Creating a new species is a bit like climbing a greased flagpole—it's hard to get started and even harder to keep going. Random genetic variations may introduce slight differences between two groups but, without some means to keep them apart, sexual interbreeding will quickly remix the genes and obliterate the differences. Accidents of geography—the rising of mountains or a course-changing river, for instance—can provide physical isolation, which then enables genetic divergence through the accumulation of mutations, either through natural selection or genetic drift.In contrast, speciation without geographic separation relies on the direct action of natural selection to complete the speciation process by strengthening behavioral differences, a process called reinforcement. One of the most powerful means of completing speciation is through the evolution of mate discrimination.Drosophila. They identify two loci that influence the likelihood that a female will choose to mate with a conspecific male, rather than one of a closely related species.A study by Daniel Ortiz-Barrientos and colleagues focuses on the genetic underpinnings of mate discrimination in Drosophila pseudoobscura and D. persimilis exist together along the west coast of the United States (sympatry), but separately elsewhere . When together, they hybridize and produce sterile males. While D. pseudoobscura males will court females of both species, females prefer conspecific males. This female preference is stronger in sympatric females, an enhancement that presumably evolved by the direct action of natural selection to prevent females from wasting their reproductive efforts producing sterile sons. This variation allowed the authors to conduct a series of genetic crosses among flies of the same species but from different locations. Because the daughters of discriminating D. pseudoobscura females were just as discriminating as their mothers, Ortiz-Barrientos and colleagues concluded that female mating discrimination was inherited as a dominant trait. Further crosses showed that genes responsible for female preference were on the X and fourth chromosomes, and high-resolution mapping refined their locations sufficiently to allow the identification of likely candidate genes. While more work remains to be done, the most promising genes in both regions appear to be involved with olfaction. The fact that one of them, CG13982, is known to up-regulate the other, bru-3, strengthens the case that these are indeed promising candidates.D. pseudoobscura based on the combined response to auditory and olfactory cues. The first of these two layers, weak, or “basal” mating discrimination, has previously been associated with a set of traits for acoustic recognition and mapped to chromosomal regions that are inverted between the two species. Such inversions prevent recombination from purging alleles, thereby contributing to hybrid male sterility. As a consequence, when these species interbreed, they inexorably produce sterile males. The second layer, elucidated in the current study, is “reinforced” mating discrimination, which appears to be related to olfactory cues. This additional system of discrimination helps the first layer to fully eliminate the inevitable cost of producing sterile males. Once the nascent species have started up that slippery pole, reinforced discrimination could provide the traction needed to reach its top.According to their findings, the authors propose a novel model of mating discrimination in


Caenorhabditis briggsae, strain-specific variation in ray pattern has provided an entrée into the evolution of ray pattern. Some strains were fixed for a derived pattern. Other strains were more plastic and exhibited derived and ancestral patterns at equal frequencies.How does intraspecific variation relate to macroevolutionary change in morphology? This question can be addressed in species in which derived characters are present but not fixed. In rhabditid nematodes, the arrangement of the nine bilateral pairs of peripheral sense organs (rays) in tails of males is often the most highly divergent character between species. The development of ray pattern involves inputs from hometic gene expression patterns, TGFβ signalling, Wnt signalling, and other genetic pathways. In C. briggsae AF16 and HK104 strains exhibited a wide range of phenotypes including some that were more extreme than either parental strain. Transgressive segregation was significantly associated with allelic variation in the C. briggsae homolog of abdominal B, Cb-egl-5. At least two genes that affected different elements of ray pattern, ray position and ray fusion, were linked to a second gene, mip-1. Consistent with this, the segregation of ray position and ray fusion phenotypes were only partially correlated in the RILs.Recombinant inbred lines (RILs) constructed from crosses between the variant The evolution of ray pattern has involved allelic variation at multiple loci. Some of these loci impact the specification of ray identities and simultaneously affect multiple ray pattern elements. Others impact individual characters and are not constrained by covariance with other ray pattern elements. Among the genetic pathways that may be involved in ray pattern evolution is specification of anteroposterior positional information by homeotic genes. Caenorhabditis elegans and Oscheius tipulae, cells in the vulval equivalence group that do not participate in vulval development are eliminated through fusion with the multinucleated syncytial epidermis . In C. elegans, unc-22 is located on chromosome IV and is linked to several genes known to regulate homeotic gene expression patterns, e.g. lin-49 which encodes a bromodomain protein thought to be involved in chromatin remodeling . These modeling . mip-1 mC. briggsae strains were constructed in which HK104 DNA, in the region of mip-1, has been introgressed into an otherwise AF16 background. This was accomplished through a series of backcrosses that were initiated by mating HK104 males to mip-1 (AF16) hermaphrodites. For each introgressed strain, F1 through F6 males were crossed to mip-1 (AF16) hermaphrodites. Wild-type F7 hermaphrodites were selected and propagated by self-fertilization. From each F7 hermaphrodite, multiple wild-type F8 hermaphrodites were picked. Homozygous strains were established from F8 hermaphrodites that segregated only wild-type progeny. From each set of backcrosses, a single introgressed strain was retained. These introgressed strains were scored for the position of ray 3 and for ray 3–4 fusions . Moreover, evidence for a gene affecting only ray fusion was obtained from comparisons of ray patterns exhibited by the mip-1 introgressed strains. Hence, variation in ray pattern evolution is not wholey constrained by the specification of ray identities and other ray pattern elements, such as neurotransmitter usage, also may vary independently. A similar pattern of partial constraint and flexibility has been observed for variation in eyespots size in the butterfly Bicyclus anynana [Ray pattern in  fusions . Derivedentities ,22-25,30 anynana .C. briggsae have been described [C. briggsae-like ray pattern also has been reported for C. clavopapillata [C. clavopapillata has not been observed since its first description and some authors have considered it and C. briggsae to be synonymous [C. clavopapillata is distinct from C. briggsae, the posterior position of ray 3 and its frequent fusion with ray 4 may be ancestral for these two species. This would make the C. elegans-like ray pattern in haplotype 2 an atavistic character. It should be possible to discriminate between these two models either through the characterization of additional C. briggsae strains or through the redescription and molecular characterization of C. clavopapillata.Two haplotypes of escribed  and variescribed . Strainsapillata , anothernonymous . If C. cC. briggsae. The relatively slight difference between the AF16 and HK104 phenotypes hid a wealth of genetic variation. This was evident in the nearly continuous variation exhibited in RILs for both the position of ray 3 and its fusion with ray 4. This variation included many RILs with phenotypes more extreme than either parental strain. Such transgressive segregation is common in both plants and animals and is thought to arise through fluctuating selection and/or through stabilizing selection at minor QTL when directional selection at major QTL overshoots a phenotypic optimum [C. elegans, ray 3 precursor cells are born at the junction of the mab-5 and egl-5 expression domains [C. elegans ray pattern to phenocopy that of C. briggsae [Cb-egl-5 and variation in ray pattern. As Cb-egl-5 and Cb-mab-5 are closely linked, variation in either or both of these genes may be responsible for the observed association. Alternatively, linkage to Cb-egl-5 may be coincidental, and ray pattern variation in C. briggsae may not result from variation in homeotic gene expression patterns. The best test of these alternative models will be the high resolution mapping of the allelic variants responsible for ray pattern variation.Regardless of their evolutionary history, haplotypes 1 and 2 provided an entrée to the genetics of morphological variation of ray pattern in  optimum . The tra domains ,47. Thes domains  and somebriggsae ,26. We hmip-1 marker assisted introgression studies. The C. elegans homolog of mip-1, unc-22, is not linked to the the homeotic gene cluster and linkage of mip-1 to Cb-egl-5 and Cb-mab-5 is unlikely. There appear to be at least two loci that affect ray pattern linked to mip-1. One of these affected both the placement of ray 3 and its fusion with ray 4. The other affected only ray fusion. These associations were not transgressive, i.e. the allelic variants linked to mip-1 antagonized the allelic variants linked to Cb-egl-5. If the transgressive segregation linked to Cb-egl-5 does result from allelic variation in one or more homeotic genes, then the antagonistic variation linked to mip-1 may be in genes that regulate homeotic gene expression patterns. A direct test of this hypothesis is possible. Several genes required for proper regulation of homeotic gene expression patterns have been identified in C. elegans [C. briggsae homologs of these genes could be tested for association with ray pattern variation in the RILs. Ideally, these tests would be integrated into a genome wide screen for variant loci with effects on ray pattern. This will require the enhancement of genetic resources available for C. briggsae.Loci not linked to the homeotic gene cluster also must be involved in ray pattern variation. Direct evidence for this was obtained from the  elegans ,37,38,49Caenorhabditis provides a powerful model for the study of morphological evolution. Macroevolutionary comparisons between species and microevolutionary analyses of variation within species are possible. Augmenting these approaches is a detailed understanding of the genetic and cellular basis of ray pattern development in C. elegans. In C. briggsae, intraspecific variants have been characterized that affect the expression of ancestral and derived ray patterns. These variants have a complex