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Published in final edited form as: Science. 2012 Feb 24;335(6071):943–947. doi: 10.1126/science.1215193

Evolution of Shape by Multiple Regulatory Changes to a Growth Gene

David W Loehlin 1,*,†, John H Werren 1,†
PMCID: PMC3520604  NIHMSID: NIHMS417568  PMID: 22363002

Abstract

Genetic changes responsible for morphological differences between species are largely unknown. Such changes can involve modifications of growth that are relevant to understanding evolution, development and disease. We identified a gene that induces male-specific wing size and shape differences between Nasonia wasp species. Fine-scale mapping and in situ hybridization reveals that changes in at least three regions (two strictly in non-coding sequence) around the gene unpaired-like (upd-like) cause changes in spatial and temporal expression of upd-like in the developing wing and corresponding changes in wing width. Upd-like shows homology to the Drosophila unpaired gene, a well-studied signaling protein that regulates cell proliferation and differentiation. Our results indicate how multiple changes in the regulation of upd-like are involved in microevolution of morphological and sex-specific differences between species.

INTRODUCTION

The diversity of animal size and shape has fascinated scientists for centuries (1, 2). Changes in morphology, such as the beaks of birds, toes of lizards and hands of humans, underpin diversification and adaptation into different ecological niches (3, 4, 5). Yet the genes and genetic changes responsible for animal shape differences between species remain poorly understood (6, 7, 8). Genetic studies have revealed some genes and pathways required for growth and regulation of size and shape during development, many of which are also involved in human disease such as cancer and diabetes (9). These include signaling genes that regulate organ-specific patterning and growth (e.g,, Wnt, Hippo, JAK/STAT), nutritional and hormonal regulators of body size (TOR and insulin signaling), and effector genes of cell growth and proliferation (cell cycle, apoptosis, protein synthesis) (9, 10). However, it is not yet clear whether evolutionary changes in these genes or others underlie size and shape differences between animal species (7).

The parasitoid jewel wasp Nasonia is an emerging as a model system for investigating the genetics of species differences in development and morphology. The Nasonia genus consists of four closely related species, each of which has evolved a distinct male wing size (8,11–13). The greater than twofold difference in male wing size between N. vitripennis and N. giraulti (Fig. 1A) provides a tool for investigating the evolution of growth regulation. We isolated the two largest effect quantitative trait loci (QTL) for this male wing size difference (11, 14); wing-size1 (ws1) (14) and widerwing (wdw) (11), which increase wing size through different mechanisms. The giraulti allele of ws1 increases overall male wing size relative to N. vitripennis. The genetic change underlying ws1 has been mapped to a 13.5kb noncoding interval containing the 5’-UTR of the sex-determining factor doublesex (15, 16) where changes in doublesex expression level in the developing wing are correlated with the wing size difference (8). doublesex is a major sex-determining factor in animals, found from humans to worms, which provides the downstream signal of somatic sex (15) and which also can have a role in growth regulation (8, 16).

Figure 1. Wdw maps to the unpaired-like locus and affects its expression.

Figure 1

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a: Male N. vitripennis and N. giraulti have diverged in wing size. b: Genes in the 200kb region around wdw in N. vitripennis, including a Maverick transposable element that is absent in N. giraulti and does not appear to affect wing size (18). c: Recombinant genotypes (left) and phenotypes (right) map the full wdw effect to a 115kb region. The wing width of haploid males of two genotypes of interest was measured using crosses (Fig. S1) (17) between the original wdw introgression (solid orange bar) and: (i) N. vitripennis, (ii) an introgression recombinant on the left, (iii) an introgression recombinant on the right, and (iv) an introgression recombinant on both sides (wdw-135k). White: N. vitripennis genotype. Orange: N. giraulti genotype. Gray: genotype between markers is unknown. Dark blue bars and *: Statistically significant difference in wing width between genotypes, corrected for multiple testing (Bonferroni). Light blue bars: no significant difference. DFDen: Denominator degrees of freedom from REML ANOVA. Error bars show standard error. d: RNA in situ hybridization reveals that the wdw introgression (wdw-135k line) has broader upd-like expression than N. vitripennis, consistent with the larger wing size. No expression pattern is seen for neighboring genes NV21595 and hmm1064384. Arrowheads denote boundaries of upd-like expression. Quantification of the size of the expression domain is in Fig. S3. Scale bars: 200um.

