D. serrata genome and drove D. serrata mFAS RNAi under the direct control of an oenocytespecific enhancer (fig. S2), obtaining two independent lines of transgenic D. serrata flies carrying this construct (mFAS RNAi-1 and mFAS RNAi-2). mbCHC levels were reduced in males and females of both lines, indicating that the role of mFAS in producing mbCHCs is conserved among Drosophila (Fig. 2A and table S1). To assess whether mbCHCs contribute to desiccation resistance in D. serrata, we subjected D. serrata mFAS-RNAi and wild-type flies to a desiccation assay. We found that the D. serrata mFAS-RNAi flies exhibited reduced desiccation
Valine
resistance (Fig. 2B). To investigate whether the loss of desiccation resistance was due to the specific loss of mbCHCs, we applied a mixture of purified synthetic mbCHCs to D. serrata mFASRNAi flies. The application of synthetic mbCHCs significantly increased resistance (fig. S3B), indicating that the low resistance of mFAS-RNAi flies was due to the specific loss of mbCHCs. We also applied synthetic mbCHCs to D. birchii flies and observed increased desiccation resistance (fig. S3B), suggesting that the comparatively low desiccation resistance of this species may be due at least in part to its low levels of mbCHCs. The D. birchii mFAS gene (GenBank JX627578) contains a complete coding sequence, and cDNA could be isolated from adults, indicating that the gene is intact and functional. However, mFAS expression was not detectable in D. birchii oenocytes by in situ hybridization, suggesting that transcription in these cells is reduced or absent (Fig. 3). mFAS is expressed in the oenocytes of all other Drosophila species examined, including D. serrata (fig. S4). Thus, mFAS expression in oenocytes appears to have been lost in the D. birchii lineage. To determine whether loss of mFAS expression is due to cis-regulatory changes, we made green fluorescent protein (GFP) reporter constructs with segments of the mFAS gene from both species and compared their activity when transformed into D. melanogaster. Both 5′ and intronic mFAS sequences from D. birchii exhibited lower activity
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Fig. 1. Closely related Drosophila species differ in mbCHC levels, whose synthesis is controlled by the mFAS gene. (A) D. serrata and D. birchii occupy different habitats, but their ranges overlap on the east coast of Australia. The habitat generalist D. serrata produces higher levels of mbCHCs (29%) than the rainforest specialist D. birchii (3%). (B) The biosynthetic pathways for CHC production in insects. Two different forms of FAS are responsible for the synthesis of methyl-branched and straight-chained CHCs in Drosophila (18). (C) RNA in situ hybridization of the three Drosophila FAS genes on adult D. melanogaster males. CG3523 transcript was detected in the fat body. CG17374 and CG3524 transcripts were detected in the oenocytes (arrowheads), the site of CHC synthesis. Insets show right side of second tergite (abdominal segment). For the CG3523 inset, the fat body was removed. Expression of all three FASs is sexually monomorphic. (D) www.sciencemag.org
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REPORTS type females were given the choice between a wildtype male and either a mFAS RNAi-1 or mFASRNAi-2 male, they preferred males with normal production of mbCHCs over males with reduced production (Table 1) [this effect was dependent on the age of the flies (see table S2)]. To determine whether increasing the level of mbCHCs also influenced mating success, we perfumed wild-type males with either 2Me-C26 or 2Me-C28 (table S3). We found that males perfumed with 2Me-C26 exhibited increased mating success, whereas 2Me-C28 had no detectable effect (Table 1). Finally, we investigated whether perfuming mFAS-RNAi males with either 2MeC26 or 2Me-C28 increased male mating success
D. serrata
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than the homologous regions from D. serrata (fig. S5), indicating that the reduction in D. birchii mFAS expression is partly due to multiple cisregulatory changes at the locus. The ecological divergence of the rainforestrestricted D. birchii and widely distributed D. serrata involved reductions of desiccation resistance, mFAS expression, and mbCHCs. mbCHCs are one factor that has been correlated with male mating success in D. serrata (13, 14), and thus could have played a role in reproductive isolation between the species. To determine whether mbCHCs directly affect mating in D. serrata, we conducted a series of mate-choice experiments in which mbCHC levels were manipulated. When single wild-type males were presented with a choice between a wild-type female and a mFAS-RNAi female, we did not observe any mating preference (n = 105; c2 = 0.77; P = 0.38). This suggests that D. serrata males do not discriminate on the basis of mbCHC levels in females. In contrast, when single wild-
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Fig. 2. Loss of mbCHCs strongly reduces desiccation resistance in D. serrata. (A) Oenocytespecific RNAi knockdown of mFAS in D. serrata results in almost complete loss of mbCHCs (results shown in males). (B) Oenocyte-specific RNAi knockdown of mFAS in D. serrata results in a dramatic reduction of desiccation resistance in both male (t test; df = 17; t = 26.7, P = 6.4 × 10−12) and female (df = 18; t = 65.7; P = 1.8 × 10−17) mFAS RNAi-1 flies. The mean time to mortality of 10 flies in each of n = 9 to 10 vials per genotype was compared with t tests (two-tailed, unpaired, unequal variance). Mean T SEM is shown.