genetic basis involving multiple genes. Some of these genes exhibit transgenic segregation, some affect all of elements of ray pattern, and some that affect only a subset of ray pattern elements. At least one gene that affects ray pattern variation in C. briggsae is linked to the homeotic gene cluster. Thus, ray pattern variation may result from altered expression patterns of homeotic genes. Further characterizations of the genetics of ray pattern variation will test this model and will address interactions between different genes that impact ray pattern in C. briggsae.Ray pattern in C. briggsae strains AF16, and HK104 are available from the Caenorhabditis Genetics Center [E. coli strain OP50. Recombinant inbred lines (RIL) were constructed starting with HK104 males mated to sperm-depleted AF16 hermaphrodites [s Center . These shrodites . Each RIRay pattern phenotypes were scored using differential interference contrast optics at a magnification of 400× . Right aCb-egl-5, CAGGGAGCGGACAACTTCAAAGG and GGACACAGCCCAGGATTAGCGAC, respectively, were designed based on genome sequence data from C. briggsae strain AF16 [Forward and reverse primers for PCR amplifications ain AF16 . AmplifiCb-egl-5 allelic variation with ray pattern variation were determined online at [mip-1 introgressed strains were compared using reciprocal chi-squared tests using Excel v10.1.0 .Pearson's product moment correlation cofficients between ray pattern elements in RILs and Wilcoxon nonparametric tests of the association between nline at . Frequennline at . Ray patdll = distalless; en = engrailed; sal = spaltRIL = recombinant inbred line; QTL = quantitative trait loci; SEB: Planned and supervised all research activities. Participated in construction and genotyping of RILs. Reviewed all statistical analyses. Wrote manuscript.CRD: Constructed, genotyped, and characterized ray pattern phenotypes of RILs. Statistical analyses of the segregation of ray pattern phenotypes and association of Cb-egl-5 variation with ray pattern variation. Reviewed manuscript.JCB: Constructed and characterized phenotypes of mip-1-assisted introgressed lines. Statistical analyses of ray pattern phenotypes of mip-1 introgressed lines. Reviewed manuscript.Cb-egl-5 genotypes and ray pattern phenotypes of RIL derived from AF16 × HK104.AF16 × HK104 RIL ray pattern data. Click here for file


Drosophila, the endemic Hawaiian species offer some of the most dramatic examples of morphological and behavioral evolution. The advent of the Drosophila grimshawi genome sequence permits genes of interest to be readily cloned from any of the hundreds of species of Hawaiian Drosophila, offering a powerful comparative approach to defining molecular mechanisms of species evolution. A key step in this process is to survey the Hawaiian flies for characters whose variation can be associated with specific candidate genes. The wings provide an attractive target for such studies: Wings are essentially two dimensional, and genes controlling wing shape, vein specification, pigment production, and pigment pattern evolution have all been identified in Drosophila.Within genus Drosophila. The image collection, available at FlyBase.org, includes 53 of the 112 known species of “picture wing” Drosophila, and several species from each of the other major Hawaiian groups, including the modified mouthparts, modified tarsus, antopocerus, and haleakalae (fungus feeder) groups. Direct image comparisons show that major wing shape changes can occur even between closely related species, and that pigment pattern elements can vary independently of each other. Among the 30 species closest to grimshawi, diverse visual effects are achieved by altering a basic pattern of seven wing spots. Finally, we document major pattern variations within species, which appear to result from reduced diffusion of pigment precursors through the wing blade.We present a photographic database of over 180 mounted, adult wings from 73 species of Hawaiian Drosophila, despite their generally low levels of DNA sequence divergence. In several independent lineages, highly complex patterns are derived from simple ones. These lineages offer a promising model system to study the evolution of complexity.The database highlights the striking variation in size, shape, venation, and pigmentation in Hawaiian  Drosophila are endemic to Hawaii, yet current evidence suggests they arose from a single introduction to the Hawaiian Island chain roughly 26 million years ago Drosophila an important model system for analysis of evolutionary processes at the species level.Nearly 1000 species of Drosophila grimshawi genome has been sequenced grimshawi sequence should permit the amplification of nearly any gene of interest from a range Hawaiian species. Identified sequence differences can then be correlated with phenotypic variations among the species, providing insights into molecular mechanisms of evolution. To make the most of this opportunity, it is important for researchers to have access to uniformly collected phenotypic data from numerous species. The data can be used to identify characters that show interesting patterns of variation, and for which candidate genes can be hypothesized. The Drosophila wing is an attractive target for such candidate-based studies, since wing development has been analyzed in great detail in D. melanogasterThe grimshawi, the prepattern is faintly visible upon eclosion, marked by an arrangement of dark versus light wing hairs. In the first day or two after eclosion, pigment precursors travel through the wing veins and diffuse into the intervein regions, allowing further darkening of the cuticle into clearly visible spots. In this model, the spots must contain localized enzymes that are waiting to convert the precursors to melanins. This enzyme prepattern is most likely formed by localized expression of pigmentation genes in response to the wing's basic patterning machinery. Wing spot evolution would then involve changes in either the upstream patterning genes, or the downstream pigmentation genes. Changes in patterning genes would tend to be pleiotropic, altering other features of the wing, so this explanation is unlikely when only pigment changes are observed. Thus, the favored explanation is that mutations occur in the cis-regulatory regions of the pigmentation genes, bringing them under control of existing, region-specific activators or repressors Wing pigment spots occur in highly reproducible, species-specific, two-dimensional patterns, and their genetics and development are beginning to be understood. True et al. yellow locus have provided multiple examples of regulatory mutations controlling the evolution of wing spots. The Yellow protein is required to pigment the cuticle, and ectopic Yellow causes dark pigmentation in a wild type background. This Yellow-dependent pigmentation is strongly enhanced by removal of Ebony protein  yellow and ebony genes have been co-opted during evolution to produce wing spots: a male-specific wing spot in D. biarmipes is presaged by increased Yellow and decreased Ebony protein levels, and the extent of the spot is controlled in part by engrailed regulation of yellow via a novel cis-regulatory element Drosophila, and yellow has at least two distinct regulatory elements that can be co-opted to produce spots Studies of the Drosophila. Unfortunately, these pigment patterns have not been photographically documented in the literature, apart from a few sporadic examples .These studies provide the framework required to understand the evolution of complex pigment patterns in the Hawaiian Drosophila species. Mounted wings were digitally photographed under uniform conditions to allow for comparisons between specimens, and the photos have been made available for download at FlyBase grimshawi genome to gain further molecular insights into morphological evolution.Here we present a photo database documenting the wings of 73 Hawaiian Drosophila arose from an introduction of a continental species to an island (now subsided) that predates Kauai, the oldest of the current high islands The endemic Hawaiian Drosophila. The original photographs and the montages are available in the Hawaiian Drosophila Wing Database at FlyBase adiastola subgroup of picture wing species . Each box represents a unique inversion genotype or karyotype present in the designated species (abbreviated to 3 letters). Circles represent inversion genotypes that do not match any species in the database; these are only included when they constitute nodes in the tree. The actual inversion names have been omitted for simplicity; see Carson grimshawi subgroup lacks these inversions (since D. grimshawi is the standard); the glabriapex subgroup has 4b, the planitibia subgroup has Xik and 4b, and the adiastola and primaeva subgroups have Xo 2c, Xik, and 4b. Relationships among the four subgroups can be obtained by connecting the trees at these points, as summarized in primaeva based on DNA and biogeographic evidence The picture wing group is divided into four major subgroups named for representative species: diastola , planitianitibia , glabriaabriapex  and grimrimshawi . A nearlrimshawi –5. D. adiastola subgroup. These species are particularly notable for the intricate and subtly graded pigment patterns of the wings. In addition, much of this group shows pronounced sexual dimorphism, and so clavisetae , likely as an adaptation that provides mechanical support for larger wings. This adaptation arose independently in the planitibia subgroup (below)spectabilis, the pigment spots are expanded and fused, giving the appearance of a black wing with light spots. The most extreme wing shape change in this collection (and perhaps in the genus) is seen in truncipenna, in which the male wings are blunted at the tips giving a nearly rectangular appearance. The female wing is slightly blunted as well, but the selection pressure on this phenotype appears to be focused on the males. The hamifera wing is perhaps the most divergent overall, with an exceptional combination of large size, distorted shape, and complex, dimorphic pigmentation. The males and females share a dark spot over the proximal part of longitudinal veins L2–4, but the rest of their patterns appear to be almost completely unrelated.primaeva/attigua specimen; these two species are considered to form their own subgroup at the base of the picture wing clade . The wings may be dimorphic in shape (longiseta) and pigmentation (stigma). The stigma wing pattern closely matches those of the Asian species D. biarmipes and elegans, which have been recently analyzed by Gompel et al. The  species  are sexuhaleakalae group  can “slide” to different positions along the proximodistal axis, generating a rainbow-like pattern in the overlay; the other spots remain largely fixed. The proximal and distal borders of this spot can vary independently, as shown by the aligned close-ups of L2 . The overlay shows that the posterior compartment also differs, with L4 and L5 diverging strongly in tanythrix.Species within one group can vary substantially in shape, as noted above for cilifera wings shows that sexual dimorphism is achieved by varying only a