Positional cloning and identification of wdw

Widerwing (wdw) alters male-specific wing shape as well as wing size, by changing cell numbers differentially across the width of the male wing, but with no effect on female wing size (11). The male wing size phenotype was initially mapped to a 1Mb region by introgressing chromosome segments from N. giraulti into N. vitripennis. Recombination mapping using generated recessive lethal mutations linked to the introgression (Fig. S1) (17) mapped wdw further to 115kb (Fig. 1). During this process, we broke the region into intervals differentially affecting male wing width, which we made into inbred lines for further analysis (18). We also created a 135kb introgression (wdw-135k line) of N. giraulti sequence in N. vitripennis, resulting in males with wide wings which are not significantly different in wing width from the original wdw introgression line (Fig. 1).

Only one gene, NV21594, occurs within the 115kb wdw region (Fig. 1) (18), suggesting that it could be the gene behind the wing size difference. However, it is also possible that genes adjacent to the 115kb region are responsible, as their expression patterns could be encoded in cis-regulatory elements within this region. We tested the expression of NV21594 and adjacent genes NV10313, NV21595 and hmm1064384 in wings using RT-PCR. We detected expression of all but NV10313 in male wings (18). We further investigated whether NV21594 or adjacent genes have spatial expression patterns in developing wings using RNA in situ hybridization (17). Whereas NV21595 and hmm1064384 do not show a spatial pattern of expression (Fig. 1) (18), NV21594 shows a clear spatial expression pattern that differs between N. vitripennis and the N. giraulti introgression. Specifically, it is expressed in the tip of the N. vitripennis prepupal male wing but is expressed much more broadly along the wing edge both in the giraulti allele of wdw in a vitripennis background (wdwgV) and in N. giraulti male wings (Figs. 1, S3, S4) (18). Thus, the correlation between expression pattern and wing size suggest NV21594 as the likely candidate for wdw.

Through searches to other arthropod genomes, we have identified genes with homology to NV21594 in the holometabolous insect orders Hymenoptera, Coleoptera, and Diptera (Fig. S5) (18), which include the Drosophila melanogaster gene unpaired (upd). The upd candidates from these orders share five conserved peptide motifs, including an unusual short motif (WxNPCG) that is diagnostic for upd candidate protein genes in Nasonia, Apis, Tribolium and D. melanogaster (Table S1) (18). We therefore refer to NV21594 as unpaired-like (upd-like). The upd candidates occur in clusters with two paralogous genes (upd-like2 and upd-like3) in each of these taxa, as is also found in D. melanogaster. These proteins are also highly alpha helical (Fig. S5), typical of cytokines (21). Upd in Drosophila regulates cell proliferation as a ligand of the JAK-STAT pathway, and is thus a reasonable candidate for a wing growth gene. Upd is thought to play the same role in Drosophila as the mammalian cytokine Interleukin-6 (IL-6), which is involved a number of human diseases (21).

The wdwg introgression both increases wing width (by 25%) and expands the length of the upd-like expression domain along the wing edge (by 159%) relative to N. vitripennis (Fig. S3). It may be that wdwg introgression could be causing the observed expansion in upd-like expression, but it is also possible that this expansion is simply a byproduct of the larger wing. We thus examined upd-like expression using the large winged ws1g QTL, which increases wing width by 30% (8). Although ws1g and wdwg have wings similarly wider than N. vitripennis, upd-like expression in ws1g is vitripennis-like (Fig. S4). Specifically, expression is absent from the posterior wing margin and significantly contracted relative to wdwg, with a 54% smaller expression edge length (Fig. S3). These results suggest that the wdw region is a driver of both upd-like expression and male wing size. In females, the wdwg introgression does not affect wing size (11), and upd-like is expressed in a broad curve in both introgression and N. vitripennis female wings (Fig. S6). This indicates that upd-like is not a male-specific gene, but rather that its expression is regulated in a male-specific fashion. The dependence of both male wing size and expression pattern on the wdw allele strongly indicates that upd-like is the gene that mediates the wing size difference.

