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Adaptation and The Origin of Species. Author(s): Douglas W. Schemske Reviewed work(s): Source: The American Naturalist, Vol. 176, No. S1, Darwinian Thinking: 150 Years after The Origin A Symposium Organized by Douglas W. Schemske (December 2010), pp. S4-S25 Published by: The University of Chicago Press for The American Society of Naturalists Stable URL: http://www.jstor.org/stable/10.1086/657060 . Accessed: 16/02/2012 17:18 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. The University of Chicago Press and The American Society of Naturalists are collaborating with JSTOR to digitize, preserve and extend access to The American Naturalist. http://www.jstor.org vol. 176, supplement the american naturalist december 2010 Adaptation and The Origin of Species Douglas W. Schemske* Department of Plant Biology and W. K. Kellogg Biological Station, Michigan State University, East Lansing, Michigan 48824 Online enhancements: appendix tables. abstract: As reflected in the title of his masterwork On the Origin of Species, Darwin proposed that adaptation is the primary mechanism of speciation. On this, Darwin was criticized for his neglect of reproductive isolation, his lack of appreciation for the role of geographic barriers, his failure to distinguish varieties from species, and his typological species concept. Two developments since Darwin, the biological species concept of Ernst Mayr and the methods of Coyne and Orr for estimating the contribution of different barriers to the total reproductive isolation, provide a framework for reconciling Darwin’s view on the primacy of adaptation in speciation with later proposals that emphasize reproductive isolation. A review of the few studies that have estimated the contributions of multiple isolating barriers suggests that habitat isolation and other barriers that operate before hybrid formation are much stronger than intrinsic postzygotic isolation. In light of these data, I suggest that Darwin’s focus on adaptation in the origin of species was essentially correct, a conclusion that calls for future studies that explore the links between adaptation and speciation, in particular, ecogeographic isolating barriers that result from adaptive divergence in habitat use. The recent revival in thinking about ecological factors and adaptive divergence in the origin of species echoes Darwin’s much-criticized “principle of divergence” and suggests that the emerging views from today’s naturalists are not so different from those espoused by Darwin some 150 years ago. Keywords: adaptation, Darwin, isolating barriers, reproductive isolation, speciation. Introduction According to my view, varieties are species in the presence of formation, or are, as I have called them, incipient species. How, then, does the lesser difference between varieties become augmented into the greater difference between species? (Darwin 1859, p. 111) Since the first publication in 1859 of Charles Darwin’s On the Origin of Species, the topic of speciation has been the subject of great interest and debate. The major problem of speciation, succinctly expressed by Darwin above, is to * E-mail: [email protected]. Am. Nat. 2010. Vol. 176, pp. S4–S25. 䉷 2010 by The University of Chicago. 0003-0147/2010/176S1-52252$15.00. All rights reserved. DOI: 10.1086/657060 identify the ecological and evolutionary mechanisms that cause populations to achieve the minimum level of divergence required for their classification as full species. This requires answers to several challenging questions: What criteria distinguish species from varieties? Can species arise within populations without extrinsic barriers? What are the primary factors that contribute to the origin of species? To Darwin, the processes responsible for the origin of species were essentially the same as those for the origin of adaptations. Invoking his theory of natural selection, Darwin reasoned that adaptation to distinct habitats caused populations to diverge in ecologically relevant traits and that the degree of morphological differentiation among populations was the primary criterion for species status. Much has been written about Darwin’s views on speciation, largely focused on Darwin’s shortcomings. For example, it has been suggested that Darwin’s typological species concept, where species are diagnosed largely by their morphological characteristics, is too subjective (Mayr 1992; Futuyma 1998). Darwin has also been criticized for failing to acknowledge the role of geographic barriers in the divergence of populations (Mayr 1959, 1992, 1994). As discussed recently by Mallet (2008), these and other considerations have led to the conclusion by many that Darwin’s On the Origin of Species contributed little to solving the problem of speciation. In criticizing Darwin’s principle of divergence as an explanation for the origin of species, Mayr (1942, p. 147) states, “It is thus quite true, as several recent authors have indicated, that Darwin’s book was misnamed, because it is a book on evolutionary changes in general and the factors that control them (selection, and so forth), but not a treatise on the origin of species. Obviously it was impossible to write such a work in 1859, because the whole concept of the species was too vague at that time.” Similarly, Coyne (1992, p. 511) suggested, “Despite its title, On the Origin of Species made few inroads on this problem,” and Coyne and Orr (2004, p. 9) assert that Darwin’s perspective on speciation in The Origin “is seen by most modern evolutionists as muddled or wrong.” Noor and Feder (2006, p. 852) suggest that “Darwin had only vague insights into the speciation pro- Adaptation and The Origin cess itself, viewing it as a later stage in a continuum from adaptive divergence among ‘varieties’ within species.” How could the same person who made the most significant contribution in the history of evolutionary biology, that is, the theory of natural selection, utterly fail to appreciate species and speciation? Did Darwin overreach in giving The Origin its title? I agree with Reznick (2010, p. 137), who concluded “that Darwin did, in fact, write a book about speciation.” Given Darwin’s protracted deliberations as he prepared his notes for publication (see below), it would be surprising if he had misrepresented one of the focal points of his life’s work. On this 150th anniversary of the publication of Darwin’s seminal achievement, I discuss how his views on species and speciation compare to those that have emerged since The Origin. My main theme is the relationship between adaptation and speciation. I attempt to reconcile Darwin’s proposal that ecological differentiation is the primary mechanism of speciation with the neo-Darwinian emphasis on the evolution of reproductive isolating factors. Despite many claims to the contrary, I suggest that these two perspectives are essentially one and the same. I begin with a historical treatment of how The Origin came to be and then consider Darwin’s work on species and speciation and summarize recent studies that investigate the relative importance of different isolating barriers. I conclude that Darwin’s view of ecological differentiation as the major factor in speciation is essentially correct. Nevertheless, further research is needed in a number of areas, and these are outlined in the concluding remarks. On the Origin of The Origin To understand the development of Darwin’s views on natural selection and speciation, it is important to recognize that The Origin was not the treatise that Darwin had planned (Stauffer 1975). Instead, it was a hurriedly prepared “abstract,” written on the heels of his communications with Alfred Russel Wallace, who in 1858 surprised Darwin with a letter outlining his independent discovery of the principle of natural selection. The remarkable circumstances that led to the publication of The Origin are summarized below. After his five-year journey around the world on the HMS Beagle (1831–1836), Darwin returned to England to transcribe his notes and deliberate on his discoveries. Progress was slow. Darwin wrote early drafts of his ideas on evolution in 1842 and 1844, but these were published only after his death, by his son Sir Frances Darwin (Darwin 1909). With financial security, Darwin was free to explore his many related interests. Before the publication of The Origin, Darwin published well-known books on coral reefs (1842) and volcanic islands (1844) based on observations S5 he made while on board the Beagle, as well as two monographs on the taxonomy of barnacles (1851, 1854). Describing his plan in a letter to William Darwin Fox in 1855, Darwin wrote, “I am hard at work at my notes collecting and comparing them, in order in some two or three years to write a book with all the facts and arguments, which I can collect, for and versus the immutability of species” (Stauffer 1975, p. 5). Many of Darwin’s associates encouraged him in this effort, with geologist Charles Lyell playing a key role. In 1856, Darwin wrote to Lyell, “I have found it quite impossible to publish any preliminary essay or sketch; but I am doing my work as completely as my present materials allow without waiting to perfect them. And this much acceleration I owe to you” (Stauffer 1975, p. 9). Later that year, Darwin wrote to Lyell, “I am working very steadily at my big book” (Stauffer 1975, p. 9). His goal was never reached. Darwin had completed 10 chapters for Natural Selection, his “big book,” when, on June 18, 1858, he received a letter from the naturalist Alfred Russel Wallace describing views on evolution that were remarkably similar to his own. This led to the famous communication of their ideas to the Linnean Society later that year (Darwin and Wallace 1858). In January of 1859, Darwin wrote to Wallace, “I look at my own career as nearly run out. If I can publish my Abstract and perhaps my greater work on same subject, I shall look at my course as done” (Darwin 1887, 2:146–147). Darwin’s On the Origin of Species was published shortly thereafter, on November 24, 1859. In addition to this “abstract” from Natural Selection, Darwin also published The Variation of Animals and Plants under Domestication (1868), which represents the first section of his manuscript. Darwin never published Natural Selection, but a version edited by the historian Robert C. Stauffer was published in 1975, under the title Charles Darwin’s Natural Selection: Being the Second Part of His Big Species Book Written from 1856 to 1858 (Stauffer 1975). This invaluable resource is the result of Stauffer’s careful and painstaking transcription of more than 1,000 pages of Darwin’s largely illegible handwriting. As stated by Mayr (1992, p. 343), in Natural Selection, “Darwin presented his reasoning and his evidence in far greater detail than in the Origin of Species. This permits a new and indeed more definitive analysis.” Nevertheless, it is largely unappreciated that the ideas and examples presented in The Origin often differ substantially from those in his unpublished manuscript. My references to Darwin’s On the Origin of Species, hereafter The Origin, are primarily from the first edition (Darwin 1859), which, with all other editions, is available online at http://darwin-online.org.uk/. Reference to material in S6 The American Naturalist Natural Selection is from Darwin’s unpublished “big book,” as transcribed by Stauffer (1975). Species and Speciation With the historical context of Darwin’s work as background, I discuss three core aspects of his theory on the origin of species that continue to be debated today: (1) the relationship between adaptation and speciation, (2) the importance of geographic barriers, and (3) species concepts. To this end, I contrast Darwin’s views on these topics, drawing on his writings in The Origin and Natural Selection, with post-Darwinian perspectives. Any attempt