subset of pattern elements . The wing pattern in hamifera can become altered in an even more complex manner. Compared to the typical pattern . Thus, comparisons among these species could provide insights into the evolution of complexity.We show several examples in which, along a known lineage, species exhibit increasing pigment pattern complexity or gain/loss of discrete pattern elements. It will be extremely informative to sequence candidate loci such as is group . The commplexity . These tadiastola subgroup. Evidence suggests the basal primaeva wing gave rise to the simple, wave-like pattern of ornata, and the more derived species have extensively modified this pattern along different branches of the adiastola subgroup  availability of genomic DNA for comparative sequence analysis; (2) ability to grow larvae for studies of gene expression and developmental biology; (3) ability to make transgenic flies; (4) ease of performing transmission and quantitative genetics D. melanogaster. DNA is available from most of the species pictured here, and cloning genes of interest will be greatly facilitated by the high sequence identity levels among the Hawaiian species. Carson's chromosome phylogeny was derived by analysis of larval chromosomes, indicating that larvae can be cultured from nearly every picture wing species D. melanogaster proteins (not shown). Transgenic Hawaiian Drosophila have been produced by injecting P element DNA into D. hawaiiensis embryos mimica) and ornate  wing patterns, although grimshawi lay eggs at a greater rate than mimica. For optimal egg collection, specialized substrates are required  Stocks of mimica, grimshawi, and several other endemic Hawaiian species are available at the Tucson Drosophila Species Stock Center. Genetic markers are not currently available, although we have demonstrated that visible mutations can be isolated and maintained in grimshawisilvestris vs. heteroneura coloration and head shape D. grimshawi, for example, can hybridize with balioptera, bostrycha, crucigera, disjuncta, pilimana, and others Each of these milestones has been reached in the picture wing flies, albeit with more effort than required for Flies were collected from banana or mushroom baits, or by netting, at previously described locations on Kauai, Oahu, Molokai, Maui, and Hawaii (Big Island) disjuncta, grimshawi, hemipeza, heteroneura, planitibia, and silvestris, specimens were taken from laboratory stocks maintained at the Univ. of Hawaii at Manoa instead of from the field. Picture wing stocks are cultured at 18°C. Oviposition occurs in vials of Wheeler-Clayton medium Clermontia . Once larval activity is observed, cornmeal-molasses-agar medium is added to the vials. Vials with third instar larvae are placed in a gallon jar half filled with damp, coarse sand. Larvae tunnel into the sand to pupate, and adults crawl back out upon eclosion. Newly eclosed adults require 2–3 weeks to reach sexual maturity; females especially require 3–4 weeks before mating and egg laying begins. Temperature and humidity regulation, culture media specific to larval and adult nutritional requirements, sterile sand as the pupation medium, etc., make laboratory husbandry of the Hawaiian Drosophila species significantly more complex than D. melanogaster. However, a number of laboratories in the U.S. as well as internationally have been successful in maintaining laboratory stocks of Hawaiian Drosophila and have been able to conduct genetic and behavioral studies on these species.For algaia, cyrtoloma, clydonia, differens, diminuens, hamifera female, hirtipalpus, truncipenna, and virgulata, the flies were recently dead when the wings were removed. Older, pinned specimens were found to be rather unsuitable for the project since their pigmentation had faded. The wings were permanently mounted in Euparal  between a slide and coverslip, taking care to avoid damage and folding. Slides were incubated overnight at 37°C to allow bubbles to dissipate, and stored in the dark. The wings were all photographed in one session under uniform conditions, using a digital camera mounted on a dissecting scope and illuminated with an overhead ring light. Raw images were adjusted in Photoshop using the “Warming Filter 81” command to neutralize the background toward gray, and contrast was restored using “Curves”. All adjustments were performed to make the backgrounds uniform across images, so that the wings are as directly comparable as possible. In Drosophila system is used here for simplicity and longitudinal veins L1–5 are defined in In most cases wings were removed from live flies; for


Study of Darwin's unpublished works, freely available on-line through the American Natural History Museum, reveals the origins of his thoughts on evolution. Journal of Researches [The Voyage of the Beagle was his most famous—until Darwin, pressured by the arrival in 1858 of A. R. Wallace's manuscript on evolution through natural selection, stopped working on his “Big Species Book,” [On the Origin of Species by Means of Natural Selection. Or the Preservation of Favoured Races in the Struggle for Life [Origin of Species that changed the world, establishing Darwin as one of the great thinkers in Western cultural history.Depending on how you count them up, Charles Darwin published just over twenty books in his lifetime. His first—the searches , also kns Book,”  and wrotfor Life . In betwOrigin of Species. All have been subsequently published, and are now freely available online, constituting the initial components of the Darwin manuscript project page of the American Museum of Natural History Digital Library of Evolution. The Darwin manuscript project complements the museum's exhibition, Darwin, opening 19 November 2005; further analysis of these works can be found in my companion volume to the exhibition [So much is well-known. Far less appreciated is the fact that Darwin wrote several other books, all on evolution, none of which were published in his lifetime. Together, they form a series that preserves the “evolutionary” history of Darwin's ideas from their very inception to their most mature form—while also revealing the more prosaic development of Darwin's written rhetoric of the hibition .Origin of Species are found in the pages of these notebooks—maddeningly interspersed in near-chaotic fashion with all manner of geological and biological observations and notations gleaned from the literature, and Darwin's already burgeoning correspondence and conversations. Indeed, only the “principle of divergence” came along later to add to Darwin's themes and arguments.The first of these books is actually a series of notebooks. I include them because they are the foundational writings for all Darwin's later, more discursive, discussions of evolution. Many of the themes—and, indeed, some of the original language—of the familiar passages of the Beagle in 1836—recording various latitude, longitude, and depth soundings—the last third of the notebook seems to have been filled out after Darwin returned to England in late 1836, early 1837. Historians still disagree whether or not—or the degree to which—Darwin had tumbled to the idea of evolution while still on the Beagle. I fully agree with Kohn et al. [Ornithological Notes, discussing the differentiation of “varieties” of mockingbirds and tortoises on various islands in the Galapagos and concluding that “if there is the slightest foundation for these remarks to zoology of Archipelagoes—will be well worth examining; for such facts would undermine the stability of Species” [Beagle arrived home. But nothing else unambiguously written while still aboard ship has as yet turned up to support this view.The “Red Notebook”  is the fOrigin of Species, Darwin had been greatly struck by “certain facts of the distribution of the inhabitants of South America” that “throw some light on the origin of species.” [Darwin was a fully committed evolutionist by the time the evolutionary passages of the “Red Notebook” were written. As he would subsequently write in the topic sentence of the pecies.” . ElsewheOrigin of Species, is unexpectedly a saltationist in the “Red Notebook.” He thinks, given the lack of intergradations between fossil forms, or his rheas, that new species must arise suddenly from ancestral species. He maintains this view to some degree in Notebook B, first of the four famous “Transmutation Notebooks” [Darwin, famous for his views of gradual evolution through natural selection in the tebooks” , begun iThere is grandeur in this view of life.Systema Naturae [2, he writes  “I think,” [First of these new expected patterns is the nested set of taxa already recognized and embodied in Linnaeus's  Naturae . We now  think,”  and sketOrigin of Species), he formulated “natural selection.” As David Kohn [But Darwin wanted more: he was constantly searching for a mechanism. Finally, in Notebook D, after having read Thomas Malthus and learned for the first time that more organisms are born to each species each generation than can possibly survive and reproduce . Darwin had been using the expression “my theory” to mean “evolution.” But now, the expression “my theory” more specifically means “evolution by natural selection.” It is in 1839, toward the end of the series of “Transmutation Notebooks,” that Darwin takes his next logical, if not fateful, step: in page 118 of Notebook E, he exhorts himself to rederive his original patterns in terms of his ideas on how natural selection works to produce evolutionary change. He is by now far beyond his initial attraction to saltational evolution: rather natural selection must produce finely gradational change. This puts him at odds with his very first evolutionary pattern, as Darwin is aware that paleontologists see little evidence of such change in their collections of fossilized plants and animals. He writes : “My very theory requires each form to have lasted for its time: but we ought in same bed if very thick to find some change in upper & lower layers.