Multiple noncoding intervals around upd-like contribute to the wing size difference

Several recombinants within the 115kb wdw region had intermediate wing sizes, indicating that it is likely that multiple functional sequence changes contribute to the full wdw phenotype. Experimental crosses between recombinant lines that differ in giraulti sequence within the 115kb region around upd-like (Fig. S1) identified three subregions within the 115kb wdw region that differentially contribute to wing size (Fig. 2). The middle subregion (wdw-B) contains both noncoding differences as well as a few coding changes in the first exon of upd-like. However, two of the three subregions, wdw-A and wdw-C, contain exclusively noncoding DNA. We created a wdw-A recombinant line (wdw-A-50k) that contains only 50kb of giraulti noncoding sequence spanning the wdw-A subregion. These wdw-A-50k males have significantly larger wings than N. vitripennis (Fig. 2), suggesting that noncoding changes in the region are sufficient to alter wing size regardless of whether the upd-like protein coding sequence comes from N. giraulti or N. vitripennis.

Figure 2. Multiple intervals within the wdw region affect wing size.

Figure 2

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The wdw phenotype splits into at least three subregions (light dotted lines), each with significant effects on wing size. Heavy dotted lines mark sequence differences between three pairs of adjacent recombinant lines which best define these subregions. Also shown are two recombinants (wdw-A-50k and LB3-125, also used in Figs. 3 and 4) where recombination has isolated the wdw-A and wdw-C subregions.

The three wdw subregions appear to each regulate distinct spatial parts of the wing width difference. We measured changes in width along the wing Anterior-Posterior (AP) axis in our recombinant lines (Figs. 3, 4, S7) and documented that the full wdwg introgression increases wing width in each sector along the AP axis, with a particularly strong effect in the anterior-central sector (Fig. 3). The three subregions subdivide this pattern. Wdw-A only affects wing size in the anterior half of the wing, and appears to contribute primarily to the size of the anterior-central sector (Figs. 3, S7). In contrast, wdw-B and wdw-C have broader effects on wing width, and their specific effects appear to depend on the genotype at other subregions. Specifically, while the spatial effects of wdw-A are similar whether we compare recombinants from the left side or from the right side of the region, the spatial effects of wdw-B and wdw-C (significant differences (Fig. S7) across all sectors or modestly spatially restricted effects) depended on the direction of the comparison. This suggests either that the subregions interact epistatically to produce the full wing size difference or that there are additional modifier QTL, most likely located between wdw-B and C. For subsequent analysis of wdw-C, we focused on a recombinant line (LB3-125) that contains giraulti sequence only at wdw-C. This line has a fairly uniform effect, a 7.4±0.2 um – 11.0±0.9 um (7–15%) increase in each wing sector relative to N. vitripennis (Fig. 4).

Figure 3. Anterior-specific effects of the wdw-A subregion.

Figure 3

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a: The original wdw introgression affects the size of sectors across the width of the adult wing, whereas the wdw-A subregion only affects the anterior-central sector. The width of four anterior-posterior (A-P) sectors was measured for the genotype pairs indicated on the left (17). i: original wdw introgression compared with N. vitripennis, ii: wdw-A-50k compared with N. vitripennis. Dark blue bars and *: Statistically significant difference in sector width between genotypes, corrected for multiple testing (Bonferroni). Error bars show standard error. b: Diagram of the adult wing sectors used in part a and Fig. 4a. c: Illustration of Nasonia wing imaginal disc, which develops in a similar orientation to the adult wing. d: Adult wings reveal anterior-specific expansion in wdw-A. Dashed line marks position of median vein (inferred A-P boundary). Dotted lines mark wing edges. e: wdw-A also regulates anterior-specific expression of upd-like in prepupae. Dashed line marks wing tip (A-P boundary, Fig. S3). Arrowheads denote boundaries of upd-like expression. Scale bars: 200um.

Figure 4. Temporal regulation of upd-like by wdw-C.

Figure 4

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a: A recombinant line containing the giraulti allele of wdw-C (LB3-125) is wider than N. vitripennis in each of four sectors across the adult wing. b: Expression of upd-like before and after pupation reveals a temporal shift in wdw-C. In N. vitripennis, upd-like expression in the tip of the wing is visible only in gray larvae and prepupae. In pupal wings, expression is only apparent in the proximal posterior wing (arrow). In wdw-135k, expression is similar to N. vitripennis in gray larvae but expands apically in prepupae and is active later into pupation. In wdw-C (LB3-125), expression is vitripennis-like in the prepupa and giraulti-like in the pupa, suggesting that this subregion controls temporal regulation of upd-like. Control gene wingless is expressed at all stages (Fig. S8).