to characterize Darwin’s views is fraught with difficulty. This challenge is illustrated by the following quote, communicated to James Mallet by one of his colleagues, “Sometimes I think that Darwin, at least on speciation, is like the Bible: one can buttress any view by choosing the right quotation” (Mallet 2008, p. 4). While I too am not immune to seeing in Darwin what I wish to see, I have tried here to characterize accurately the various sides of the controversies. The Relationship between Adaptation and Speciation Darwin more and more treated speciation as a process of adaptation, an aspect of the principle of divergence, completely omitting any reference to the need for the acquisition of reproductive isolation. (Mayr 1982, p. 416) If one adopted Darwin’s claim, then the problem of the origin of new species was virtually solved; every variety was at the threshold of becoming a new species, and it required only a little push of natural selection to complete the process. (Mayr 1992, p. 349) For Darwin, the origin of species was identical to the origin of adaptations within species—the production of different varieties. He therefore conflated the problem of change within a lineage with the problem of the origin of new lineages. (Coyne and Orr 2004, p. 11) Mayr and others have criticized Darwin for not making a distinction between adaptation and speciation, yet these two evolutionary mechanisms are inextricably linked (Schemske 2000; Schluter 2001, 2009; Rundle and Nosil 2005; Sobel et al. 2010). The closest Darwin came to making that association is laid out in his principle of divergence, the foundation of Darwin’s views on the origin of species (Browne 1980; Mayr 1992; Pfennig and Pfennig 2010, in this issue). His argument is that adaptation and natural selection cause varieties to diverge for ecological and morphological traits that contribute to their competitive performance and that ultimately these heritable dif- ferences are sufficient to elevate varieties to the level of species. As Pfennig and Pfennig (2010) describe, Darwin’s principle of divergence has implications for a number of problems concerning the relationships between adaptation and speciation, particularly the importance of character displacement in adaptive divergence. In chapter 4 of The Origin, Darwin (1859, pp. 127–128) summarized his principle of divergence as follows: “Natural selection, also, leads to divergence of character; for more living beings can be supported on the same area the more they diverge in structure, habits, and constitution. … Thus the small differences distinguishing varieties of the same species, will steadily tend to increase till they come to equal the greater differences between species of the same genus, or even of distinct genera.” In this same chapter, Darwin describes a theoretical scenario for the evolution of varieties and species by his principle of divergence. Interestingly, this section includes the only figure in The Origin, which, Darwin (1859, p. 116) states, “will aid us in understanding this rather perplexing subject.” The figure depicts evolution in a single large genus of organisms over the course of 14,000 generations. The assemblage consists initially of 11 species (A, B, …, L) that ultimately produce 15 descendant species: one of the ancestors (A) gives rise to eight new species, another (I) gives rise to six new species, and a third (F) gives rise to just one new species. None of the other eight ancestral species produces any living descendants. Darwin (1859, p. 120) states, “The diagram illustrates the steps by which the small differences distinguishing varieties are increased into the larger differences distinguishing species.” In Natural Selection, the principle of divergence is illustrated in detail, here for a hypothetical genus of plants initially consisting of 12 species that differ in their moisture requirements. Darwin states (Natural Selection [Stauffer 1975], p. 238), “The complex action of these several principles, namely, natural selection, divergence & extinction, may be best, yet imperfectly, illustrated by the following Diagram, printed on a folded sheet of paper for convenience of reference. This diagram will show the manner, in which I believe species descend from each other & therefore shall be explained in detail.” In this diagram, reprinted here (fig. 1), Darwin proposes that the most “moisture-loving” species (A) gives rise to three new species (a10, h10, and I10; superscripts denote stages of descent) while the least moisture-loving species (M) gives rise to one new species (m10). The new species derived from species A are proposed to occupy a wider range of conditions than those occupied by their ancestor, “so that in love of moisture & in many other respects, a1–10, h1–10, I1–10 would come to differ or diverge more & more from each other & their original parent-stock” (Natural Selection [Stauffer 1975], p. 243). Likewise, Darwin Adaptation and The Origin S7 Figure 1: Diagram I from Darwin’s Natural Selection, illustrating the “Principle of Divergence” for a hypothetical genus of plants that differ in their moisture requirements. The most “moisture-loving” species (A) gives rise to three new species (a10, h10, and I10; superscripts denote stages of descent), one of which (a10) is more aquatic than its ancestor, and the least “moisture-loving” species (M) gives rise to one new species (m10), which is more drought tolerant than its ancestor. The genus thus expands from 12 to 14 species that occupy a wider range of soil moisture environments than their ancestors (reprinted from Stauffer 1975 with the permission of Cambridge University Press). proposes that the new species m10 is more drought tolerant than its ancestor (M). The other 10 original species persist but leave no new descendants, and the original species A and M are “supplanted” by their descendants. Thus, “the genus will have become not only more divergent in character (a10 more aquatic than A; & m10 more droughtenduring than M) but numerically larger” (Natural Selection [Stauffer 1975], pp. 244–245), expanding from 12 to 14 species (fig. 1). It is worth noting that Darwin does not explain here whether adaptive divergence is prompted by a change in the environment or, instead, competitive interactions favor the partitioning of shared features in the current environment. In Natural Selection, Darwin summarized his views on how natural selection leads to adaptively differentiated varieties and species: A little reflexion will show the extreme importance of this principle of divergence for our theory. I believe all the species of the same genus have descended from a common parent; & we may call the average amount of difference between the species, x; but if we look at the contemporaneous varieties of any one species, the amount of difference between them is comparatively extremely slight & may be called a. How thus can the slight difference a be augmented into the greater difference x; which must on our theory be continually occurring in nature, if varieties are converted into good species? The process feebly illustrated in our diagram, I believe, explains this; namely the continued natural selection or preservation of those varieties, which diverge most in all sorts of respects from their parent-type. (Stauffer 1975, p. 243) Darwin was criticized for failing to understand the distinction between adaptation and speciation. Here he makes it clear that these subjects are inseparable. Speciation and Geographic Barriers Darwin’s basic oversight was that he failed to partition isolation into extrinsic geographical-ecological barriers and intrinsic isolating mechanisms. (Mayr 1982, p. 414) Darwin established the principle of divergence in order to substantiate a theory of sympatric speciation. (Mayr 1994, p. 529) The idea of sympatric speciation was, of course, Darwin’s. (Coyne and Orr 2004, p. 125) The prevailing opinion among many evolutionary biologists is that Darwin believed that species could often arise within populations without geographic isolation, that is, via sympatric speciation (Mayr 1992, 1994; Coyne and Orr S8 The American Naturalist 2004). Understanding Darwin’s position on the role of geographic isolation in speciation is challenging. In discussing this point, Mayr (1994, p. 529) stated that in The Origin, Darwin “omitted most of his putative evidence and condensed his argument to such an extent that it is almost impossible to follow his reasoning.” In addition, Darwin’s views on isolation changed over the years, being influenced by his efforts to understand the nature of inheritance (Vorzimmer 1965), by the development of his principle of divergence (see above), and through communications with colleagues and critics (Sulloway 1979; Mayr 1982). Nevertheless, Darwin did voice substantial support for the idea that geographic isolation is often an important factor in speciation. For example, in a letter to J. D. Hooker in 1844, Darwin writes, “The conclusion, which I have come at is, that those areas, in which species are the most numerous, have oftenest been divided and isolated from other areas, united and again divided; a process implying antiquity and some changes in the external conditions. This will justly sound very hypothetical. I cannot give my reasons in detail; but the most general conclusion, which the geographical distribution of all organic beings, appears to me to indicate, is that isolation is the chief concomitant or cause of the appearance of new forms (I well know there are some staring exceptions)” (Darwin 1887, 2:28). In The Origin, Darwin (1859, p. 105) suggested that geographic isolation was important but not the main factor in speciation, stating, “Although I do not doubt that isolation is of considerable importance in the production of new species, on the whole I am inclined to believe that largeness of area is of more importance, more especially in the production of species, which will prove capable of enduring for a long period, and of spreading widely.” From this and other statements in this section, it appears that Darwin is mainly speaking to how “largeness of area” might provide increased opportunities for incipient species to come into contact and therefore compete for similar resources. Natural selection might then favor the evolution of traits that increase competitive ability, that is, divergence of character (Pfennig and Pfennig 2010), leading to speciation. In this regard, Darwin (1859, p. 106) states, “Each new form, also, as soon as it has been much improved, will be able to spread over the open and continuous area, and will thus come into competition with many others.” To emphasize that geographic isolation contributes to this process, Darwin suggests (p. 106), “Moreover, great areas, though now continuous, owing to oscillations of level, will often have recently existed in a broken condition, so that the good effects of isolation will generally, to a certain extent, have concurred.” Darwin’s clearest statements on the role of geographic isolation are found in Natural Selection (Stauffer 1975), for example, “I am inclined to believe, that wherever very many individuals of a freely crossing & highly locomotive animal existed, the retardation of any selected modification from crossing would be so strong, that it could hardly be overcome, without indeed the tendency to vary in some particular direction was extremely strong. Hence I infer that some degree of isolation would generally be almost indispensable” (p. 255). Darwin notes that in birds and mammals, varieties and the most similar species “generally inhabit distinct areas” (p. 256). This is a prescient description of geographic speciation, and it portends the later observation by Jordan (1905) that the most closely related species typically have abutting distributions, as would be expected if they arose in allopatry (Barraclough