—good objection to Origin of Species.After discovering natural selection in Notebooks D and E, Darwin turns renewed attention to both variation and the process of artificial selection in embryonic form— the analogy to what he would soon call “natural selection” by this time clear. The theme that varieties are incipient species—perhaps the most pervasive of Darwinian argumentative themes—is found in these notebooks , as are Particularly striking is Darwin's invocation of the travails of astronomers who labored so hard  to establish the laws of gravitation governing the behavior of celestial bodies. Darwin was not only fearful of attack on religious grounds, he also knew all too well that the only competing theory to explain the origin and diversity of life was in fact Judeo–Christian creationism. In Notebook B (page 101) , Darwin Origin of Species some 21 years later. Here we have not only the analogy with scientific law replacing creationist belief in astronomy, but also the origin of the famous phrase “there is grandeur in this view of life” [This passage is “ancestral” to Darwin's most famous passage—concluding the of life” ,11,12. WFoundations of the Origin of Species [In 1909, Francis Darwin (Charles and Emma's seventh child), on the 100th anniversary of his father's birth, published  Species . The boo Species  and the  Species .The “Sketch” is Darwin's earliest known  attempt to write out his evolutionary theory in essay form. The fact that it was never intended for publication, but rather served as a first-shot “dry-run” in setting out his views, is amply demonstrated by the sometimes elliptical, almost notebook-like passages with incomplete sentences and occasional reminders to himself on how to develop his arguments further. .The 1842 “Sketch” is an exciting read. Darwin is effectively organizing his thoughts and putting them in more coherent form for the first time. He adopts a two-part structure  a succinct statement of his theory of the mechanisms of evolution, and a longer part 2 (seven chapters) the application of his ideas of evolution through natural selection to the, by now, familiar patterns of the biological world —and the persistent problems with the fossil record).It is in the second chapter of part 1 that we see the fateful two words “natural selection” as a subhead of a section that lays out by far his most coherent description of the process to date: “DeCandolle's war of nature—seeing contented face of nature—may be well at first doubted; we see it on borders of perpetual cold. But considering the enormous geometrical increase in every organism and as every country, in ordinary cases, must be stocked to full extent, reflection will show that this is the case. Malthus on man—in animals no moral restraint—they breed in time of year when provision most abundant, or season most favourable….The unavoidable effect of this is that many of every species are destroyed either in egg or young or mature….In the course of a thousand generations infinitesimally small differences must inevitably tell….Nature's variation far less, but such selection far more rigid and scrutinizing” .The bulk of Darwin's 1842 text integrates all he has read in books, monographs, and correspondence about variation, artificial selection, patterns of geographic distribution of animals and plants, and gradation between varieties and distinct species—the main topics of his notebooks. He continues the hypothetico-deductive theme begun in Notebook B—showing that such patterns should be expected as the natural outcome of the evolutionary process.Origin of Species itself: “There is a simple grandeur in the view of life with its powers of growth, assimilation, and reproduction, being originally breathed into matter under one or a few forms, and that whilst this our planet has gone circling on according to fixed laws, and land and water, in a cycle of change, have gone on replacing each other, that from so simple an origin, through the process of gradual selection of infinitesimal changes, endless forms most beautiful and most wonderful have been evolved” [His “Recapitulation and Conclusion” in the 1842 “Sketch” is a brilliant, impassioned summary of his ideas. It ends with the passage, already adumbrated in Notebook D, that remains virtually identical, not only in 1844 but in the evolved” .For the most part, Darwin's fervent intellectual search is over with the conclusion of the 1842 “Sketch.” The 1844 “Essay,” at 198 pages, is a much longer manuscript; with exactly the same structure and sequence of topics, it is essentially a smoothed out version of its predecessor—written completely in essay form now, all sentences complete, with no personal notes and queries to interrupt the flow of ideas. The bulk of it, for the most part, consists of vastly more examples bolstering Darwin's points throughout.That said, the 1844 version of his ideas is far less exciting to read than the 1842 manuscript. It is very much as if the excitement is muted by the sheer bulk of the material reviewed—and probably as much by the fact that the ideas are no longer so novel to Darwin himself. Freshness is lost to familiarity and the sheer weightiness of his verbiage.The Origin of Species. This, his most famous book, was fresh and new to its readers in 1859, so successful had Darwin been in keeping his views private. But to anyone who has had the privilege of reading the 1830s notebooks, and the early manuscripts , the Origin of Species reads like a mature work in both the best and worst sense of the term. He has honed his arguments beautifully, but the ideas, no longer fresh in his own mind, are just not as enthrallingly expressed as when he was younger and much closer to their inception.Much the same can be said of Darwin's so-called abstract of his views—Natural Selection [Origin of Species, it is true that, at least as far as the discussion of the Principle of Divergence is concerned, the discussion in this last of Darwin's unpublished-in-his-lifetime books is more cogent and complete than the Origin of Species itself. The Origin of Species, one is tempted to conclude, written as it was in such haste, relied heavily on the earlier manuscripts.  In many ways, the unpublished versions hold more rewards than what the Origin of Species—the book that shook the world—offers the modern reader.Darwin's Principle of Divergence—poorly understood by modern scholars—melds nascent ecological theory with various models of the origination of new species. Darwin was developing those ideas around the time he started writing his “Big Species Book,” eventually published (second part only) as election . And thoother books to pick themes and trace their development over time. Seldom has the history of ideas, so important as Darwin's evolution by natural selection, been so faithfully preserved as it has in this virtual “fossil record” preserved in this magnificent series of Darwin's other books.There is much more that can be said. Darwin's thoughts about the relative importance of isolation, for example, changed over the years. It is possible with this treasure trove of Darwin's Natural Selection (1856-1858) are freely available online at http://darwinlibrary.amnh.orgThe "Red" and "Transmutation" notebooks (1836-1839), the "Sketch" (1842), the "Essay" (1844), and


Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occuring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-β signaling pathways as putative morphogens for eyespot development.Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development.Saturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Wingless and TGF-β ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution. The wings of butterflies and moths display a wealth of color patterns that provide excellent material for investigating the evolution of pattern formation in a simple, two-dimensional system. Pattern elements consisting of one or more concentric rings of colored scales, the eyespots, can occur at different positions in the wing in different lineages and also display different morphologies and, thus, make intriguing subjects for investigating questions of homology . When eyIdeally, tests of homology should include not only comparisons at the level of the phenotype but also of the genes and developmental processes underlying that phenotype ,5. This Research on eyespot developmental mechanisms has mostly focused on the border eyespots of nymphalid butterflies. Two and a half decades ago, Nijhout proposed that a group of signaling cells, the focus, organizes the differentiation of butterfly eyespot patterns by producing a long range diffusible morphogen that is interpreted in a threshold-like fashion by the surrounding epidermal cells . When thBicyclus anynana , ectopic eyespots consisting of a ring of gold scales, sometimes containing a black central disc, appear on the adult wing following pupal wing wounding [The sink model, in particular, is derived from the observation that eyespot mimics, containing rings of differently colored scales but no central white pupil, can differentiate on the wing following pupal epidermal wounding -9. The wwounding . The genDistal-less (Dll), Notch (N), engrailed (en), hedgehog (hh), cubitus interruptus (ci), patched (patch), spalt   appear to be involved in the differentiation of the central signaling cells [A series of genes have been associated with nymphalid eyespot development. In the larval stage, ng cells -19. Lateng cells ,21. The B. anynana. We also follow the expression of these proteins around wound sites in the pupal wing to determine the extent to which ectopic eyespots share developmental pathways with normal eyespots. Finally, we compare gene expression patterns in the discal-cell eyespots of the moth species Antheraea polyphemus and Saturnia pavonia  and in the uniformly-colored, non-pupilated, spots of the cabbage white butterfly Pieris rapae, from a more basal butterfly family , to determine to what extent they are sharing similar developmental pathways.Here we investigate whether the Wingless (Wg) and Transforming Growth Factor-beta (TGF-β) signaling pathways are involved in differentiating the border eyespots, as the ligands in these pathways are known morphogens in other systems (reviewed in ). Using en-expressing scale-building cells is visible around the eyespot focus  and present until 16 h after pupation , whereas Dll was expressed in the intervenous stripes  after wounding . Also, wg and decapentaplegic (dpp), a ligand from the TGF-β family, in the nymphalid butterfly Precis coenia using in situ hybridizations [B. anynana eyespot centers from the early pupa until around 16–22 h after pupation, a critical developmental period when pupil transplants and ablation experiments were originally shown to affect the eyespot phenotype [Until functional data is collected for Wg and TGF-β family members, these ligands remain excellent candidates for association with focal signaling and eyespot differentiation due to their temporal and spatial localization. The presence of Wg and pSmad along the wing margin and their absence from the eyespot foci during the larval stage is in agreement with previous reports for the expression of izations . Both Wghenotype . The genDll, en, and sal was only observed in scale-building cells around the wound site from 12 h onwards after the wounding operation. These patterns of gene expression mimic the temporal and spatial patterns of genes expressed around the natural eyespot centers where Dll, En and Sal only appear in scale-building cells at least 12 h after pupation  in the larval wing stage, and they do not have pSmad or wg expression at their centers during the early pupal stage. These spots, however, share the expression of the transcription factor, Sal, which is associated in both B. anynana and P. rapae with the area of black scales in the adult wing. In addition, in the late larval wings of both species, pSmad is first visible along the wing margin  may be simultaneously needed to activate one or more sal cis-regulatory modules along a continuous stripe on the wing.The restriction of Sal in the he veins ,34 may bhe veins . These gsal, and perhaps two of the same pre-existing and thus, homologous, gene circuits, e.g. pSmad being upstream of sal activation, and sal being upstream of black scale differentiation. These two circuits may also be ancestral to