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Fig. 3. mFAS is not expressed in D. birchii oenocytes. RNA in situ hybridization reveals that, whereas mFAS shows a sexually monomorphic expression pattern in D. serrata oenocytes (left), it is undetectable in either sex in D. birchii (right). To confirm that the lack of transcript detection was not due to any defect of the D. birchii RNA probe, we showed that this probe detected mFAS expression in D. serrata (center). Arrowheads point to oenocytes; open arrowheads indicate no visible expression.
and found that 2Me-C26 again increased male mating success. Together, our results indicate that mbCHCs, whose production is controlled by the mFAS gene, affect mating success as well as desiccation resistance, and thus function as a dual trait in D. serrata. To determine whether the difference in mbCHC levels might also contribute to premating isolation between D. serrata and D. birchii, we investigated whether manipulating the level of mbCHCs in D. birchii could break the interspecies mating barrier. We perfumed D. birchii males and females with synthetic mbCHCs before introducing them to D. serrata females and males, respectively. In neither case did we observe any copulations. Importantly, however, we also observed that no males of either species even attempted to court females of the other species, a necessary preamble before females exhibit a choice. These observations suggest that courtship by males is also governed by cues that differ between the species, such that modulation of mbCHCs alone is not sufficient to surmount the barrier to mating. This is not surprising because numerous factors contribute to courtship and mating among Drosophila species, and these traits may also be subject to rapid divergence (18, 19). Although restoring mbCHCs to D. birchii was not sufficient to surmount its reproductive isolation from D. serrata, the demonstrated role of mbCHCs as a component of mate choice in D. serrata and the loss of mbCHCs in its rainforestrestricted sibling suggest a plausible scenario for mbCHCs in ecological speciation. That is, as populations of the common ancestor of these two species diverged ecologically, and if the rainforestadapting population lost mbCHCs and mFAS expression before or during speciation (perhaps due to relaxed constraints on desiccation resistance), then such changes would likely result in increasing reproductive isolation between the populations. Subsequent changes at other genetic loci would then complete and/or reinforce speciation (fig. S6).
Table 1. mbCHCs affect mating success in D. serrata. (A) Three-day-old wild-type D. serrata males had significantly higher mating success than mFAS-RNAi flies in female choice assays where single wild-type females were given a choice between the two genotypes. (B) Wild-type and mFASRNAi-2 males were perfumed with 2Me-C26 and 2Me-C28 individually. Females are given a choice between a perfumed male and a nonperfumed male with the same genotype. Perfuming with 2Me-C26 significantly increased mating success of both wild-type and mFAS-RNAi flies. (A) Wild-type (WT) males have greater mating success than mFAS-RNAi males n WT chosen RNAi chosen df c2 RNAi-1
WT versus mFAS WT versus mFASRNAi-2
79 89
55 54
24 35
1 1
12.165 4.056
P
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of March 11, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/343/6175/1148.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2014/02/12/science.1249998.DC1.html This article cites 27 articles, 6 of which can be accessed free: http://www.sciencemag.org/content/343/6175/1148.full.html#ref-list-1 This article appears in the following subject collections: Evolution http://www.sciencemag.org/cgi/collection/evolution
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on March 11, 2014