and Vogler 2000). Furthermore, Darwin recognized that in more sedentary organisms or those, like plants, that rely less on cross-fertilization, divergence could proceed without complete isolation, “though in such cases, isolation, at least partial isolation at first, would be favourable to their natural selection” (Natural Selection [Stauffer 1975], p. 256). In 1876, responding to criticism from Moritz Wagner that he had neglected to appreciate the role of geographic isolation, Darwin stated, “I do not believe that one species will give birth to two or more new species, as long as they are mingled together within the same district. Nevertheless I cannot doubt that many new species have been simultaneously developed within the same large continental area; and in my ‘Origin of Species’ I endeavoured to explain how two new species might be developed, although they met and intermingled on the borders of their range. It would be a strange fact if I had overlooked the importance of isolation, seeing that it was such cases as that of the Galapagos Archipelago, which chiefly led me to study the origin of species” (Darwin 1887, 3:159). The evolution of species from varieties by the process of natural selection in Darwin’s description of speciation seems perfectly consistent with the idea that geographically isolated populations may incrementally acquire reproductive isolating barriers during adaptive divergence. Although Mayr (1992) criticized Darwin for not appreciating the important of geographic barriers in speciation, in his paper titled “Ecological Factors in Speciation,” Mayr (1947, p. 263) states, “It appears to me that there is no real conflict between those authors who stress the ecological aspects of speciation … and those, who, like myself, have stressed geographical aspects.” He suggests that those who emphasize ecological factors in the origin of species “generally accept sympatric speciation as an integral part of ecological speciation” (Mayr 1947, p. 263). Although that is indeed a common view, there is no reason to invoke sympatric speciation whenever ecological factors are thought to play a primary role in speciation. Indeed, Coyne and Orr (2004, p. 179), staunch advocates of the primacy of allopatric speciation, state that “virtually all barriers can Adaptation and The Origin be considered ecological in the sense that they may arise from environmentally imposed selection.” Darwin clearly believed that under some circumstances, divergent natural selection could occur between varieties that are at least partially sympatric, perhaps elevating them to species by divergent natural selection (Pfennig and Pfennig 2010). Yet I can find no evidence that Darwin believed that varieties arise in sympatry and/or that species typically evolve from sympatric varieties, as his critics have sometimes charged. As Sulloway (1979, p. 48) concluded, “without some form of isolation—whether complete, partial, or behavioral—Darwin fully understood, and repeatedly insisted, that speciation would be impossible.” Species Concepts A species, as we now see it, is characterized not only by being different, but also by being distinct from other species (separated by a gap, reinforced by isolating mechanisms). In his more nominalistic treatment of species in the late 1850s Darwin was concerned only with difference, the first criterion of this dual characterization. (Mayr 1992, p. 349) In The Origin, Darwin apparently felt that species were not real. (Coyne and Orr 2004, p. 10) Part of the challenge in studying speciation is to develop and implement criteria for distinguishing “species” from other categories, such as varieties, subspecies, races, and ecotypes. This is rarely a problem in sympatric taxa because the maintenance of morphological and genetic differences other than simple genetic polymorphisms is clear evidence that sympatric populations function as distinct species. The main problem is with allopatric taxa: are the observed genetic/and or morphological differences sufficient to regard populations as distinct species? Darwin (1859, p. 52) was unable to provide clear guidelines in these situations: “It will be seen that I look at the term species, as one arbitrarily given for the sake of convenience to a set of individuals closely resembling each other, and that it does not essentially differ from the term variety, which is given to less distinct and more fluctuating forms.” It is understandable that this unambiguous statement is used to support the suggestion by many authors that Darwin saw no difference between varieties and species and hence was unable to make any real contribution to our understanding of the origin of species (Mayr 1959, 1992, 1994; Futuyma 1998; Coyne and Orr 2004). Yet, later, in discussing species of large genera, Darwin (1859, pp. 57–58) reaches a different conclusion, stating, “the amount of difference between varieties, when compared with each other or with their parent-species, is much less than that between the species of the same genus. But S9 when we come to discuss the principle, as I call it, of Divergence of Character, we shall see how this may be explained, and how the lesser difference between varieties will tend to increase into the greater differences between species.” It thus seems that Darwin recognized the importance of species in both evolutionary and taxonomic terms. He spent years carefully studying the taxonomy of barnacles, and Mayr (1992, p. 347) describes Darwin’s travails in trying to assign species status in these organisms, “I have gnashed my teeth, cursed species, & asked what sin I had committed to be so punished.” Would Darwin have engaged in these studies if he had no appreciation for species? Darwin also devoted a long section in The Origin to the difficulties that he and other naturalists had in attempting to distinguish species from varieties (chap. 2, the section titled “Doubtful Species”). As one example, he refers to the difficulties presented by the wide variability seen in oaks. It is worth noting that using morphological traits to delimit species boundaries in difficult groups like oaks remained a challenge long after Darwin’s observation (Burger 1975; Van Valen 1976), and it has taken new and sophisticated molecular genetic approaches to resolve taxonomic and evolutionary questions in this group (Howard et al. 1997; Dodd and Afzal-Rafi 2004; Lexer et al. 2006). We now recognize that Darwin’s taxonomic struggles to classify species were not simply a consequence of subtleties in distinguishing “good” species. If speciation is the gradual accumulation of genetic, ecological, and morphological differences, we should sometimes find intermediate stages in speciation where closely related groups are substantially differentiated but are not yet species (Grant 1957; Hendry 2009). As Grant (1957, p. 75) concluded, “Where species originate from races by a continuous and gradual process of evolution, some populations at any given instant will be in a halfway stage between race and species.” It seems that Darwin recognized this intermediate stage in speciation, hence his difficulties in drawing a clear line between species and varieties. Darwin also recognized that whether crosses between varieties or putative species could create fertile hybrids was a poor criterion for assigning species status. As discussed by Mayr (1992, p. 353), Darwin’s perspective on this topic was apparently influenced by his conversations with botanists, who recognized that many “good” plant species could hybridize, “They convinced him … that sterility was not the secure species criterion he had once thought.” In Natural Selection (Stauffer 1975, p. 97), Darwin states, “there are great difficulties … in taking lesser fertility in the offspring as an unerring guide what forms to call species.” Darwin’s views were echoed by Huxley (1859, p. 8) in his commentary on The Origin: S10 The American Naturalist But is it not possible to apply a test whereby a true species may be known from a mere variety? Is there no criterion of species? Great authorities affirm that there is—that the unions of members of the same species are always fertile, while those of distinct species are either sterile, or their offspring, called hybrids, are so. It is affirmed not only that this is an experimental fact, but that it is a provision for the preservation of the purity of species. Such a criterion as this would be invaluable; but, unfortunately, not only is it not obvious how to apply it in the great majority of cases in which its aid is needed, but its general validity is stoutly denied. trinsic or intrinsic factors (Cronquist 1978). The same can be said of species concepts based on phylogenetic differences: the criterion of “fixed diagnosable differences” for diagnosis of species under the phylogenetic species concept (Cracraft 1989) can be met only if there is a barrier to gene flow between groups. Thus, Darwin’s approach to the problem of species, that is, a search for biologically meaningful discontinuities between groups, is not so different from current taxonomic practices. The importance of hybrid sterility in the study of species and speciation is still a matter of debate. For example, in discussing Darwin’s rejection of hybrid sterility as a criterion for species status, Mallet (2008) applies a narrow definition of reproductive isolation, treating intrinsic postzygotic isolating mechanisms such as hybrid sterility and inviability as being the exclusive forms of reproductive isolation. He suggests that “it is sufficient to say here that we now know that a great deal of the evolution of reproductive isolation has nothing to do with speciation; instead it occurs long after speciation has occurred” (Mallet 2008, p. 12). The implication is that reproductive isolation is synonymous with hybrid sterility and inviability and that Darwin was right to reject reproductive isolation as a criterion for species. What Darwin really said is that given the wide variation observed in the success of crosses between species, hybrid inviability and sterility are not sufficient in their own right to distinguish varieties from species. As suggested by Reznick (2010), a major goal of The Origin was to present the theory of evolution by natural selection as an alternative to the prevailing view of species as the products of special creation. Hence, Darwin’s view of varieties and species as being interconnected by many small differences was perhaps more important than establishing just how species originate or how to distinguish “species” from everything else. Most evolutionists now define species as reproductively isolated groups and regard hybrid sterility as just one of many possible isolating barriers (Dobzhansky 1937, 1951; Mayr 1942, 1947; Coyne and Orr 2004). Darwin correctly did not treat hybrid sterility as the sole criterion for species status, but he also failed to appreciate the role of other reproductive isolating barriers (Reznick 2010). For practical reasons, reproductive isolation per se is rarely used as a criterion for diagnosing species status. More often, morphological and genetic discontinuities between populations serve as proxies for measures of reproductive isolation. Even staunch defenders of the morphological species concept recognize that differences between populations can arise only if gene flow is limited by ex- The study of speciation from an evolutionary perspective is now based almost entirely on reproductive isolation and thus differs importantly from Darwin’s approach to the problem (J. A. Coyne, personal communication). Darwin attempted to delineate the boundaries between varieties and species by their morphological divergence. That these differences were often subtle and the interpretation of the differences arbitrary was a constant source of frustration to Darwin. The biological species concept (BSC) proposed by