the butterfly-moth lineage split but our current lack of data for the expression of these genes in the saturniids does not allow us to examine this proposition. In any event, because the mechanism of how sal is eventually activated in the pupal stage in Pieris and Bicyclus scale-building cells is proposed to be different for each system , that could lead to a similar focus signaling mechanism, but whether the systems have independently evolved in their non-homologous positions by the co-option of an ancestral focus gene circuit into a novel position , display these discal-cell eyespots, making their appearance at the base of the tree more parsimonious. Second, it was previously shown that Dll is also expressed at a discal-cell location in a nymphalid butterfly [Our mapping of the co-option of a ree Fig. , rather utterfly . Denser Dll, en and sal) may, at least in part, have been co-opted from the wound repair genetic circuitry, as the two developmental processes share many temporal and genetic similarities. Future work on wound healing across a broader range of species should help verify this hypothesis.The genetic circuitry involved in nymphalid eyespot production, with its putative signaling ligands (Wg and TGF-βs) and several response genes . Pupation times for P. rapae and B. anynana were scored by means of time-lapse photography with a Kodak 290 digital camera, inside the 27°C and 80% humidity environmental chamber. The time-lapse was set to take pictures every 30 minutes with a time stamp on each photo. We dissected roughly 200 pairs of pupal wings and 100 larval wings from B. anynana, 60 pairs of pupal forewings and 50 larval forewings from Pieris, 12 larval wings from S. pavonia and 80 larval wings from A. polyphemus. Larval and pupal wings were fixed and stained following the protocol in reference [Drosophila., thus serving as an indicator of TGF-β activity on the butterfly wing. The Wg polyclonal antibody was developed against a conserved epitope in the region between amino acids 25–75 of the Human Wnt-1 protein . The epitope is proprietary and therefore only a range of amino acids was provided for where the epitope is located. In this 50 a.a. region there is 35.3% identity among amino acids when compared to Bombyx mori, the closest species with a fully sequenced wg gene. Within the same region there is a smaller group of 12 amino acids (#64–75) that have 75% identity with the Bombyx sequence. We used Alexa Fluor 488 anti-mouse with either Alexa Fluor 594 anti-goat or Texas-Red anti-rabbit secondary antibodies . Wings were mounted on glass slides in Slow Fade medium , covered with a glass coverslip, sealed with clear nail polish, and stored at -20°C until observation under FITC and Texas Red filters on a Leica DMIRE II microscope at 50X, 100X and 200X magnifications.eference . We usedeference ) for alleference ), rabbiteference ) named heference ) and goaIn order to induce a wound and healing response we pierced individual pupal wings at one or two of three different sites with a fine tungsten needle . Operations were performed between 5 h and 20 h after pupation (AP), covering the time period where most ectopic eyespots are produced, between 12 h and 18 h AP . Pupae wPieris and Bicyclus using combinations of secondary antibodies only. These stainings, apart from the ones mentioned explicitly in the text in the context of the wound healing experiments, yielded very dark preparations with no particular patterns of fluorescence.Images shown represent the best specimens for each treatment but similar patterns were observed in at least 3 other specimens of each species, and often many more. Control stainings were also performed for all the developmental stages of P. rapae and the wounding experiments, and drafted the final manuscript. GG collected the Wingless data, part of the pSmad data for B. anynana and P. rapae, and put together the first draft of the manuscript. SS collected part of the data for pSmad in B. anynana and, together with NG, collected part of the data for the wound healing experiments. DR helped draft the manuscript. All authors read and approved the final manuscript.AM conceived the study, collected the data for the moths, part of the data for


Sphenodon), archaic reptiles of enormous zoological significance as the sole representatives of a once widespread reptilian order.The sex of many reptiles is determined by the temperature an embryo experiences during its development. Three patterns of temperature-dependent sex determination (TSD) have been defined, but one pattern where only males are produced above an upper temperature threshold (Type IB) is controversial. Here we report new data on the relationship between constant temperature incubation and sexual phenotype in two species of tuatara , males were produced above a pivotal temperature of 22.0°C. The pivotal temperatures and scaling parameters differed between species (p < 0.001). The thermosensitive period (TSP), where temperature influences gonad morphogenesis, occurs between 0.25 and 0.55 of embryonic development. While it is possible that the more common female→male→female (FMF or Type II) pattern exists, with a second pivotal temperature above 23–24°C, we review several lines of evidence to the contrary. Most notably, we show that in S. punctatus, the warmest natural nests during the TSP produce predominantly males.In both species, the pattern observed with constant incubation temperatures from 18 to 23°C (or 24°C) supported a female→male (FM or Type IB) pattern of TSD: in S. guntheri because current patterns of global warming could exacerbate the male bias already present in the relic population.An FM pattern of TSD could be currently adaptive in promoting sexual size dimorphism in tuatara. However, an FM pattern has particularly serious consequences for  The re1 - l))] The authors declare that they have no competing interests.S. guntheri and drafted the manuscript; NJN conducted the laparoscopies, and helped to draft the manuscript. AC conducted experiments on S. punctatus that provided additional support for the FM TSD pattern. SP performed the statistical analysis, SNK coordinated fieldwork and assisted with husbandry of juvenile tuatara, and CHD conceived the study at Victoria University of Wellington. All authors read, commented on, and approved the final manuscript.NJM carried out the work on


While studying the local flora and fauna of the Amazon jungle in the 1860s, Henry Walter Bates made a striking discovery. Butterflies inhabiting a particular geographic region sported the same wing patterns—even though they were unrelated species. Bates proposed that nonpoisonous species had mimicked the patterns of noxious species that predators avoided, thus gaining a selective advantage. Today, scientists can use the tools of genomics and genetics to investigate the mechanism of convergent evolution—the emergence of similar physical traits (or phenotypes) in unrelated species. Such empirical studies have provided insight into a longstanding controversy spawned by evolutionary theorists over the origin of mimicry: does it arise gradually through the accumulation of random mutations by selection or in “phenotypic leaps” through the constraining influence of shared developmental pathways with a bias toward a particular phenotype?Heliconius butterflies allowed the researchers to test the possibility that mimetic convergence results from constraints in the regulation of butterfly color patterns.Recent molecular studies found evidence that regulation or recruitment of the same genes or gene variants may explain convergent evolution. In a new study, Mathieu Joron, Chris Jiggins, and colleagues took a different approach by investigating the molecular basis of both convergent and divergent phenotypes. The involvement of the same genomic loci in convergent phenotypes suggests that developmental constraints give rise to these shared phenotypes. The presence of a multitude of convergent and divergent phenotypes in the wing patterns of Heliconius butterflies, including Müllerian mimics . Two species, H. melpomene and H. erato, are distantly related yet have identical wing patterns. Both species radiated into over 30 races (or subspecies) in parallel, with the two species (“co-mimics) displaying a single pattern locally. The third species, H. numata, is closely related to H. melpomene but has radically different wing patterns, with up to seven different variations in a single region. Each of these variations mimics a different species of another butterfly genus, Melinaea.The researchers worked with three species of H. melpomene, variation in white and yellow patterns is controlled by at least three genomic loci—N, Yb, and Sb—that are tightly linked, or inherited together. Red pattern elements are controlled by another linked loci pair—B and D. In H. erato, the Cr locus produces patterning effects similar to the interaction of N, Yb, and Sb in H. melpomene, while an unlinked locus, D, appears to control red pattern variation much like the B–D pair does in H. melpomene. In polymorphic H. numata, all the mimicked color patterns derive from a single locus, P, thought to be a “supergene” .In H. melpomene from different regions in Ecuador were crossed with an H. melpomene subspecies stock from French Guiana to produce second-generation offspring. Offspring were then genotyped to identify genes responsible for the resulting color patterns and to map the relevant major color-patterning loci—N, Yb, and Sb loci for H. melpomene crosses, Cr for H. erato, and P for H. numata—in individual offspring.The researchers crossed different races of each of the three species to explore the genetic basis of the variations. For example, two different subspecies of H. melpomene  and H. erato(Cr) are tightly linked to genetic markers that occupy the same position in both species. Similarly, the locus P, which controls whole-wing variation in H. numata, is also linked to the same series of markers.Using molecular markers developed in the region of the pattern genes, they found that the three loci controlling color pattern variation for each species inhabit the same genomic location. Indeed, the elements controlling white and yellow pattern variation in Heliconius butterflies. Rather than a constraining role, this locus provides what the researchers call a “jack-of-all-trades flexibility.” It presumably functions as a “developmental switching mechanism” for natural selection, they explain, by responding to a wide range of mimetic pressures to produce radically divergent, locally adapted wing patterns. Now researchers can begin to identify and determine the modus operandi of the genes at the center of what has been called a “developmental hotspot” to better understand how they drive the adaptive evolution of mimetic color pattern shifts. For more on the evolution of mimicry in butterflies, see the Primer .These results, Joron et al. conclude, suggest that a single conserved locus is responsible for producing wing pattern diversity in