Wdw-A causes an increase in wing width in the anterior half of the wing. It also causes an anterior-specific change in upd-like expression, which is broad and giraulti-like in the anterior of wdw-A (wdw-A-50k line) prepupal wings yet contracted and vitripennis-like in the posterior (Figs. 3, S3). We verified that this was a shift in the anterior expression pattern and not a shift in the A-P compartment boundary by costaining with an antibody against the posterior compartment-specific protein Engrailed (Fig. S3).

In contrast to wdw-A’s spatial effects, wdw-C (LB3-125 recombinant line) increases wing width across each A-P axis region (Fig. 4). To our surprise, upd-like expression in prepupal wings is vitripennis-like in this line (Fig. 4), despite the increase in adult wing size. An alternative mechanism that could produce a change in wing size is a temporal expansion of upd-like expression. Indeed, a broad giraulti-like expression pattern emerges in wdw-C pupal wings at a stage where expression in the wing tip is absent in N. vitripennis (Fig. 4) (18). Together, these observations suggest that wdw-C is a late-acting regulatory element with a broad effect.

We inferred the effects of wdw-B even though we did not genetically isolate it as cleanly as wdw-A and wdw-C. Upd-like has a broad expression pattern in recombinants bearing giraulti alleles at wdw-B+C (LB3-208 line), not significantly different from the full introgression (Figs. S3, S9). These data suggest that this subregion controls the early expression pattern while wdw-C controls the late expression pattern. This expression pattern also suggests that both wdw-A and wdw-B activate upd-like expression in anterior cells, perhaps in an overlapping pattern.

Interspecific patterns of unpaired-like expression and wing size

Male wing size and upd-like expression were compared in three Nasonia and two large-winged outgroup species, the closely related Trichomalopsis sarcophagae and more distant Muscidifurax raptorellus (Fig. 5). Small male wings appear to be derived, occurring in both N. vitripennis and N. longicornis, even though N. longicornis is more closely related to N. giraulti (12, 22). The reduced male wing width in N. longicornis appears to be due, in part, to evolution at the wdw locus: introgression of the wdwl region into N. vitripennis (wdwlV) increases adult wing size by 11%, intermediate between N. vitripennis and wdwgV (11). Contraction of the upd-like expression pattern has occurred in both N. vitripennis and N. longicornis males (Fig. 5). Furthermore, the N. longicornis upd-like expression pattern change is also caused by evolution at wdw, as the wdwlV introgression has a contracted expression pattern relative to wdwgV (Figs.5, S3).

Figure 5. Evolution of wing size and unpaired-like expression.

Figure 5

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a: Adult wings and upd-like expression in prepupal wings from Nasonia males and outgroup species. Both N. vitripennis and N. longicornis males have a contracted expression pattern. The species cladogram is adapted from (29). b: A N. longicornis introgression of upd-like into N. vitripennis also shows a contracted expression pattern.

Although the smaller wing size and upd-like expression in N. vitripennis and N. longicornis could be the result of a hybridization event between the species followed by introgression of the wdw region, the data do not support this. Sequence divergence in the three subregions is consistent with the species tree; there is 1.2% divergence between N. longicornis and N. giraulti, less than the respective 2.4% and 2.8%, between these species and N. vitripennis (18). Furthermore, analysis of phylogenetic tree topology with a sliding window across the wdw region, as well as examination of parsimony-informative sites, revealed no evidence of sequence introgression between N. vitripennis and N. longicornis (18). These results indicate that regulatory evolution of upd-like probably occurred in two separate Nasonia lineages, either by parallel reductions in N. vitripennis and N. longicornis or by reduction in an ancestral Nasonia lineage followed by expansion in the N. giraulti lineage. This observation plus the role of three subregions that we have identified between N. vitripennis and N. giraulti suggests that unpaired-like is a hotspot for wing size evolution in Nasonia.