Ernst Mayr made reproductive isolation the principal criterion for evaluating the taxonomic and evolutionary status of different populations. Mayr (1942, p. 120) defined species as “groups of actually or potentially interbreeding natural populations, which are reproductive isolated from other such groups.” The BSC has revolutionized the way we think of species and speciation. Although the BSC is limited in its applicability to sexual, primarily outcrossing taxa, it is clearly superior to the morphological species concept that Darwin accepted. The BSC provides a general framework for assessing the taxonomic and evolutionary status of populations. However, until recently, there were no specific guidelines for how the BSC could or should be implemented to study the factors contributing to the origin of species. Here I consider three critical questions in the study of speciation that are motivated, in large part, by the BSC and review a method proposed by Coyne and Orr (1989) that holds great promise for answering them. First, do populations constitute different species under the BSC? A standard approach for evaluating species status is to experimentally cross different populations: if such crosses fail to yield viable and fertile hybrids, the populations are clearly different species under the BSC. Yet, as Darwin noted (see above), this criterion is not always sufficient, as some “good” species are capable of producing fertile hybrids. Mayr (1947, p. 278), concluded, “Most isolating mechanisms between closely related species that have been studied thoroughly were found to be multiple. There always seem to be involved (a) differences in the ecological requirements, (b) reduction of the mutual sexual stimulation, and (c) reduction in the number and the Understanding Speciation after Darwin Adaptation and The Origin viability of the offspring.” Thus, when any single barrier is insufficient to prevent gene flow, we need a method that can quantify the total isolation that is currently, or potentially, achieved through the action of all possible barriers. Second, what is the contribution of each of the possible isolating barriers to the current total isolation? For the most part, studies of speciation have investigated putative isolating barriers without regard to their actual contribution to the total isolation. One common practice is that isolating barriers that can function only in sympatry, for example, mate choice, gametic isolation, and postzygotic isolation, are commonly measured without consideration of the geographic distributions of species. Consider a recent study of intrinsic postzygotic isolation between two allopatric subspecies of fruit flies, Drosophila pseudoobscura pseudoobscura and D. pseudoobscura bogotana. Phadnis and Orr (2009) found that a single segregation distorter gene causes hybrid sterility between these taxa, and concluded that this is “a strong reproductive barrier between these young taxa” (p. 376). While this is true, given that these taxa are allopatric, it seems improbable that hybrid sterility is a significant barrier to contemporary gene flow, although it may have contributed to their initial divergence (see below). Such intrinsic genetic incompatibilities between allopatric taxa might best be regarded as potential isolating barriers that could become realized if the two taxa become wholly or partly sympatric because of changes in their geographic ranges. Without a method for estimating the contributions of different isolating barriers to the total isolation, we risk ascribing speciation to isolating barriers that may have little or no actual contribution to current or past isolation. Finally, which barriers were in place at the time of speciation, that is, when gene flow between taxa essentially ceases? This is one of the most pressing problems in the study of speciation. It is one thing to find that a given barrier explains most of the current isolation between biological species or that it might prevent gene flow if allopatric populations were forced into sympatry, but this is no assurance that it had the same importance at the time of speciation. Answers to these and other questions about the origin of species can be obtained via the components-of-isolation method (CIM) developed by Coyne and Orr (1989) and implemented in their landmark papers on speciation in Drosophila (Coyne and Orr 1989, 1997). The CIM estimates the contributions of different individual isolating barriers to the total reproductive isolation between populations or species. The conceptual advance of the CIM is the recognition that the barriers isolating species or S11 populations act sequentially throughout the life cycle.1 One can think of different potential isolating barriers as filters that act at different life-history stages to reduce gene flow. Later-acting barriers can eliminate only genes that were not removed by the action of earlier barriers. Hence, the contributions of later-acting barriers are “discounted” by those that precede them. Coyne and Orr (1989) developed the CIM to compare the contributions of premating, sexual isolation (Pre), and intrinsic postzygotic isolation (Post) to the total isolation between species pairs of Drosophila. Assuming that the two barriers act sequentially, Coyne and Orr estimated the total isolation between species (T) as T p Pre ⫹ (1 ⫺ Pre) Post. (1) Ramsey et al. (2003) showed that this approach could be generalized to multiple barriers. The absolute contribution (ACn) of a component of reproductive isolation (RI) at stage n in the life history is ( 冘 ) n⫺1 ACn p RIn 1 ⫺ ACi , (2) ip1 and for m barriers the total isolation is 冘 m Tp ACi . (3) i⫺1 The CIM provides empirical estimates of (1) the individual barrier strength, (2) the absolute contribution of each barrier to the total isolation, and (3) the total isolation. To illustrate, consider how the CIM might be used to assess the degree of reproductive isolation between two populations and to estimate the current importance of the various isolating barriers. We assume that estimates of the strength of individual barriers are obtained for habitat isolation, premating isolation, postmating isolation, and postzygotic isolation. For simplicity, we also assume that barrier strength is symmetrical between the taxa and that barriers evolve at the same rate, such that each barrier has the same individual strength, with a hypothetical value of 0.80. Thus, in this example, 80% of the potential gene flow between populations is eliminated by each barrier (fig. 2A). No single barrier is sufficiently strong to isolate the populations to a level that would satisfy the BSC, yet when the four barriers are considered together, the total isolation is very strong (T p 0.998; fig. 2B). The CIM thus gives an answer to the first question 1 When I asked Coyne about the source of this method, he remarked, “It was my idea, … but it’s obvious, isn’t it?” S12 The American Naturalist Figure 2: Hypothetical example applying Coyne and Orr’s (1989) components-of-isolation method, with modifications from Ramsey et al. (2003), for estimating the contribution of sequential reproductive barriers to the total reproductive isolation. A, Individual barrier strengths are the same (0.80) for each barrier. B, Absolute contribution of each barrier to the total isolation. Total isolation is the sum of the absolute contributions for all barriers. See text (eqq. [2], [3]) for methods. above: Do populations meet the criteria for species status under the BSC? This could prove particularly useful in comparisons of allopatric populations where potential isolating barriers are not “tested” in sympatry. Furthermore, the CIM answers the second question above: What is the contribution of each of the possible isolating barriers to the current total isolation? As illustrated in the example (fig. 2B), we find that habitat isolation, the earliest-acting barrier, is responsible for most of the total isolation and that subsequent barriers have progressively lesser roles. This is despite the fact that each barrier has evolved at the same rate and has the same individual strength. Finally, when combined with estimates of time since divergence, the CIM has the potential to answer the last question above: Which barriers were in place at the time of speciation? This is an issue with all categories of barriers. Consider, for example, the challenge in trying to assess the historical importance of intrinsic postzygotic barriers in cases where these barriers are currently strong. Were such barriers also strong at the time of speciation? An example where this is probably the case is polyploid speciation, because intrinsic postzygotic isolation is often nearly complete at the time of polyploid formation. Yet new polyploids also display substantial ecological differentiation from their ancestors (Ramsey and Schemske 2002). Thus, even in polyploid speciation, intrinsic postzygotic isolation may not be the primary isolating barrier (Sobel et al. 2010; see below). By estimating the contributions of those barriers that allow nascent populations to achieve complete reproductive isolation, the CIM may offer a way to answer Darwin’s (1859, p. 111) main question, “How, then, does the lesser difference between varieties become augmented into the greater difference between species?” Although I suggest that the CIM of Coyne and Orr (1989) provides a valuable framework for evaluating current and historical barriers to gene exchange, there are some unresolved issues concerning its implementation. For example, Martin and Willis (2007) and Lowry et al. (2008b) suggest that one cannot treat different barriers as evolutionarily independent and that reciprocal differences, that is, asymmetries between the parental taxa in the magnitude of gene flow, must be considered when estimating the overall effects of different barriers. On the basis of these and other concerns, Martin and Willis (2007, p. 78) concluded that “quantitative measures of either total isolation or the proportional contributions of its components appear to be unattainable.” However, they present no alternative, and for reasons outlined above, I suggest that the CIM is currently the best approach to evaluate the importance of multiple barriers to the total isolation between populations and species. Nevertheless, it is critical that future studies investigate how the CIM and other methods can be developed for making these estimates (see below). Reproductive Isolation in Nature What is the total reproductive isolation between species in nature, and what are the primary isolating barriers? To address this question, I review here empirical studies that have investigated multiple barriers, largely through implementation of the CIM, to estimate the current total isolation and its components. This is one approach to evaluating Darwin’s view that adaptive divergence caused by natural selection is a major factor in the origin of species. However, it is important to note that the studies reviewed here examined current barriers and thus do not necessarily reflect the importance of those barriers that were in place at the time of speciation. Specifically, I compare the importance of early barriers, such as habitat and premating isolation, to that of late-acting barriers, such as postmating prezygotic isolation and postzygotic isolation. Adaptation and The Origin S13 This is admittedly an imperfect test of Darwin’s principle of divergence, as it is well understood that ecological adaptation can contribute to the evolution of late-acting barriers, such as intrinsic postzygotic isolation (see below). Nevertheless, a finding that ecological barriers can serve as a major isolating barrier would support the idea that adaptive divergence is a critical factor in the origin of species, as Darwin suggested. A Case Study: Adaptation and Speciation in Mimulus To illustrate how