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REPORTS peak QTL marker, respectively (Fig. 2C) (21). Furthermore, the tail-selected F3 individuals contained none of the shared TE insertions identified in the epiRIL population (Fig. 2D and table S6). We thus conclude that the epiRIL QTL are most likely caused by the heritable (ddm1-2 induced) loss of DNA methylation in the QTL intervals. Next, we searched for putative causal DMRs in the RL and FT QTL intervals. We analyzed the methylomes (~165–base pair resolution) of the 123 epiRILs and their founders (19) and required candidate DMRs to be in approximate linkage disequilibrium with the peak QTL marker and displaying clear differences in DNA methylation states and expression levels between the WT and the ddm1-2 founder lines (figs. S5 to S8). Our search revealed 325 candidate DMRs within the FT QTL intervals (chr1, 53; chr4, 16; chr5, 256), which mapped to 44 unique genes (including promoter regions), 153 annotated TE sequences, and 77 intergenic regions (Fig. 3, A and C, and tables S9, S10, and S12). For the RL QTL intervals, we detected 506 candidate DMRs (chr 1, 122; chr2, 367; chr4, 17), mapping to 71 unique genes (including promoter regions), 261 annotated TE sequences, and 122 intergenic regions (Fig. 3, B and C, and tables S9, S11, and S13). Further analysis of these candidate DMRs did not identify any obvious flowering time and root length genes (tables S10 and S11), which could be consistent with the lower amplitude of phenotypic variation observed among the epiRILs than among highly contrasted accessions (16). However, we cannot rule out that the candidate DMRs are in LD with causal DMRs that could not be called with our method. Ultimately, finemapping approaches and targeted manipulation
of selected DMRs will be required to identify causal regions. Our analysis of the epiRILs demonstrates that induced DMRs can be stably inherited independently of DNA sequence changes and function as epigenetic quantitative trait loci (QTLepi). Phenotypically, the detected QTLepi have all the necessary properties to become targets of natural or artificial selection. Taking advantage of the singlenucleotide resolution methylomes of 138 natural accessions (4) (table S14), we could show that about 30% of the heritable DMRs identified in the epiRIL population overlap with naturally occurring DMRs among these accessions (figs. S9 and S10). Therefore, these epiRIL DMRs may have been historical targets of epimutations in the wild, either through trans-induced ddm1-like mutation events or else through still unknown mechanisms. This finding indicates in turn that DMRs could also act as QTLepi in natural populations and thus constitute a measureable component of the so-called “missing heritability.” This possibility may have deep implications on how we delineate and interpret the heritable basis of complex traits. References and Notes 1. J. A. Law, S. E. Jacobsen, Nat. Rev. Genet. 11, 204–220 (2010). 2. S. Feng et al., Proc. Natl. Acad. Sci. U.S.A. 107, 8689–8694 (2010). 3. A. Zemach, I. E. McDaniel, P. Silva, D. Zilberman, Science 328, 916–919 (2010). 4. R. J. Schmitz et al., Nature 495, 193–198 (2013). 5. S. R. Eichten et al., Plant Cell 25, 2783–2797 (2013). 6. H. Heyn et al., Genome Res. 23, 1363–1372 (2013). 7. D. Weigel, V. Colot, Genome Biol. 13, 249 (2012). 8. C. Becker et al., Nature 480, 245–249 (2011). 9. R. J. Schmitz et al., Science 334, 369–373 (2011). 10. F. Johannes, V. Colot, R. C. Jansen, Nat. Rev. Genet. 9, 883–890 (2008).
A Single Gene Affects Both Ecological Divergence and Mate Choice in Drosophila Henry Chung,1 David W. Loehlin,1 Héloïse D. Dufour,1 Kathy Vaccarro,1 Jocelyn G. Millar,2 Sean B. Carroll1* Evolutionary changes in traits involved in both ecological divergence and mate choice may produce reproductive isolation and speciation. However, there are few examples of such dual traits, and the genetic and molecular bases of their evolution have not been identified. We show that methyl-branched cuticular hydrocarbons (mbCHCs) are a dual trait that affects both desiccation resistance and mate choice in Drosophila serrata. We identify a fatty acid synthase mFAS (CG3524) responsible for mbCHC production in Drosophila and find that expression of mFAS is undetectable in oenocytes (cells that produce CHCs) of a closely related, desiccation-sensitive species, D. birchii, due in part to multiple changes in cis-regulatory sequences of mFAS. We suggest that ecologically influenced changes in the production of mbCHCs have contributed to reproductive isolation between the two species. he evolution of traits with dual roles in ecological divergence and mate choice could cause populations to become reproductively isolated through local adaptation (1, 2).