Axel Meyer discusses how evolution frequently converges on similar mimetic patterns in different species. Fascination with flora and fauna usually starts early in life as an all-encompassing childhood pastime. Growing up in Germany in the 1960s and 1970s, I developed an affinity for natural history as a child, inspired by famous television naturalists, such as Jacques Cousteau and Bernhard Grzimek, as well as by role models closer to home. As a child, it seemed quite natural to observe, experiment with, and collect all kinds of animals, dead or alive, and their parts  for my private “Wunderkammer,” or cabinet of curiosities.Lolita, was also a distinguished lepidopterist who specialized in the systematics of the butterfly family Lycaenidae. Nabokov's obsession with butterflies also started early in life and arguably influenced his thinking and writing for the rest of it. He published numerous scholarly papers in recognized entomology journals, mostly on species from Europe and North America, and he was inordinately proud that a species of butterfly (Cyllopsis pyracmon nabokovi) was named after him. Nabokov was particularly fascinated by the mapping of spots on butterfly wings.Literature Nobel laureate Vladimir Nabokov, probably most famous for his notorious novel The diversity of life, both within and between species, often lights the fire of childhood fascination, fostering a naïve juvenile obsession and the desire to collect. But as one becomes more familiar with the natural world, one also notices curious similarities between organisms. This similarity might simply, and rather uninterestingly, be a reflection of close evolutionary relationships. But more intriguing evolutionary questions and lessons emerge when the similarity exists between distantly related species. Convergence, the term used to describe this type of similarity, is arguably more interesting than plain diversity. The paleontologist Simon Conway Morris  has madeConvergence teaches us that the seemingly unbounded creativity of evolution through natural selection and the diversity it produces are not without limits but sometimes seem to follow predictable patterns verging on rules. This is also evident in one of the prime examples of diversity in vertebrates, the species flocks of cichlid fishes of Eastern Africa. Although each of the three large East African Lakes contains hundreds of endemic species of cichlids ,3, the oRedundancy in the outcome of natural selection is most evident in the beautiful examples of mimicry in butterflies . MimicryDrosophila developmental genetics—paved the way and has led to a deeper understanding of both the genetic underpinnings and the evolutionary implications of butterfly color evolution. The Bicyclus system from Africa analyzed by Paul Brakefield from Leiden University  and Sean Carroll from the University of Wisconsin Madison  [Papilio mimicry system investigated by Frederik Nijhout from Duke University [Evolution clearly repeats itself in mimicry. The “why” and “how” is at the center of a field of research that combines comparative genomics and evolutionary developmental approaches. Through a variety of technical approaches in comparative developmental genetics and the construction of genetic linkage maps that can identify chromosome regions and sometimes even specific genes that control phenotypic traits, it has now become possible to bridge the wide gap between natural history and developmental biology. During the past decade, the development and evolution of color patterns in butterfly wings has become one of the best-studied examples in which knowledge from molecular developmental biology—with lessons and tools from  States) , and theiversity   yielded very interesting new results. The crosses, genomic analyses of bacterial artificial chromosome clones, and genetic maps demonstrate that color pattern divergence has a relatively simple genetic basis—few regions of the genome control most of the dramatic color-pattern differences between races. That the same genetic region is used across species boundaries to create similar (mimetic) colors in some and divergent patterns in other species demonstrates that phenotypic similarity (and divergence) can be potentially controlled by the same set of genes.Two species of butterflies, n et al.  in this Heliconius butterflies that show that a locus, Yb, controls the presence of a yellow wing band in H. melpomene. Interestingly, the Yb locus maps to the same genomic location as the Cr locus in its congener and co-mimic H. erato, which also sports a yellow band on its wings. Evolution appears to have recruited the same gene region repeatedly and in parallel, resulting in the similar phenotypes of these Müllerian mimics. Natural selection apparently pushed for similar phenotypes, yet the conservative nature of the underlying developmental genetic mechanisms constrained (in the sense that the same genes and pathways are used) the outcome of selection as well.Joron et al.  present H. numata—in which some morphs can look quite different from both H. melpomene and H. erato—the same genetic locus appears to be involved in bringing about a phenotype that mimics that of the distantly related butterflies of the genus Melinaea. So, although this gene locus is involved in similar wing color patterns in H. melpomene and its co-mimic H. erato, it acts as a “supergene” in conjunction with other unlinked color-pattern loci to control the development of quite different wing color patterns in different populations of H. numata. It is remarkable and warrants further investigation that the same genomic region is controlling not only convergent wing color patterns in H. erato and H. melpomene, but also divergent patterns in H. numata. So, genomic conservatism at an upstream supergene and seemingly unconstrained downstream genes combined to simultaneously produce mimetic similarity and novel color patterns across species of butterflies.What makes this study  even morHeliconius species, could be used to identify the expected downstream target genes of this supergene. These approaches might reveal how the genetic architecture of downstream genetic pathways, which are involved in the expression of similar or divergent color patterns during wing development, might have diverged and whether some of their components show signatures of Darwinian selection. As Joron and co-workers' investigation [Future comparative genomic work, for example on expressed sequence tags and DNA microarrays of the developing wings of different tigation  beautifu


Heliconius is gaining the attention of an ever-widening audience  has long been the subject of genetic analysis audience .Heliconius erato and Heliconius melpomene groupings and noted repeated mimetic convergence between them. However, within those groupings, he failed to distinguish species, races, and hybrids. In the mid 1950s, William Beebe and associates initiated studies of life history, behavior, systematics, and genetics of Heliconius at Simla in Trinidad. There, Michael Emsley elucidated biogeographic details of the system erato and melpomene each generate similar arrays of hybrid phenotypes, many of which would be sufficiently distinct to warrant separate species status when viewed out of context.Early in the 20th century, Oxford's pre-eminent evolutionist and student of insect color patterns, Edward B. Poulton, urged Harry Eltringham to study taxonomic relationships of a spectacularly colorful, mimetic, and diverse set of specimens pouring into European museums from field collectors across the Neotropics. Eltringham H. erato and H. melpomene at Simla established the framework of classification of pattern loci in general use today erato and melpomene. Thus, if viewed only through the lens of structural genes not manifest in the visible phenotype, few of the many races described for these species would be delimited. Later research in Peru on selection and gene flow in parallel interracial hybrid zones by James Mallet In the 1960s, genetic studies of B/D locus, which controls the presence/absence of red patterns, and the Yb/Cr locus, which controls the presence/absence of a yellow bar, respectively map to homologous linkage groups between the co-mimics H. melpomene and H. erato, although co-mimetic phenotypes evolved independently. In other words, convergent evolution in wing patterning between species involved the same genetic intervals, and, since synteny between distantly related Lepidoptera is conserved cis-regulatory or trans-regulatory changes responsible for these polymorphisms Several teams have been busy in recent years trying to relate underlying allelic variation in color pattern observed in laboratory crosses and in natural hybrid zones to changes occurring in the genome. Classic genetic mapping previously showed that these adaptive polymorphisms in four different radiations were linked to homologous intervals Heliconius system forms a unique replicated natural experiment to study the genetics of adaptive traits: allowing comparison between parapatric races of different phenotypes, between geographically distant races of similar phenotypes, and finally, between different species (co-mimics) across parallel inter-racial hybrid zones. The papers in this issue exploit this system by seeking signatures of selection across previously identified genetic intervals B/D and Yb/Cr in hybrid zones where populations of different phenotypes are admixed. Indeed, in these species mimicry ring structure on both sides of a hybrid zone imposes a strong positive frequent-dependent selection favoring common wing patterns H. melpomene and H. erato wing pattern loci, whereas unlinked regions of the genome showed no deviation from neutrality. Also, both studies form a consistent set of observations at a finer genomic scale by looking for haplotypes statistically associated with a certain phenotype. They found a rapid decay in linkage disequilibrium in these species, yet they did not identify completely fixed differences that would pinpoint wing pattern genes with confidence. However, both studies implicated a kinesin-motor gene (kinesin) as a B/D candidate gene, since it was close to a hotspot of genotype-by-phenotype association and also showed a higher expression level correlating with red pattern phenotypes. Similarly, both studies identified a “parallel” peak of genotype-to-phenotype association between polymorphism in a Leucine-Rich Repeat gene (LRR) and the Yb/Cr phenotypes. Finally, although the two studies are somewhat complementary in their design, they do not always converge in their results. For instance, while Baxter et al. sampled three geographically distant pairs of admixing populations, Counterman et al. focused their geographical sampling on a narrow area with a sharp transition in wing phenotypes where numerous generations of recombination have had the opportunity to break down variation around causative switch genes. In this latter study , several hotspots of pattern association were observed in addition to kinesin and LRR. This raises the possibility that loci involved in pattern variation in each zone of a wing consist of several functional sites, whether they are coding or regulatory changes. While puzzling at first sight, this observation is consistent with the notion that these loci are supergenes with multiple wing patterning effects Drosophila that tightly linked mutations of small effect participate in shaping an allele of major effect Heliconius wing patches and bands The Heliconius butterflies are clearly emerging as a premier model system for such integrative research (e.g., Heliconius wing patterns still seem like a rainforest under a shroud of fog. Only a few canopy trees are visible as we fly over. It should be exciting when the clouds lift.Understanding the evolution of diversity will surely involve better integration of ecology, behavior, population genetics, and developmental biology, leading to new models of species diversification that incorporate well-characterized selective environments, adaptive peaks, and how networks of genes determine important phenotypes. ch e.g., . The stu