the CIM can be implemented, I begin with a description of the relationship between adaptation and speciation in two closely related species of monkeyflowers, Mimulus lewisii and Mimulus cardinalis (section Erythranthe, Phrymaceae). These species were first studied by Hiesey et al. (1971) and have been the subject of numerous field and laboratory experiments designed to investigate the ecological genetics of adaptation and speciation (Bradshaw et al. 1995, 1998; Schemske and Bradshaw 1999; Ramsey et al. 2003; Bradshaw and Schemske 2003; Angert and Schemske 2005; Angert 2006; Angert et al. 2008). The bee-pollinated M. lewisii has pale pink flowers with contrasting yellow nectar guides and a wide corolla with inserted anthers and stigma (fig. 3A). Mimulus cardinalis is hummingbird pollinated and has red flowers, a narrow tubular corolla, reflexed petals, and exserted anthers and stigma (fig. 3B). In the Sierra Nevada of California, M. lewisii is found primarily at high elevations and M. cardinalis at low elevations; the two species are sympatric in a narrow band at midelevations (fig. 3C; Hiesey et al. 1971). Reciprocal-transplant experiments conducted at sites that span the geographic ranges of the two species in the Sierra Nevada (fig. 3C) demonstrate strong local adaptation (fig. 3D; Angert and Schemske 2005) because of physiological differences between the species in their temperature tolerances and flowering phenologies (Angert 2006; Angert et al. 2008). Hence, the fact that the geographic ranges of these species are largely nonoverlapping is due to adaptive differentiation and represents “ecogeographic isolation” (Schemske 2000; Ramsey et al. 2003; Sobel 2010), that is, the reduction in gene flow between populations or species that is due to heritable differences in their geographic distributions. F2 hybrids produced by intercrossing M. lewisii and M. cardinalis were used in field and laboratory studies to examine the floral traits that influence pollinator visitation and the genetic basis of these traits. Experiments with these hybrids conducted near the zone of sympatry showed that the absolute and relative visitation rates by bees are much lower in plants with high petal carotenoid concentration (Schemske and Bradshaw 1999). Genetic mapping studies Figure 3: Studies of adaptation and speciation in two closely related monkeyflowers, the bee-pollinated Mimulus lewisii (A) and the hummingbird-pollinated Mimulus cardinalis (B; photos: D. W. Schemske, H. D. Bradshaw). C, Elevational distribution of the species in the Sierra Nevada Mountains in California (modified from Angert and Schemske 2005). Mimulus lewisii is found from mid-elevations to the timberline, and M. cardinalis is found from low to mid-elevations: the two species are sympatric in a narrow band at mid-elevations (gray bar). Boxes indicate the locations of experimental gardens used in reciprocal transplant studies: Jamestown (JA), Mather (MA), White Wolf (WW), and Timberline (TI). D, Results of reciprocal transplant experiments (modified from Angert and Schemske 2005). E, Wild-type (WT) flower of M. lewisii. F, Flower of the M. lewisii near-isogenic line (NIL) in which the genomic region containing the M. cardinalis allele for petal carotenoid concentration was introgressed into M. lewisii. (E and F reprinted from Bradshaw and Schemske 2003.) S14 The American Naturalist of the F2’s determined that this trait is controlled largely by a single quantitative trait locus (QTL; Bradshaw et al. 1998). To examine further the genetic control of pollinator specificity, near-isogenic lines (NILs) were produced in which the QTL containing the M. cardinalis allele for petal carotenoid concentration was introgressed into M. lewisii, and vice versa (Bradshaw and Schemske 2003). The NILs are thus a reasonable approximation of a single flower color mutation at the petal carotenoid QTL. Field experiments with the NILs showed that the petal carotenoid substitution had major effects on pollinator visitation (Bradshaw and Schemske 2003). In comparison to the M. lewisii wild type (fig. 3E), the M. lewisii NIL (fig. 3F) had much lower bee visitation and increased hummingbird visitation, while the M. cardinalis NIL had much lower bee visitation than the wild type. Phylogenetic studies and the predominance of bee pollination in the genus suggest that hummingbird pollination is the derived condition in M. cardinalis (Beardsley et al. 2003). Thus, the finding that the M. lewisii NIL has both lower bee visitation and higher hummingbird visitation than the wild type suggests that the single adaptive QTL for petal carotenoid concentration played a major role in the transition from bee to hummingbird pollination (Bradshaw and Schemske 2003). Given these findings of adaptive divergence in preferred habitat and pollinators, it is of interest to determine how these factors contribute to speciation. Are these and other ecological factors sufficient to prevent gene flow? A comprehensive study of multiple isolating barriers in this system using CIM found that ecogeographic, pollinator, gametic, and intrinsic postzygotic barriers were individually strong (Ramsey et al. 2003) but that ecogeographic and pollinator isolation, in that order, made by far the largest contributions to the total isolation. These data were reanalyzed here, with revisions made to three barriers, as outlined in table A1 in the online edition of the American Naturalist. First, new results from ecological niche modeling caused a revision of the estimate of ecogeographic isolation upward from 0.587, reported by Ramsey et al. (2003) and acknowledged there to be an underestimate, to 0.97 (Sobel 2010). Second, by including an estimate of expected values, the barrier strength for pollinator isolation was revised from 0.976 to 0.952. Finally, the negative estimates for F1 biomass reported by Ramsey et al. (2003) were revised with a newly proposed method where barrier strengths are bounded between ⫺1 and ⫹1, following Sobel (2010). Although these revisions do alter somewhat the quantitative estimates, the general conclusions from Ramsey et al. (2003) are unchanged. Of the eight barriers investigated, three prezygotic barriers (ecogeographic, pollinator, and gametic) and two postzygotic barriers (hybrid pollen fertility and hybrid seed mass) were much stronger than the others (fig. 4A). The total isolation was estimated as 0.9999 (as compared to 0.9995 in Ramsey et al. 2003), meeting the criterion of the BSC that these taxa constitute reproductively isolated groups. Applying the CIM for estimating the components of isolation illustrates the overriding importance of barriers that act early in the life history. Ecogeographic and pollinator isolation combined cause strong isolation (10.99), while the contribution from all other barriers combined is !0.01 (fig. 4B). Thus, despite substantial reductions in the pollen and seed fertility of F1 hybrids (fig. 4A), these postzygotic barriers contribute little to the total isolation (fig. 4B) because earlier barriers block most of the potential gene flow before hybrids are produced. The ease of crossing the two species in the lab and the high fertility of F1 hybrids led early workers to question whether these taxa were valid species under the BSC. Hiesey et al. (1971, p. 24) stated, “From a biosystematic point of view we are inclined to regard the Erythranthe section as being composed of only two biologically distinct species,” of which M. lewisii and M. cardinalis were viewed as conspecifics. Yet these same authors clearly understood that M. lewisii and M. cardinalis were almost completely reproductively isolated in nature: “It is evident that the combined effect of ecological isolation and different pollinating agents serves as an extremely effective barrier to natural intercrossing between these morphologically very distinct but genetically highly interfertile species” (Hiesey et al. 1971, p. 23). As Darwin recognized (see above), the observation that two populations can form hybrids in experimental crosses is not proof that they should be considered the same species. The Mimulus study is an obvious case in point. Interspecific hybrids are easily produced in the lab but are rarely observed in nature (Ramsey et al. 2003). The two species are almost completely reproductively isolated, largely by strong ecological barriers, and, as final proof of their status as biological species, maintain their distinct characteristics in sympatry. Comparative Studies The results obtained for M. lewisii and M. cardinalis illustrate that ecological differences between these two species are currently the major isolating barriers, consistent with Darwin’s principle of divergence. To determine whether this is a general pattern, I reviewed studies in plants and animals that had estimated multiple barriers. The findings presented here reveal a general pattern of strong contributions from early barriers, such as habitat and premating isolation, and weak evidence for a major role of postzygotic factors, but further research using a variety of different approaches is needed to evaluate the generality of these conclusions. Adaptation and The Origin S15 Figure 4: Estimates of the individual strength of eight isolating barriers (A) and their contribution to the total isolation (B) between Mimulus lewisii and Mimulus cardinalis, from the components-of-isolation method (CIM) of Coyne and Orr (1989) as modified by Ramsey et al. (2003). Data are from Ramsey et al. (2003), with revisions of the individual barrier strengths for ecogeographic isolation, pollinator isolation, and F1 biomass (see text and table A1 in the online edition of the American Naturalist). Values presented are the means of the estimates of individual barrier strength for each species (A) and the mean contribution to the total isolation (B). See text and table A1 for details of the calculations and the data. Barriers are defined as follows: (1) ecogeographic isolation, heritable differences in the geographic range of populations or species due to local adaptation; (2) pollinator isolation, the reduction in interspecific gene flow in sympatry due to pollinator specificity; (3) gametic isolation, the reduction in F1 hybrid formation due to gametic competition and/or the failure of hybrid zygotes to form mature seed; (4) germination, the percentage difference in seed germination between F1 hybrids and their parents; (5) flowering, the percentage difference in survival to flowering between F1 hybrids and their parents; (6) biomass, the percentage difference in biomass between F1 hybrids and their parents; (7) pollen, the percentage difference in the pollen viability between F1 hybrids and their parents; and (8) seed mass, the percentage difference in the seed mass between F1 hybrids and their parents. Plants. Drawing on the review of Lowry et al. (2008b) and subsequent studies, I compiled data on barrier strength in plants for studies that examined multiple intrinsic isolating barriers and estimated the strength of at least one barrier in each of four different categories: (1) habitat isolation, (2) premating isolation, (3) postmating isolation, and (4) postzygotic isolation. Habitat isolation is defined here as the reduction in gene flow caused by differential adaptation to habitat, either on the scale of the geographic range (ecogeographic isolation) or at the scale of local populations (microhabitat). Premating isolation is the reduction in gene flow in sympatry due to temporal isolation (differences in flowering time), pollinator isolation, floral mechanical isolation, and/or the mating system. Adopting a strict definition of postmating isolation is problematic, because the failure to produce viable seed in interpopulation crosses could be due to gametic competition and/ or the failure of fertilized ovules to develop. The studies reviewed here differed in how