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However, the direct contribution of ecological divergence to speciation is uncertain, in part because relatively few systems have been investigated experimentally, and the genes affecting
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
F. Johannes et al., PLOS Genet. 5, e1000530 (2009). Z. Lippman et al., Nature 430, 471–476 (2004). S. Tsukahara et al., Nature 461, 423–426 (2009). T. Kakutani, K. Munakata, E. J. Richards, H. Hirochika, Genetics 151, 831–838 (1999). F. K. Teixeira et al., Science 323, 1600–1604 (2009). V. Latzel, Y. Zhang, K. Karlsson Moritz, M. Fischer, O. Bossdorf, Ann. Bot. 110, 1423–1428 (2012). F. Roux et al., Genetics 188, 1015–1017 (2011). F. Johannes, M. Colomé-Tatché, Genetics 188, 215–227 (2011). M. Colomé-Tatché et al., Proc. Natl. Acad. Sci. U.S.A. 109, 16240–16245 (2012). E. S. Lander, D. Botstein, Genetics 121, 185–199 (1989). Materials and methods and supplementary text are available as supporting material on Science Online.
Acknowledgments: We thank O. Bossdorf, C. Richards, T. Day, and K. Verhoeven for their input on an earlier version of this report. This work was supported by grants from the Netherlands Organization for Scientific Research (to F.J., R.C.J., R.W., and M.C.-T.); a University of Groningen Rosalind Franklin Fellowship to M.C.-T.; the Agence Nationale de la Recherche (ANR-09-BLAN-0237 EPIMOBILE to V.C. and P.W.; ANR-06GPLA-010 TAG, Investissements d’Avenir ANR-10-LABX-54 MEMO LIFE, and ANR-11-IDEX-0001-02 PSL* Research University to V.C.); and the European Union (EpiGeneSys FP7 Network of Excellence number 257082, to V.C.). S.C. and M.E. were supported by Ph.D. studentships from the Ministère de l’Enseignement Supérieur et de la Recherche, with additional support from the Fondation pour la Recherche Médicale (to S.C.). Sequence reads for the 52 epiRILs and parental lines are deposited at the European Bioinformatics Institute under accession number ERP004507.
Supplementary Materials www.sciencemag.org/content/343/6175/1145/suppl/DC1 Materials and Methods Supplementary Text Figs. S1 to S11 Tables S1 to S14 References (22–28) 5 November 2013; accepted 22 January 2014 Published online 6 February 2014; 10.1126/science.1248127
dual traits have not been identified. Insect cuticular hydrocarbons (CHCs) are potential dual traits (3). CHCs seal the cuticle, protecting against desiccating environments (4), and can also act as pheromones that influence mate choice and mating success (5). Whereas specific CHC molecules act as pheromones (6, 7), no specific CHC or class of CHCs has been demonstrated to be involved directly in desiccation resistance. One class of CHCs, methyl-branched CHCs (mbCHCs), which have melting points above ambient temperature, are hypothesized to help maintain a barrier against evaporative water loss (8). D. serrata, a habitat generalist found outside of and on the fringes of the rainforest on the east coast of Australia, is relatively desiccation resistant and produces relatively large amounts of mbCHCs (29% of all CHCs) (Fig. 1A). In contrast, its close relative D. birchii, a habitat specialist found exclusively in the humid rainforest, is ex1 Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA. 2Department of Entomology, University of California, Riverside, CA 92521, USA.
*Corresponding author. E-mail: [email protected]
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REPORTS tremely sensitive to desiccation and produces very low amounts of mbCHCs (9–11) (3% of all CHCs) (Fig. 1 and fig. S1A). D. serrata and D. birchii show prezygotic isolation, which appears to be mediated by chemical signals (12). Notably, mbCHC levels are one factor correlated with male mating success in D. serrata (13, 14). If mbCHCs contribute to both mating success and desiccation resistance, then mbCHCs would constitute a dual trait, and the loss of mbCHCs in D. birchii could have contributed to its reproductive isolation from D. serrata. To explore this scenario, we identified the gene(s) responsible for the differences in mbCHCs between D. serrata and D. birchii. Because the levels of both major mbCHCs (2Me-C26 and 2MeC28) are lower in D. birchii than in D. serrata (10) (fig. S1), we reasoned that the underlying genetic differences between these two species must have occurred early in the fatty acid synthase (FAS) pathway responsible for mbCHC synthesis (Fig. 1B). Biochemical studies have identified two forms of FASs in insects, one for producing unbranched CHCs such as alkanes, monoenes, and dienes, and the other for producing mbCHCs (15). The D. melanogaster genome contains three putative FASs: CG3523, CG3524, and CG17374 (16). In situ hybridization of RNA probes for all three FASs in adult abdomens revealed that CG3523 is expressed only in the adult fat body, whereas CG17374 and CG3524 are both expressed in adult
B