DISCUSSION

This study determined the genetic basis of wdw, a major component of the male-specific wing size difference between N. vitripennis and N. giraulti. Our data indicate that upd-like causes the spatial changes in cell proliferation and growth within the wing and that upd-like is a hotspot for size and shape evolution in Nasonia. Unpaired (upd) in D. melanogaster is a ligand for the JAK/STAT pathway (20). This pathway’s role in cell proliferation makes it a plausible target of morphological evolution, though it is not yet known whether upd-like mediates its effects in Nasonia through JAK/STAT or other pathways. Nasonia upd-like appears to be particularly susceptible to wing growth-altering changes, indicating that the gene might have a specialized role in specifying organ size in the growth gene network. Considering that many size and shape differences between animals are due to differences in cell numbers (9), upd-like genes could be hotspots of size and shape evolution in other species as well.

Using a phenotype-based positional cloning approach, we have identified two different major QTL genes (wdw and ws1) that are responsible for sex specific differences in wing development between closely related Nasonia species. Based on what is known about fly wing development (19, 23, 24), the alternative candidate gene approach would not have predicted the role of either upd or dsx in Nasonia wing size. Nevertheless, both genes appear to be homologs of functionally conserved developmental regulatory genes, which is consistent with the hypothesis that core developmental genes tend to be involved in morphological evolution (25, 26). It has also been argued that non-coding cis-regulatory changes could play a central role in developmental differences between species (27, 28). Our findings support this view, and further implicate growth-regulating genes in organ-specific size and shape evolution.

Supplementary Material

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ACKNOWLEDGEMENTS

We thank L. Gu and J. Sysol for volunteer assistance with mapping, M. Rosenberg for sharing the Nasonia wingless plasmid, and H. Jasper, J. D. Lambert, M. Welte, D. Stern, D. Presgraves, D. Wheeler, R. Edwards, A. Avery and M. Clark for advice and technical assistance. This work was supported by a NSF Doctoral Dissertation Improvement Grant DEB-0910017 to D. Loehlin and NIH grants 5RO1 GM070026-04 and 5R24 GM084917-04 to J. Werren. Sequences are deposited at GenBank with accessions JQ082366-JQ082369.