they classify postmating barriers to F1 hybrid formation. Ramsey et al. (2003) and Kay (2006) have strong evidence of gametic isolation as a cause of reduced interspecific seed production and treated this as a prezygotic barrier, while Lowry et al. (2008b) considered a reduction in F1 seed production after mating to be a postzygotic barrier. G. Chen (personal communication) estimated both gametic isolation and intrinsic isolation as factors in the failure of interspecific seed set. Here I classify postmating barriers as the reduction in F1 hybrid formation that is due to gametic isolation. Finally, postzygotic isolation is defined as the reduction in F1 hybrid formation after interpopulation crosses (but see above) and/or the percentage difference in the relative fitness of F1 hybrids as compared to the parents for extrinsic (ecological unfitness of F1 hybrids) and/or intrinsic (F1 hybrid genetic incompatibilities) isolation. Of the six taxon pairs in Lowry et al. (2008b, table 1) that met this criterion, Iris fulva–Iris brevicaulis and Phlox cuspidata–Phlox drummondii were excluded from further analysis. In the former pair, individual barrier strengths were reciprocally negative for three of the six barriers in- S16 The American Naturalist vestigated (immigrant inviability, F1 seed set, and extrinsic postzygotic isolation; Lowry et al. 2008b). This would mean that each taxon and the F1 hybrids perform better in the habitat of the other taxon and that the F1 hybrids produce more seed than both parents. If these are in fact different biological species, other important isolating barriers were probably missed. For the Phlox species pair, a quantitative estimate of premating isolation was available for only one of the species. Although the study of geographic adaptation in ecotypes of Mimulus guttatus by Lowry et al. (2008a) lacked an estimate of barrier strength for postmating isolation, it was included in the analysis because premating isolation was complete; hence, the contribution of postmating isolation to the total would be 0 regardless of its individual strength. Two additional taxon pairs not in the review by Lowry et al. (2008b) were included in the analysis; Husband and Sabara’s (2004) study of reproductive isolation between diploid and tetraploid populations of Chamerion angustifolium and a study of the Neotropical gingers Costus villosissimus and Costus allenii (G. Chen, unpublished data). In total, seven taxon pairs were used in the comparative analysis of reproductive isolation presented here. In addition to those discussed above for M. lewisii and M. cardinalis, several corrections and improvements to the original data were made before calculating the parameters presented here (table A1). The studies differed in the number of individual barriers investigated, so for purposes of comparison, I first calculated the strength of isolation for each of the four barrier categories discussed above, that is, habitat, premating, postmating, and postzygotic isolation. This was accomplished by applying the CIM approach to each category (Lowry et al. 2008b; G. Chen, personal communication). I then estimated the total isolation and the contribution to the total isolation (eq. [3]). Table A1 gives the individual strengths and contributions to the total isolation for all measured barriers, and table A2 gives the comparable results for the four barrier categories. Although there is considerable scatter, the individual strength of early-acting barriers such as habitat and premating isolation is typically greater than that for postzygotic isolation (fig. 5A). For all taxa considered together, the mean barrier strength was highest for habitat isolation (0.7482) and premating isolation (0.7840), followed by postmating (0.5685) and postzygotic isolation (0.3914). The mean total isolation estimated by the CIM for the seven pairs of plant taxa is 0.9899 (range 0.9591–1.00), indicating strong barriers to gene flow. In those cases where the estimated total isolation is incomplete (i.e., !1.00), it seems highly probable that one or more isolating barriers were missed. That partially sympatric taxa such as Costus pulverulentus and Costus scaber (Kay 2006) and Figure 5: Summary of individual isolating barriers and the components of the total isolation for plant studies that estimated multiple isolating barriers. Numbers refer to the taxa studied; T p total isolation. 1, Mimulus lewisii–Mimulus cardinalis: T p 0.9999. 2, Costus pulverulentus– Costus scaber: T p 0.9970. 3, Costus allenii–Costus villosissimus: T p 0.9917. 4, Chamerion angustifolium, diploids and tetraploids: T p 0.9950; 5, Ipomopsis aggregata–Ipomopsis tenuituba: T p 0.9871; 6, Iris fulva–Iris hexagona: T p 0.9591 ; 7, Mimulus guttatus, coastal and inland: T p 1.00. A, Mean barrier strength for four composite barriers. The filled symbols represent the grand mean across all seven taxa. B, Contribution to the total isolation. The filled symbols represent the grand mean across all seven taxa. See text and tables A1, A2 in the online edition of the American Naturalist for details. Adaptation and The Origin diploid and tetraploid C. angustifolium (Husband and Sabara 2004) retain their genetic and morphological discontinuities despite an estimated total isolation that is !1.00 strongly implicates unmeasured barriers. For example, neither of these studies estimated extrinsic postzygotic isolation. However, even where all possible barriers are examined, the total isolation estimated may be inadequate to maintain independent gene pools, that is, if Nem 1 1. In this regard, estimates of historical patterns of gene flow using molecular genetic markers (Wakeley and Hey 1997; Counterman and Noor 2006; Strasburg and Rieseberg 2008) would be useful for evaluating the population genetic consequences of different levels of total isolation as estimated by the CIM (see below). On average, the contribution to the total isolation drops substantially with each successive stage of isolation (fig. 5B). For all taxa considered together, the mean contributions are 0.7482, 0.2056, 0.0309, and 0.0053 for habitat, premating, postmating, and postzygotic isolation, respectively. A decline in the contribution to the total isolation from early to late in the life history is expected because late-acting barriers can prevent only the gene flow remaining after the action of earlier barriers (fig. 2). However, the greater strength of earlier barriers for the plant studies reviewed here (fig. 5A) also contributes to the observed decline in barrier strength. For six of the seven studies included here, the contribution from habitat isolation was higher than that of any other stage. The one exception is the I. fulva–Iris hexagona species pair, for which there is no estimate of ecogeographic or microhabitat isolation using spatial distribution data, although a reciprocal-transplant study showed only weak habitat isolation (immigrant inviability in table 1 of Lowry et al. 2008b). That habitat isolation has by far the strongest contribution to the total isolation is consistent with Darwin’s focus on adaptation to different habitats as a major cause of speciation, yet it must be emphasized again that current barrier strengths may differ from those at the time of speciation. Six of the seven studies obtained estimates of intrinsic postzygotic isolation, but in only two cases is this barrier strong (table A1). The two exceptions are the M. lewisii– M. cardinalis species pair, where the strength of postzygotic isolation is strong (10.50) for F1 pollen viability and F1 seed mass (fig. 4A), and diploid and tetraploid C. angustifolium, where intrinsic postzygotic isolation is 0.87, stronger than all other measured barriers (table A1). Nevertheless, even in these two systems the contribution of intrinsic postzygotic isolation to the total isolation is low (!0.1; tables A1, A2) because strong habitat and pollinator isolation stop virtually all gene flow before F1 hybrid formation. As exemplified by Husband and Sabara’s (2004) study S17 of diploid and tetraploid C. angustifolium, crosses between ploidy levels typically produce sterile hybrids. This potential isolating barrier evolves immediately with polyploidization (Ramsey and Schemske 1998, 2002). These observations led Schluter (2001) to suggest that polyploid speciation is nonecological. However, strong postzygotic isolation may actually hinder polyploid establishment. Consider that the first polyploids are almost certainly rare. Thus, in the case of sexual and outcrossing taxa, new polyploids will mate primarily with their ancestors and produce largely sterile offspring. All else being equal, this negative frequency dependence, that is, minority-cytotype exclusion (Levin 1975), will cause the rapid extinction of new polyploids unless there are other isolating barriers in place. Spontaneous polyploids often differ substantially from their ancestors in a number of ecological and physiological traits (Ramsey and Schemske 2002) that may cause immediate habitat isolation. In this regard, it is interesting that Husband and Sabara (2004) found that habitat isolation had the strongest contribution to the total isolation between diploid and tetraploid populations of C. angustifolium. When considered individually, intrinsic postzygotic isolation was strong, yet this barrier had only a small contribution to the current total isolation (fig. 5; table A1). Regrettably, despite its importance, polyploid speciation is almost entirely unexplored and deserves further attention. The C. villosissimus–C. allenii species pair is a striking example of Darwin’s proposal for the origin of plant species by adaptation to different soil moisture environments (Darwin’s diagram depicted in fig. 1). These two species of Neotropical gingers are each other’s closest relatives (Kay et al. 2005), use the same pollinators (euglossine bees), and are often found within close proximity but occupy very different habitats. Costus allenii inhabits dense forest understory along streams, while C. villosissimus is found in drier sites with less canopy cover (G. Chen, personal communication). That these differences reflect local adaptation was confirmed by extensive reciprocal-transplant experiments (G. Chen, unpublished data). The two species are easily crossed to produce fully fertile F1 hybrids, yet their strong local habitat differentiation (microhabitat isolation 1 0.90; table A1) results in a microallopatric distribution that greatly reduces gene flow. Laboratory and field experiments show that differences in drought tolerance and the timing of seed germination contribute to their spatial isolation (G. Chen, personal communication) and indicate that local adaptation to edaphic factors has played an important role in adaptation and speciation in these taxa. As further evidence of the importance of adaptive habitat differentiation in the origin of plant species, Sobel (2010) employed ecological-niche modeling (see Sobel et S18 The American Naturalist al. 2010) to examine the role of environmental factors such as temperature, precipitation, and geology to the spatial distributions of 12 pairs of phylogenetically independent sister species in the genus Mimulus. He found strong or complete ecogeographic isolation in seven of the species pairs and moderate ecogeographic isolation in five pairs. The average individual strength of ecogeographic barriers was 0.641. By contrast, the mean barrier strength for pollen viability of F1 hybrids, a common measure of intrinsic postzygotic in plants, was just 0.004 (Sobel 2010). These data strongly suggest that habitat isolation has been more important than intrinsic postzygotic isolation in the speciation of these young taxa. In their review of 19 plant systems, some of which are included here, Lowry et al. (2008b) found that the absolute strength of prezygotic barriers was, on average, twice