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D. melanogaster oenocytes (Fig. 1C), where CHCs are produced (17). RNA interference (RNAi)–mediated knockdown of CG3524 and CG17374 in D. melanogaster by a ubiquitously expressed driver (tubulin-GAL4) was lethal. An oenocyte-specific driver (oenoGAL4) (17) inducing CG17374 RNAi was also lethal, whereas oenoGAL4-driven RNAi knockdown of CG3524 produced viable adult flies in which mbCHC production was strongly and specifically reduced (Fig. 1D). This suggests that CG3524 is a mbCHC-specific FAS (mFAS). RNAi-mediated knockdown of CG17374 in oenocytes after adult flies eclosed yielded viable adults with unchanged CHC profiles, whereas knockdown of CG3524 in adults reduced mbCHCs, thus confirming the specific role of CG3524 as mFAS. We then isolated the orthologous gene in the D. serrata genome and drove D. serrata mFAS RNAi under the direct control of an oenocytespecific enhancer (fig. S2), obtaining two independent lines of transgenic D. serrata flies carrying this construct (mFAS RNAi-1 and mFAS RNAi-2). mbCHC levels were reduced in males and females of both lines, indicating that the role of mFAS in producing mbCHCs is conserved among Drosophila (Fig. 2A and table S1). To assess whether mbCHCs contribute to desiccation resistance in D. serrata, we subjected D. serrata mFAS-RNAi and wild-type flies to a desiccation assay. We found that the D. serrata mFAS-RNAi flies exhibited reduced desiccation
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resistance (Fig. 2B). To investigate whether the loss of desiccation resistance was due to the specific loss of mbCHCs, we applied a mixture of purified synthetic mbCHCs to D. serrata mFASRNAi flies. The application of synthetic mbCHCs significantly increased resistance (fig. S3B), indicating that the low resistance of mFAS-RNAi flies was due to the specific loss of mbCHCs. We also applied synthetic mbCHCs to D. birchii flies and observed increased desiccation resistance (fig. S3B), suggesting that the comparatively low desiccation resistance of this species may be due at least in part to its low levels of mbCHCs. The D. birchii mFAS gene (GenBank JX627578) contains a complete coding sequence, and cDNA could be isolated from adults, indicating that the gene is intact and functional. However, mFAS expression was not detectable in D. birchii oenocytes by in situ hybridization, suggesting that transcription in these cells is reduced or absent (Fig. 3). mFAS is expressed in the oenocytes of all other Drosophila species examined, including D. serrata (fig. S4). Thus, mFAS expression in oenocytes appears to have been lost in the D. birchii lineage. To determine whether loss of mFAS expression is due to cis-regulatory changes, we made green fluorescent protein (GFP) reporter constructs with segments of the mFAS gene from both species and compared their activity when transformed into D. melanogaster. Both 5′ and intronic mFAS sequences from D. birchii exhibited lower activity
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Fig. 1. Closely related Drosophila species differ in mbCHC levels, whose synthesis is controlled by the mFAS gene. (A) D. serrata and D. birchii occupy different habitats, but their ranges overlap on the east coast of Australia. The habitat generalist D. serrata produces higher levels of mbCHCs (29%) than the rainforest specialist D. birchii (3%). (B) The biosynthetic pathways for CHC production in insects. Two different forms of FAS are responsible for the synthesis of methyl-branched and straight-chained CHCs in Drosophila (18). (C) RNA in situ hybridization of the three Drosophila FAS genes on adult D. melanogaster males. CG3523 transcript was detected in the fat body. CG17374 and CG3524 transcripts were detected in the oenocytes (arrowheads), the site of CHC synthesis. Insets show right side of second tergite (abdominal segment). For the CG3523 inset, the fat body was removed. Expression of all three FASs is sexually monomorphic. (D) www.sciencemag.org
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REPORTS type females were given the choice between a wildtype male and either a mFAS RNAi-1 or mFASRNAi-2 male, they preferred males with normal production of mbCHCs over males with reduced production (Table 1) [this effect was dependent on the age of the flies (see table S2)]. To determine whether increasing the level of mbCHCs also influenced mating success, we perfumed wild-type males with either 2Me-C26 or 2Me-C28 (table S3). We found that males perfumed with 2Me-C26 exhibited increased mating success, whereas 2Me-C28 had no detectable effect (Table 1). Finally, we investigated whether perfuming mFAS-RNAi males with either 2MeC26 or 2Me-C28 increased male mating success