Footnotes

SUPPORTING ONLINE MARTERIAL

www.sciencemag.org

Acknowledgements

Materials and Methods

Supporting Text

Figs. S1 to S9

Tables S1 to S3

REFERENCES

  • 1.Galilei G. In: Dialogues concerning two new sciences. Crew H, De Salvio A, translators. New York: MacMillan; 1914. [Google Scholar]
  • 2.Darwin C. On the origin of species by means of natural selection, or the preservation of favored races in the struggle for life. London: John Murray; 1859. [Google Scholar]
  • 3.Losos JB, Jackman TR, Larson A, Queiroz K, Rodriguez-Schettino L. Contingency and determinism in replicated adaptive radiations of island lizards. Science. 1998;279:2115–2118. doi: 10.1126/science.279.5359.2115. [DOI] [PubMed] [Google Scholar]
  • 4.Abzhanov A, Protas M, Grant BR, Grant PR, Tabin CJ. Bmp4 and morphological variation of beaks in Darwin’s finches. Science. 2004;305:1462–1465. doi: 10.1126/science.1098095. [DOI] [PubMed] [Google Scholar]
  • 5.McKechnie SW, et al. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol. Ecol. 2010;19:775–784. doi: 10.1111/j.1365-294X.2009.04509.x. [DOI] [PubMed] [Google Scholar]
  • 6.Shapiro MD, et al. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature. 2004;428:717–723. doi: 10.1038/nature02415. [DOI] [PubMed] [Google Scholar]
  • 7.Stern DL, Orgogozo V. The loci of evolution: How predictable is genetic evolution? Evolution. 2008;54:2155–2177. doi: 10.1111/j.1558-5646.2008.00450.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Loehlin DW, et al. Non-coding changes cause sex-specific wing size differences between closely related species of Nasonia. PLoS Genetics. 2010;6(1) doi: 10.1371/journal.pgen.1000821. e1000821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Edgar BA. How flies get their size: genetics meets physiology. Nature Rev. Gen. 2006;7:907–915. doi: 10.1038/nrg1989. [DOI] [PubMed] [Google Scholar]
  • 10.Lecuit T, Le Goff L. Orchestrating size and shape during morphogenesis. Nature. 2007;450:189–192. doi: 10.1038/nature06304. [DOI] [PubMed] [Google Scholar]
  • 11.Loehlin DW, Enders LS, Werren JH. Evolution of sex-specific wing shape at the widerwing locus in four species of Nasonia. Heredity. 2010;104:260–269. doi: 10.1038/hdy.2009.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Raychoudhury R, et al. Behavioural and genetic characteristics of a new species of Nasonia. Heredity. 2010;104:278–288. doi: 10.1038/hdy.2009.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Werren JH, Loehlin DW. In: Emerging Model Organisms. Crotty DA, Gann A, editors. vol. 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2009. pp. 267–298. [Google Scholar]
  • 14.Weston RF, Qureshi I, Werren JH. Genetics of a wing size difference between two Nasonia species. J. Evol. Biol. 1999;12:586–595. [Google Scholar]
  • 15.Raymond CS, Murphy MW, O'Sullivan MG, Bardwell VJ, Zarkower D. Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev. 2000;14:2587–2595. doi: 10.1101/gad.834100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sanders LE, Arbeitman MA. Doublesex establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by directing cell number. Dev. Biol. 2008;320:378–390. doi: 10.1016/j.ydbio.2008.05.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Materials and methods are available as supporting material on Science Online.
  • 18.Supporting text is available as supporting material on Science Online.
  • 19.Hombría JC, Brown S, Häder S, Zeidler MP. Characterization of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 2005;288:420–433. doi: 10.1016/j.ydbio.2005.09.040. [DOI] [PubMed] [Google Scholar]
  • 20.Arbouzova NI, Zeidler MP. JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions. Development. 2006;133:2605–2616. doi: 10.1242/dev.02411. [DOI] [PubMed] [Google Scholar]
  • 21.Rawlings JS, Rosler KM, Harrison DA. The JAK/STAT signaling pathway. J. Cell Sci. 2004;117:1281–1283. doi: 10.1242/jcs.00963. [DOI] [PubMed] [Google Scholar]
  • 22.Werren JH, et al. Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science. 2010;327:343–348. doi: 10.1126/science.1178028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Mukherjee T, Hombría JC, Zeidler MP. Opposing roles for Drosophila JAK/STAT signalling during cellular proliferation. Oncogene. 2005;24:2503–2511. doi: 10.1038/sj.onc.1208487. [DOI] [PubMed] [Google Scholar]
  • 24.Robinett CC, Vaughan AG, Knapp JM, Baker BS. Sex and the Single Cell. II. There is a time and place for sex. PLoS Biol. 2010;8(5) doi: 10.1371/journal.pbio.1000365. e1000365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Werner T, Koshikawa S, Williams TM, Carroll SB. Generation of a novel wing colour pattern by the Wingless morphogen. Nature. 2010;464:1143–1149. doi: 10.1038/nature08896. [DOI] [PubMed] [Google Scholar]
  • 26.Wasik BR, Rose DJ, Moczek AP. Beetle horns are regulated by the Hox gene, Sex combs reduced, in a species- and sex-specific manner. Evol. Dev. 2010;12:353–362. doi: 10.1111/j.1525-142X.2010.00422.x. [DOI] [PubMed] [Google Scholar]
  • 27.Stern DL. Perspective: Evolutionary developmental biology and the problem of variation. Evolution. 2000;54:1079–1091. doi: 10.1111/j.0014-3820.2000.tb00544.x. [DOI] [PubMed] [Google Scholar]
  • 28.Carroll SB. Evolution at two levels: On genes and form. PLoS Biology. 2005;3:1159–1166. doi: 10.1371/journal.pbio.0030245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Raychoudhury R, Baldo L, Oliveira DC, Werren JH. Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex. Evolution. 2009;63:165–183. doi: 10.1111/j.1558-5646.2008.00533.x. [DOI] [PubMed] [Google Scholar]
  • 30.Tautz D, Pfeifle C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 1989;98:81–85. doi: 10.1007/BF00291041. [DOI] [PubMed] [Google Scholar]
  • 31.Lynch JA, Brent AE, Leaf DS, Pultz MA, Desplan C. Localized maternal orthodenticle patterns anterior and posterior in the long germ wasp Nasonia. Nature. 2006;439:728–732. doi: 10.1038/nature04445. [DOI] [PubMed] [Google Scholar]
  • 32.Loots G, Ovcharenko I, Pachter L, Dubchak I, Rubin E. rVISTA for comparative sequence-based discovery of functional transcription factor binding sites. Genome Res. 2002;12:832–839. doi: 10.1101/gr.225502. [DOI] [PMC free article] [PubMed] [Google Scholar]

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