that of postzygotic barriers, and in reviewing the components of isolation in several different plant systems, Rieseberg and Willis (2007) also concluded that early-acting barriers are the most important. The total isolation and its components were not estimated in either of these studies. Nevertheless, their findings, and the analysis presented here, strongly suggest that early-acting barriers such as habitat and premating isolation are the principle isolating barriers in plants. Animals. At present, few studies in animals have estimated multiple components of reproductive isolation. The original papers on Drosophila by Coyne and Orr (1989, 1997) examined one prezygotic barrier, mating discrimination, and two postzygotic factors, the viability and fertility of male and female F1 hybrids, all measured under laboratory conditions. They concluded that both prezygotic and postzygotic isolation increase with genetic distance between species pairs, a surrogate for time, and that prezygotic isolation evolves more quickly in sympatric than in allopatric taxa, a conclusion consistent with selection for reinforcement in sympatry. Of the species pairs investigated in their studies, 150% are allopatric (from table 1 in Coyne and Orr 1989 and table 1 in Coyne and Orr 1997), yet the barriers they examined can come into play only in sympatry. Hence, the studied barriers do not currently contribute to reproductive isolation in allopatric species, although they may become important if these taxa became sympatric. Their finding that there is little or no intrinsic postzygotic isolation for many “young” species pairs with low genetic divergence (fig. 1b in Coyne and Orr 1997) may suggest that this putative barrier evolves mainly after speciation is complete (Mallet 2006). A similar pattern is seen in Lepidoptera, in which intrinsic postzygotic isolation accumulates with time, yet the youngest species display little or no postzygotic isolation (fig. 1 in Presgraves 2002). Other studies of isolating barriers in animals also suggest that intrinsic postzygotic isolation may play little role in the origin of species. For example, in their review of intrinsic postzygotic isolation in birds, Price and Bouvier (2002, p. 2088) concluded that “genetic incompatibilities causing F1 infertility and F1 inviability in captivity often, but not always, arise after the speciation process is essentially complete.” In a study of freshwater fish (Percidae: Etheostoma), Mendelson (2003, p. 317) concluded that sexual isolation due to strong sexual selection “will tend to evolve to completion before hybrid inviability, strictly as a by-product of divergence in geographically isolated populations.” Similarly, Bolnick and Near (2005) found that intrinsic hybrid inviability in centrarchid fish, measured under laboratory conditions, does not appear until approximately six million years after divergence, and they concluded that “the evolution of hybrid inviability plays little role in driving speciation in centrachids” (p. 1765). Two recent studies investigating multiple potential barriers in animals also find a greater importance of earlyacting barriers. Matsubayashi and Katakura (2009) investigated premating, postmating, and postzygotic barriers between two closely related ladybird beetles (Henosepilachna spp.) and found that reproductive isolation is nearly complete, largely because of habitat isolation. Similarly, Dopman et al. (2009) investigated multiple potential isolating barriers between strains of the North American corn borer (Ostrinia nubilalis) and found that much of the total isolation is caused by premating barriers, particularly temporal and behavioral isolation. Summary: Reproductive Isolation in Nature The pattern that emerges from this admittedly incomplete review is that the most closely related populations and species are isolated primarily by ecological barriers that prevent hybrid formation. While the individual strength of intrinsic postzygotic factors is sometimes large, the current contribution of these late-acting barriers to the total isolation is usually very small. These findings are consistent with Darwin’s principle of divergence, that the origin of species is achieved largely through ecological adaptations. Obviously, more studies are needed to determine whether these are general patterns and whether current barriers were important at the time of speciation. Future Directions Some 150 years after publication of The Origin, substantial controversy remains over the nature of speciation. With the extraordinary achievements made since Darwin in fields such as genetics, ecology, population biology, and systematics, there is now an unprecedented opportunity Adaptation and The Origin to answer many of the most pressing and difficult questions concerning the origin of species. Here I discuss some fertile areas for further research. What Are the Traits That Contribute to Reproductive Isolation, and What Is the Genetic Basis of These Traits? Darwin emphasized the relationship between adaptive divergence and the origin of species, but as discussed above, he did not link adaptation to reproductive isolation. As a result, in large part, of the new focus on ecological speciation (Schluter 2001, 2009; Rundle and Nosil 2005), we now have a number of examples of how the traits involved in adaptive divergence contribute directly to reproductive isolation (Schemske and Bradshaw 1999; Rundle et al. 2000; Nosil 2007). However, we do not know how often the traits that are the direct targets of natural selection might also contribute to the evolution of reproductive barriers that are manifest at other stages in the life history. These indirect effects (Coyne and Orr 2004) or by-products (Sobel et al. 2010) of adaptive divergence could play an important role in speciation (Coyne and Orr 2004; Nosil et al. 2009; Sobel et al. 2010). For example, the genes involved in adaptation to copper-rich soils on mine tailings may also play a role in hybrid incompatibilities between Mimulus populations growing on and off copper mines (Christie and Macnair 1987). And in Drosophila, there is evidence that the lethality of hybrids between Drosophila melanogaster and Drosophila simulans is due to adaptive divergence in their nuclear-pore proteins (Presgraves et al. 2003). Thus, adaptive substitutions may contribute to several potential isolating barriers, including hybrid incompatibilities. Linking adaptive traits to reproductive isolation is just one step toward identifying the origin of species. The next is to investigate the genetic basis of those traits. How many genetic changes are required for speciation? How do the genes that cause reproductive isolation evolve? Do isolating barriers differ in their genetic makeup? How much reproductive isolation is achieved though the substitution of individual “speciation genes”? The remarkable discoveries in genetics since Darwin now make it possible to identify genes and genomic regions that contribute to speciation (Noor and Feder 2006; Lexer and Widmer 2008). Examples include studies of floral traits and pollinator isolation in Mimulus (Bradshaw et al. 1998; Bradshaw and Schemske (2003) and Iris (Bouck et al. 2007) and of hybrid incompatibilities in Iris (Taylor et al. 2009), Mimulus (Fishman and Willis 2006), Helianthus (Rieseberg et al. 1999), and Drosophila (Presgraves et al. 2003; Orr et al. 2004; Presgraves 2010a, in this issue). One promising approach that deserves further attention is the use of near- S19 isogenic lines (NILs) to study the relationship between traits, genes, and reproductive isolation. NILs are created by moving small genomic regions containing an allele from one species into the genome of another. The NILs and their associated wild-type lines can be employed in field or laboratory experiments to estimate the change in phenotype and the strength of isolation that might be experienced after a single substitution. For example, NILs created for petal carotenoid concentration in Mimulus (fig. 3E, 3F) revealed that substantial pollinator isolation is achieved after the substitution of what is essentially a single gene (Bradshaw and Schemske 2003). Ortı́z-Barrientos and Noor (2005) used a similar approach to verify that a single genomic region has a large effect on premating isolation in Drosophila pseudoobscura and Drosophila persimilis. Further studies using these approaches could determine the direct and indirect effects of adaptive substitutions on reproductive isolation (Nosil et al. 2009; Sobel et al. 2010) and ultimately estimate the number of genes required for speciation. What Is the “Importance” of Different Isolating Barriers at the Time of Speciation? Reproductive isolating barriers continue to evolve once speciation is complete; thus, studies of current barriers may not reflect those in place at the time of speciation. Identifying the barriers that contributed to speciation is particularly challenging if one or more barriers are currently strong enough to cause complete isolation: Were they also in place at the time of speciation, or did they evolve after speciation was complete? To answer this question will require studies that examine the full range of potential isolating barriers in nascent and recently diverged species. One possible approach is to identify pairs of sister taxa, from nascent to young species, and to then estimate the contributions of all isolating barriers to the total isolation for each pair. A regression of the contribution to the total isolation against time since divergence for each barrier gives an estimate of its rate of evolution. The importance of the different barriers present at the time of speciation could then be estimated at the point where the total isolation summed across all barriers approaches 1.00. Such studies are difficult to implement because of the many different pieces of information required, from phylogenetic studies of species relationships to field and lab studies estimating barrier strengths and the contributions of individual barriers to the total isolation. S20 The American Naturalist What Is the Role of Ecogeographic Isolation in Speciation? Although microhabitat isolation is widely acknowledged as an important isolating barrier (Coyne and Orr 2004), ecogeographic isolation, defined here as heritable differences in the geographic range of populations or species due to local adaptation, is not. This reflects a contradiction in our current approach to the study of speciation. On the one hand, most researchers acknowledge that allopatric speciation, that is, the evolution of essentially complete reproductive isolation after the geographic isolation of populations by extrinsic barriers (e.g., mountains, rivers, climate change), is probably the major mechanism of speciation (Coyne and Orr 2004). Yet during the period of geographic isolation, we may often expect the evolution of intrinsic ecological barriers that might maintain allopatry even if the original extrinsic barriers were to disappear. This ecogeographic isolation has long been recognized as a legitimate isolating barrier (Coyne and Orr 2004; Sobel et al. 2010), but until very recently it has not been included in empirical studies of speciation. If most speciation begins (and often ends) with complete geographic isolation, why would we not estimate ecogeographic isolation in allopatric species? One obvious answer is that it may be difficult to distinguish historical from biological causes of allopatry. The important distinction between history and adaptation in evaluating geographic isolation as a potential barrier was probably first recognized by Dobzhansky (1937), who noted that “the occupation of separate areas by two species may be due not only to the fact that they have developed there, but also to the presence of physiological characteristics that make each species attached to the environment” (p. 