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than the homologous regions from D. serrata (fig. S5), indicating that the reduction in D. birchii mFAS expression is partly due to multiple cisregulatory changes at the locus. The ecological divergence of the rainforestrestricted D. birchii and widely distributed D. serrata involved reductions of desiccation resistance, mFAS expression, and mbCHCs. mbCHCs are one factor that has been correlated with male mating success in D. serrata (13, 14), and thus could have played a role in reproductive isolation between the species. To determine whether mbCHCs directly affect mating in D. serrata, we conducted a series of mate-choice experiments in which mbCHC levels were manipulated. When single wild-type males were presented with a choice between a wild-type female and a mFAS-RNAi female, we did not observe any mating preference (n = 105; c2 = 0.77; P = 0.38). This suggests that D. serrata males do not discriminate on the basis of mbCHC levels in females. In contrast, when single wild-
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Fig. 2. Loss of mbCHCs strongly reduces desiccation resistance in D. serrata. (A) Oenocytespecific RNAi knockdown of mFAS in D. serrata results in almost complete loss of mbCHCs (results shown in males). (B) Oenocyte-specific RNAi knockdown of mFAS in D. serrata results in a dramatic reduction of desiccation resistance in both male (t test; df = 17; t = 26.7, P = 6.4 × 10−12) and female (df = 18; t = 65.7; P = 1.8 × 10−17) mFAS RNAi-1 flies. The mean time to mortality of 10 flies in each of n = 9 to 10 vials per genotype was compared with t tests (two-tailed, unpaired, unequal variance). Mean T SEM is shown.
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Fig. 3. mFAS is not expressed in D. birchii oenocytes. RNA in situ hybridization reveals that, whereas mFAS shows a sexually monomorphic expression pattern in D. serrata oenocytes (left), it is undetectable in either sex in D. birchii (right). To confirm that the lack of transcript detection was not due to any defect of the D. birchii RNA probe, we showed that this probe detected mFAS expression in D. serrata (center). Arrowheads point to oenocytes; open arrowheads indicate no visible expression.
and found that 2Me-C26 again increased male mating success. Together, our results indicate that mbCHCs, whose production is controlled by the mFAS gene, affect mating success as well as desiccation resistance, and thus function as a dual trait in D. serrata. To determine whether the difference in mbCHC levels might also contribute to premating isolation between D. serrata and D. birchii, we investigated whether manipulating the level of mbCHCs in D. birchii could break the interspecies mating barrier. We perfumed D. birchii males and females with synthetic mbCHCs before introducing them to D. serrata females and males, respectively. In neither case did we observe any copulations. Importantly, however, we also observed that no males of either species even attempted to court females of the other species, a necessary preamble before females exhibit a choice. These observations suggest that courtship by males is also governed by cues that differ between the species, such that modulation of mbCHCs alone is not sufficient to surmount the barrier to mating. This is not surprising because numerous factors contribute to courtship and mating among Drosophila species, and these traits may also be subject to rapid divergence (18, 19). Although restoring mbCHCs to D. birchii was not sufficient to surmount its reproductive isolation from D. serrata, the demonstrated role of mbCHCs as a component of mate choice in D. serrata and the loss of mbCHCs in its rainforestrestricted sibling suggest a plausible scenario for mbCHCs in ecological speciation. That is, as populations of the common ancestor of these two species diverged ecologically, and if the rainforestadapting population lost mbCHCs and mFAS expression before or during speciation (perhaps due to relaxed constraints on desiccation resistance), then such changes would likely result in increasing reproductive isolation between the populations. Subsequent changes at other genetic loci would then complete and/or reinforce speciation (fig. S6).
Table 1. mbCHCs affect mating success in D. serrata. (A) Three-day-old wild-type D. serrata males had significantly higher mating success than mFAS-RNAi flies in female choice assays where single wild-type females were given a choice between the two genotypes. (B) Wild-type and mFASRNAi-2 males were perfumed with 2Me-C26 and 2Me-C28 individually. Females are given a choice between a perfumed male and a nonperfumed male with the same genotype. Perfuming with 2Me-C26 significantly increased mating success of both wild-type and mFAS-RNAi flies. (A) Wild-type (WT) males have greater mating success than mFAS-RNAi males n WT chosen RNAi chosen df c2 RNAi-1
WT versus mFAS WT versus mFASRNAi-2
79 89
55 54
24 35
1 1
12.165 4.056
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