Heliconius butterflies is a longstanding example of both Müllerian mimicry and phenotypic radiation under strong natural selection. The loci controlling such patterns are “hotspots” for adaptive evolution with great allelic diversity across different species in the genus. We characterise nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium, and candidate gene expression at two loci and across multiple hybrid zones in Heliconius melpomene and relatives. Alleles at HmB control the presence or absence of the red forewing band, while alleles at HmYb control the yellow hindwing bar. Across HmYb two regions, separated by ∼100 kb, show significant genotype-by-phenotype associations that are replicated across independent hybrid zones. In contrast, at HmB a single peak of association indicates the likely position of functional sites at three genes, encoding a kinesin, a G-protein coupled receptor, and an mRNA splicing factor. At both HmYb and HmB there is evidence for enhanced linkage disequilibrium (LD) between associated sites separated by up to 14 kb, suggesting that multiple sites are under selection. However, there was no evidence for reduced variation or deviations from neutrality that might indicate a recent selective sweep, consistent with these alleles being relatively old. Of the three genes showing an association with the HmB locus, the kinesin shows differences in wing disc expression between races that are replicated in the co-mimic, Heliconius erato, providing striking evidence for parallel changes in gene expression between Müllerian co-mimics. Wing patterning loci in Heliconius melpomene therefore show a haplotype structure maintained by selection, but no evidence for a recent selective sweep. The complex genetic pattern contrasts with the simple genetic basis of many adaptive traits studied previously, but may provide a better model for most adaptation in natural populations that has arisen over millions rather than tens of years.Wing patterning in  Heliconius butterflies is a longstanding example of both Müllerian mimicry and adaptive radiation. The genetic regions controlling such patterns are “hotspots” for adaptive evolution, with small regions of the genome controlling major changes in wing pattern. Across multiple hybrid zones in Heliconius melpomene and related species, we no find no strong population signal of recent selection. Nonetheless, we find significant associations between genetic variation and wing pattern at multiple sites. This suggests patterning alleles are relatively old, and might be a better model for most natural adaptation, in contrast to the simple genetic basis of recent human-induced selection such as pesticide resistance. Strikingly, across the region controlling the red forewing band, a very strong association with phenotype implicates three genes as potentially being involved in control of wing pattern. One of these, a kinesin gene, shows parallel differences in expression levels between divergent forms in the two mimetic species, making it a strong candidate for control of wing pattern. These results show that mimicry involves parallel changes in gene expression and strongly suggest a role for this gene in control of wing pattern.The diversity of wing patterns in  One of the central outstanding questions in evolutionary biology concerns the predictability of evolution. Specifically, can we predict the number and effect size of genes involved in evolution, and the types of genetic changes that underlie particular kinds of evolution? Striking empirical examples of repeated use of the same genes in similar evolutionary changes suggest that common patterns may emerge even across distantly related taxa Heliconius butterflies offer an excellent model system in which to address these questions as their wing patterns are under simple Mendelian genetic control and subject to strong natural selection Heliconius melpomene and between closely related species H. melpomene and Heliconius erato, and a third species, Heliconius numata, with entirely different wing patterns Heliconius butterflies. However, the specific genetic changes underlying phenotypic diversity in Heliconius remain unknown.Heliconius mimicry as a system to study the population genetic patterns around loci involved in parallel phenotypic changes. Patterns of genetic variation in natural populations are commonly used to identify genes under selection, either through association studies that correlate genetic and phenotypic variation Here and in a companion paper Heliconius, however, we can take advantage of natural hybrid zones, where admixture occurs between divergent geographic races, to carry out analysis of genotype-by-phenotype association. In particular, studies of a parallel hybrid zone in Peru between races of the co-mimics H. erato and H. melpomene have estimated that selection is extremely strong, on the order of a 20–50% reduced survival of foreign colour pattern morphs Heliconius races typically differ at just two or three loci with major phenotypic effect on wing pattern Heliconius offers an unusual opportunity to study populations that are strongly morphologically differentiated but nonetheless free to exchange genes across most of the genome. Previous demographic studies of Heliconius have demonstrated genetic differentiation on a regional scale, for example between the Guiana Shield, Amazon basin and Pacific slopes, but extensive gene flow between local populations , protein binding , splicing (slu7), protein-protein interactions (leucine rich repeats or LRR), transport , basic cell function  and a transcription factor (six/sine) , and a similar pattern is seen at HmYb where sequence is available for H. erato.We characterised the ix/sine) . In addi20 genes . Overallelpomene . A direcHmYb markers for three pairwise population comparisons, two involving races of H. melpomene (amaryllis vs aglaope and melpomene vs rosina) and one pair of hybridizing species (H. pachinus vs. H. cydno) . Such a pattern was not seen at any of 16 unlinked loci studied previously for this species pair We estimated population structure at . cydno) . In all ed genes . In tota10 genes . Second,24 genes . This waH. melpomene, that differ in presence of the HmB red forewing band, were also studied for HmB linked markers H. melpomene amaryllis has a red forewing band, controlled by the dominant HmB allele, which is absent in H. melpomene aglaope  in H. m. aglaope. The lack of completely fixed differences between any of the comparisons suggests that we have not yet sampled causative sites, or that there is epistasis between the variable sites with phenotype determined by allelic combinations.The two races of  aglaope . H. m. a 5391 bp . Again, ed genes . In totaed genes . The peaCR genes . SeveralH. m. aglaope population that showed a significantly negative Tajima's D . When LD was plotted against genetic distance, there was strong LD (0.5>r2>0.9) between sites separated by a distance of up to 14 kb , or as being a nearby candidate locus for pattern specification (Mad). The same populations studied above were not available as live material, so we instead used two forms available from commercial suppliers, H. melpomene malleti and H. melpomene cythera, which have yellow and red forewing bands respectively, providing a similar comparison as that between H. m. aglaope and H. m. amaryllis. First, forewings were removed from three pupal developmental stages of H. melpomene cythera, and dissected into three wing segments, to examine spatial gene expression between proximal, medial (HmB) and distal regions . Second, whole forewings and hindwings were separately dissected from 5th instar larvae and early pupae of H. melpomene malleti and H. melpomene cythera for expression analysis ‘between races’.For the kinesin gene, with H. m. malleti having an overall higher expression than H. m. cythera  stage but no differences between different segments of the wing, consequently developmental stage made a strong contribution to gene expression prediction (Pr(β≠0) = 89.6; The most striking result was a strong contribution to the prediction of gene expression by race at the  cythera . AnalysiSlu 7 showed higher expression in later developmental stages (highest in the ommochrome only stage [OO]), but no differences between segments of the wing  = 100). Race made a contribution to the prediction of gene expression in the ‘between races’ experiment (Pr(β≠0) = 86.5), with H. m. cythera having a 1.52-fold overall higher expression than H. m. malleti  = 94), but no differences between races or wing segments  shows a very marked peak in both the H. pachinus vs. H. cydno and the H. m. amaryllis vs H. m. aglaope hybrid zones, and is also highlighted in H. eratoHmYb. In addition however, another peak around HM00008 and HM00010 is shared between the two H. melpomene inter-racial comparisons, but not with H. erato. The repeatability of these patterns across independent hybrid zones suggests that multiple associations are not merely the stochastic results of selection at a single causative site, but rather imply a role for multiple functional sites underlying wing pattern differences. This is supported by the observation of increased linkage disequilibrium between associated sites at the HmYb locus as compared to background levels, suggesting that selection is maintaining particular allelic combinations across this region , where regions of differentiation are limited to a few hundred base pairs Heliconius.Overall, the stronger signal of both haplotype structure and genotype-by-phenotype association in hedgehog and wingless signalling pathways are involved in wing eyespot formation Drosophila wing pigmentation have implicated cis-regulatory regions of shared pigment biosynthesis genes in regulating inter-specific differences HmB region, similar to the pattern already found in HmYba priori assumptions regarding the identity of the wing patterning genes. Instead we have cloned the genetic region controlling natural variation in a striking phenotype, and in doing so ruled out a role for any of the wing pattern ‘toolkit’ genes described previously. Of course, these two approaches may be complementary, and the locus that is controlling the wing phenotype may be an upstream regulator of classic ‘toolkit’ genes. Indeed, the intriguing observation that the ‘Bigeye’ pattern mutant of Bicyclus anynana maps to