231). Mayr (1947), too, recognized that geographical and ecological differences often go hand in hand, citing as an example the “geographical-ecological” races of the mistle thrush in Europe. Stebbins (1950) applied this same idea to plant distributions, and Coyne and Orr (2004) acknowledged that both micro- and macrospatial habitat isolation can contribute to speciation. Thus, it is not so much that evolutionists doubt the importance of ecogeographic isolation; it seems instead that because of the difficulty in estimating this barrier, they have simply neglected it. A number of approaches to the problem have been considered, each with its own set of advantages and disadvantages. The classic approach of reciprocal-transplant experiments is definitive but also raises conservation concerns if habitats are disrupted and/ or nonnative genotypes are introduced. Ecological-niche modeling is a new approach that has great promise for asking whether historical factors, ecological adaptations, or both cause allopatric distributions (Sobel et al. 2010). This method might prove the only means available to study species of conservation concern or those where a reciprocal transplant is impractical. Yet it requires comprehensive data on species distributions, is difficult to apply to rare species, and requires that the major determinants of the geographic distribution have been enumerated at a relatively fine spatial scale. Finally, Nosil et al. (2005) have recently proposed that “immigrant inviability” be considered as a measure of isolation due to local adaptation. They define immigrant inviability as “the reduced survival of immigrants upon reaching foreign habitats that are ecologically divergent from their native habitat” (p. 705). As discussed by Sobel et al. (2010), the procedure for estimating immigrant inviability seems no different from that for evaluating local adaption by a reciprocal-transplant experiment. One important parameter left out of all of these approaches is the migration rate between populations. By combining studies of ecogeographic isolation with estimates of migration rates, it should be possible to partition the total ecogeographic isolation into that due to failure to migrate and that due to the failure of migrants to establish. Ecogeographic isolation is essentially the product of these two parameters. This is an important area of future research. When Is Speciation Nonecological? Recently, Schluter and colleagues (Schluter 2001, 2009; Rundle and Nosil 2005) made the connection between adaptation and barriers to gene flow that Darwin missed. They define ecological speciation as “the evolution of reproductive isolation between populations or subsets of a single population by adaptation to different environments or ecological niches” (Schluter 2009, p. 737). All other means of evolution of isolating barriers, for example, uniform natural selection, genetic drift, and polyploidy, are considered nonecological mechanisms (Schluter 2001). The ecological-speciation perspective has stimulated a wave of new research on the role of ecological factors in speciation. Recent examples include studies of body size and premating isolation in sticklebacks (McKinnon et al. 2004) and host plant adaptation in walking sticks (Nosil 2007). Moreover, in a review of ecological divergence in a variety of plants and animals, Funk et al. (2006) concluded that, controlling for time, reproductive isolation is greater in those groups with the greatest ecological divergence. Yet Hendry (2009) suggests that few studies of ecological speciation have employed adequate methods. And Sobel et al. (2010) question whether the ecologicalspeciation perspective presents a false dichotomy, suggesting that the putative examples of nonecological speciation (uniform selection, genetic drift, polyploidy) also involve ecological processes (although these mechanisms Adaptation and The Origin are “nonecological” in that they do not require divergent natural selection). Nevertheless, nonecological speciation is theoretically possible. Price (2008) proposed a hypothetical scenario whereby sexual selection in birds can cause reproductive isolation between populations without ecological divergence. Evidence for such a mechanism comes from studies of Enallagma damselflies by McPeek and colleagues. They find that species are ecologically equivalent (Siepielski et al. 2010) and that coexistence of multiple species in sympatry is due largely to mate choice and divergence in mating structures (McPeek et al. 2008). Schluter (2009) suggested that even where populations experience the same selection pressures, the fixation of different adaptive mutations in different populations could lead to speciation, a mechanism termed “mutation-order” speciation. And there is also growing evidence that intrinsic postzygotic reproductive isolation (hybrid sterility and inviability) can evolve as the incidental by-product of intragenomic conflicts between selfish genetic elements and their host genomes (Johnson 2010; Presgraves 2010a, 2010b). Intragenomic conflicts, which occur without regard to ecological setting, represent a special case of mutation-order speciation in which selfish genes and host genomes experience recurrent bouts of coevolution. Studies are needed that investigate the likelihood of these and other proposed nonecological mechanisms of speciation. How Do We Best Evaluate the Strength of Isolating Barriers and Their Importance in Speciation? Here I have argued that estimating total isolation and the contribution of different barriers to the total isolation is critical for understanding the importance of current and historical isolating barriers. While acknowledging the need for such an approach, Martin and Willis (2007) identified several issues that merit further attention, including methods for evaluating asymmetries in barrier strength, comparing observed to expected isolation, and estimating total isolation if barriers are not independent. Another potential problem is in evaluating extrinsic and intrinsic postzygotic isolation: the latter is typically estimated under benign laboratory conditions, and it is not clear how one might distinguish it from extrinsic isolation, which is estimated in nature (T. Price, personal communication). These concerns highlight the need for investigating alternative approaches for estimating the importance of different isolating barriers. In particular, studies are needed that adopt a population genetic framework for estimating barrier strength, such as those proposed by Barton and Bengtsson (1986) and Gavrilets and Cruzan (1998). In addition, estimates from alternative methods must be compared, for example, estimates of total isolation using S21 the CIM of Coyne and Orr (1989) and gene flow estimates obtained from coalescent models of genetic data, as proposed by Wakeley and Hey (1997). This would allow the important test of whether isolation estimates such as those presented here are predictive of current and/or historical levels of introgression. In this regard, Sobel (2010) recently implemented such a study in two species pairs of Mimulus and, consistent with expectation, found greater introgression between the taxa that had the lowest estimate of total isolation. What Is the Fate of Populations or Species That Experience Reduced Reproductive Isolation Due to Geologic or Environmental Perturbations? Under some circumstances, young species may not possess sufficient irreversible isolation to prevent collapse if extrinsic geographic barriers are erased or environmental conditions change. One approach to this problem is to compare the total isolation that would result solely from the action of irreversible barriers, such as intrinsic genetic incompatibilities in F1 and later-generation hybrids, to that realized by all barriers. This would provide a means of identifying where populations lie along a continuum of speciation states (Hendry 2009). For example, the collapse of a sympatric stickleback species pair is thought to be a result of extensive introgression after a decline in water clarity attributed to an invasive crayfish (Taylor et al. 2006). In this system, it appears that sexual isolation achieved through visual mating cues was a reversible premating barrier and that irreversible postzygotic barriers were insufficient to prevent gene flow after the environmental perturbation. Studies are needed that examine the fate of taxa after perturbation: will they fuse, or will they maintain separate gene pools through the evolution of additional isolating barriers, that is, reinforcement (Noor 1999; Servedio and Noor 2003; Kay and Schemske 2008)? Moreover, in the case of collapse, should we regard the taxa as “good” biological species before the perturbation? Do the Rates and Mechanisms of Speciation Differ in Different Geographic Regions, and If So, What Might Be the Consequences for Broad Patterns of Community Diversity? Speciation is the source of organismal diversity, and therefore study of the origin of species should figure prominently in discussions of the factors that contribute to geographic patterns in community diversity. For the most part, this is not the case. An obvious example is the longstanding debate over the cause of the latitudinal diversity gradient (LDG). Discussions on this topic have focused almost exclusively on ecological factors that might influ- S22 The American Naturalist ence the maintenance of species and far less on species origins (Mittelbach et al. 2007; Schemske 2009). Recent hypotheses propose higher speciation rates in equatorial regions due to higher temperatures (Allen et al. 2006) or to a greater importance of biotic interactions (Schemske 2009; Schemske et al. 2009), and new phylogenetic approaches hold great promise for assessing geographic patterns in rates of speciation (Weir and Schluter 2007; Ricklefs 2009). Yet there is surprisingly little discussion of how speciation might contribute to the LDG or to other geographic patterns of diversity. Darwin, The Origin, and the Next Naturalists When the views advanced by me in this volume, and by Mr. Wallace, or when analogous views on the origin of species are generally admitted, we can dimly foresee that there will be a considerable revolution in natural history. (Darwin 1876, p. 425) In the final edition of The Origin, Darwin (1876, p. 424) speaks to the profound impact of his theory of natural selection: “I formerly spoke to very many naturalists on the subject of evolution, and never once met with any sympathetic agreement. … Now things are wholly changed, and almost every naturalist admits the great principle of evolution.” Some 150 years later, much remains to be done. What are the traits that contribute to speciation? What is the genetic basis of these traits? Which isolating barriers are most “important”? There are now unparalleled opportunities to combine ecological, phylogenetic, and molecular genetic approaches to answer these and other persistent questions. The study of speciation, as the engine of biodiversity, must be considered one of the fundamental disciplines in biology. Yet the rapid loss of intact natural habitats and the increased fragmentation of those that remain make time of the essence if we wish to make further progress in understanding the mechanisms of speciation. Darwin entrusted young naturalists to pursue the many questions about the origin of species that he left unanswered (1876, p. 423): “I look with confidence to the future,—to young and rising naturalists, who will be able to view both sides of the question with impartiality.” I too am hopeful that the next generation of naturalists will further our understanding of what Darwin (1859, p. 1) called “that mystery of mysteries.” Acknowledgments I thank G. Chen, J. “King” Coyne, D. Pfennig, D. Presgraves, J. 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