the homologous chromosome of HmYb might indicate that the gene networks uncovered in Heliconius may have a general role in butterfly wing patterning The development of scale-covered wings, pigmentation and an elaborate patterning system are evolutionary innovations of the Lepidoptera and have been cited as an example of the redeployment of conserved gene networks HmYb locus, two regions are implicated, first around the genes HM00007, HM00008 and HM00010. These genes have putative orthologues in Drosophila  about which little is known, except that HM00010 contains WD40 repeats that are likely to be involved in protein-protein interaction. More striking is the association found around HM00024 (LRR-3), which was replicated in two of the comparisons studied here and in H. erato. This gene shows similarity to Sur-8 in Drosophila, consistent with a possible role in signal transduction, and is also adjacent to HM00023, a probable non-coding transcript with complex patterns of alternative splicing that may have a regulatory function HmYb suggested by sequence similarity was the transcription factor Unkempt which, unlike in H. erato, showed no significant association with wing pattern in H. melpomene.At the HmB gene. In both H. melpomene and H. erato when yellow and red forewing band forms are compared, the former showed much higher expression levels of the putative kinesin, HM01018, in late larval and early pupal stages, suggesting a role for this locus in wing pattern specification. Members of the kinesin superfamily play diverse roles in cellular organization, using a catalytic motor to move along microtubules and transport organelles and molecules H. melpomene kinesin contains an N-terminal motor domain, yet has no sequence similarity to other eukaryotes in the C-terminal cargo domain, making it difficult to speculate regarding the function of this gene. In Drosophila, kinesin proteins can function to determine cell polarity and RNA localisation during embryogenesis, and are therefore known to play a role in pattern specification at a cellular level kinesin expression across the whole wing regulate either scale cell fate or pigment localization, and thus ultimately specify pattern formation.Against a background of multiple associated sites, and extensive LD across three genes, there is one strong candidate for the Heliconius are clustered in the genome across multiple independent hybrid zones. This adds to what is already known from crossing experiments, showing that two patterning loci, HmB and HmD, controlling the red forewing band and orange hindwing rays respectively, are tightly linked, as are HmYb and HmSb that similarly control the yellow hindwing bar and white margin Heliconius genome that play a disproportionately large role in pattern specification and indeed, in speciation. The population data described here offer novel insights into the genetic architecture of these regions. First, there is significant association of both genetic variation and long distance haplotype structure with wing pattern at both loci, indicating the influence of strong mimicry selection on the genome. Nonetheless, consistent with previous estimates of the age of H. melpomene  with the ReRep pipeline BAC clones in the H. cydno galanthus (n = 33) and H. pachinus (n = 22) were collected from Costa Rica; H. melpoomene melpomene (n = 16) and H. melpomene rosina (n = 19) were collected from Panama and Venezuela; H. melpomene aglaope (n = 30) and H. melpomene amaryllis (n = 30) were collected from Peru . rom Peru . Two admllis see . The Per2, 0.1 mM dNTP, 50 pmol of each primer, 0.25 units of Taq polymerase (Bio-Line). Thermal cycling conditions were 94°C 1 min, 35 cycles of 94°C 15 sec, annealing 30 sec, 72°C 60 sec, and a final extension of 72°C. Prior to sequencing, products were incubated with 2 units of Exonuclease 1 (NEB) and 1 unit of Shrimp Alkaline Phosphatase (Fermentas) for 40 minutes at 37°C then 80°C for 20 minutes. Sequencing was performed in 10 µl reactions using 1–3 µl of template, 1× reaction buffer, 1µl BigDye terminator v3.1 (Applied Biosystems) and 0.32 pmol primer and run in a thermal cycler for 25 cycles of 95°C 30 seconds, 50°C 20 seconds, 60°C 4 minutes. Products were then sequenced using an ABI3730 capillary sequencer, analyzed using CodonCode Aligner software and single nucleotide polymorphisms identified manually. Genes contained insertion/deletion (IN/DEL) variation were trimmed to exclude such regions from analysis, or cloned using Promega pGEM-T Easy kit before sequencing , were sequenced, along with three control genes unlinked to wing colour patterning loci  and unlinked markers were carried out for each pair of populations using a Mann-Whitney U test implemented in Minitab v15.1 (Minitab Inc.). For the H. cydno/H. pachinus comparison background levels of differentiation were obtained from 16 previously published genes Haplotypes were inferred using Phase/Unphase implemented in DnaSP HmYb in H. melpomene, 127 in H. cydno/pachinus, 40 variable sites for HmB between H. m. aglaope and H. m. amaryllis and 20 sites at unlinked loci. Colour pattern genotypes at the HmYb and HmB loci were scored as 0.0 or 1.0 representing alternative homozygotes, and 0.5 for heterozygotes. Although HmB is dominant, two hybrid individuals with the dominant red forewing HmB allele and orange rayed HmD alleles were scored as heterozygotes, as recombinants between HmB and HmD are rare. A stringent significance cut-off was calculated using a Bonferroni correction applied to all 866 sites tested (−Log10(0.05/866) = 4.24).We determined if any SNPs were associated with a colour pattern phenotype using a chi-squared linear trend test HmYb and HmB was calculated for all population samples with a sample size ≥20. This restricted the analysis to the H. m. aglaope and H. m. amaryllis populations, and gave a total of 40 sites for HmB, 24 for HmYb and 20 for unlinked loci with a rare allele frequency greater than 0.05 that were considered informative for LD analysis. Multi-allelic sites that had a minor allele with a frequency less than 0.05 were condensed to bi-allelic SNPs by merging the minor allele genotypes. LD was estimated using the commonly used composite estimate of LD method described by Weir H. m. aglaope and H. m. amaryllis populations.Linkage disequilibrium across Mad, slu7, kinesin and GPCR. RNA was extracted from forewings of H. m. cythera pupae at three developmental stages; EP , PO (Pre-Ommochrome – well developed wings with veins but no pigmentation) and OO (Only-Ommochrome – red band visible but no melanin present) HmB band and D = distal) totalling 27 samples .Quantitative real time PCR (qPCR) was used to determine the relative transcript levels of H. m. cythera and H. m. malleti individuals. The former has a red forewing band, which is absent in the latter. RNA was collected from whole forewings and hindwings of three individuals from two developmental stages (late 5th instar larvae and EP), totalling 24 samples .To compare pattern races we used SensiMix, Quantace). The reactions were subjected to 40 cycles of amplification in an Opticon 2 DNA engine (MJ Research) under the following conditions: 95°C for 15 min and then 40 cycles of 94°C for 15 sec, 55.4°–60°C for 30 sec, 72°C for 30 sec followed by a final incubation at 72°C for 10 min. Melting curves were generated between 55° and 90° with readings taken every 0.2° for each of the products to check that only a single product was generated. We sequenced at least one product from each set of primers to confirm identity and size. For most loci, the amplified fragments spanned at least one intron, ensuring that genomic DNA contamination could be identified. We used two housekeeping control genes for normalization in the ‘between wing segments’ experiment; EF1-α and RpS3A. However, as results of these two genes were consistent, only EF1-α was used in the ‘between race’ experiment. qPCRs on target and control loci were always assayed using product from the same cDNA synthesis reaction. Furthermore all samples were assayed 3 times but, because differences in gene expression between replicates were always <0.05× (see below), we report only the average value for each sample. Statistical methods for qPCR experiments are given in Total RNA (500 ng) was reverse transcribed with random hexamers and 1 µl of the resulting cDNA template (25ng) was combined with 200 nM of each primer in 25 µl of total reaction volume containing 1× SYBR Green master mix  was used to compare three sequences; Bombyx mori nscaf3026 1161033.1269872 (http://silkworm.genomics.org.cn), H. melpomene BAC clone 28L23 (CU467808) and H. erato BAC clone 31N19 (accession). Comparisons were performed using tBLASTx, limited to 200 HSPs and an expect value 1.0e-10. B. mori genes displayed are BGIBMGA011292-TA (bves), BGIBMGA011315-TA , BGIBMGA011316-TA (Slu7), BGIBMGA011317-TA (Sorting Nexin), BGIBMGA011291-TA (phosphodiesterase 10A), BGIBMGA011289-TA and BGIBMGA011290-TA (predicted Kinesin), BGIBMGA011318-TA (GPCR), BGIBMGA011288-TA , BGIBMGA011287-TA (Epoxide Hydrolase).Alignment of (0.15 MB PDF)Click here for additional data file.Figure S2HmB (red circles) and HmYb (green triangles) for combined H. m. amaryllis and H. m. aglaope populations, both for all sites (A) and with sites showing a significant association with phenotype removed (B). The only remaining comparisons showing long-range LD after removal of associated sites are also between the Slu7 and GPCR genes, in the region associated with the HmB phenotye.Decay of linkage disequilibrium with distance across the two regions. Data are shown for (0.54 MB PDF)Click here for additional data file.Figure S3H. m. amaryllis and H. m. aglaope populations, both for coding and non-coding markers in regions linked and unlinked to colour pattern.Decay of linkage disequilibrium with distance within gene markers. Data are shown for combined (0.22 MB PDF)Click here for additional data file.Figure S4H. m. amaryllis, but with expression of the dominant HmD allele from the Amazonian race controlling the red hindwing rays and basal red patch on the forewing.Phenotypes of two hybrid individuals sampled in the Peruvian hybrid zone. Both individuals are phenotypically most similar to (1.62 MB PDF)Click here for additional data file.Table S1HmB region.Annotation of genes in the (0.12 MB DOC)Click here for additional data file.Table S2Gene sequencing and quantitative PCR primers.(0.40 MB DOC)Click here for additional data file.Table S3Summary of gene regions sequenced for population genetic analysis.(0.12 MB DOC)Click here for additional data file.Table S4HmYb SNP data. Details of all sites significantly associated with phenotype at the HmYb locus.(0.05 MB XLS)Click here for additional data file.Table S5HmB and unlinked gene SNP data. All variable SNPs used in the analysis for the aglaope/amaryllis comparison at the HmB locus and at unlinked loci.(0.05 MB XLS)Click here for additional data file.Table S6Estimates of Tajima's D and nucleotide diversity estimates for each locus by population.(0.28 MB DOC)Click here for additional data file.Text S1Supplementary methods.(0.03 MB DOC)Click here for additional data file.