What Genetic Changes Would Most Likely Lead To The Evolution Of New Morphological Forms

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Evolution at 2 Levels: On Genes and Form

• Sean B Carroll

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• Published: July 12, 2005
• https://doi.org/10.1371/journal.pbio.0030245

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Citation: Carroll SB (2005) Evolution at Ii Levels: On Genes and Form. PLoS Biol 3(7): e245. https://doi.org/x.1371/journal.pbio.0030245

Published: July 12, 2005

Copyright: © 2005 Sean B. Carroll. This is an open-admission article distributed nether the terms of the Creative Commons Attribution License, which permits unrestricted employ, distribution, and reproduction in whatsoever medium, provided the original work is properly cited.

Abbreviations: MC1R, melanocortin-one receptor; Ubx, Ultrabithorax

This commodity is based on the Allan Wilson Memorial Lectures given at the University of California at Berkeley in October 2004.

In their archetype paper "Evolution at Two Levels in Humans and Chimpanzees," published exactly 30 years agone, Mary-Claire King and Allan Wilson described the great similarity between many proteins of chimpanzees and humans [i]. They ended that the small caste of molecular departure observed could not account for the anatomical or behavioral differences betwixt chimps and humans. Rather, they proposed that evolutionary changes in anatomy and way of life are more oft based on changes in the mechanisms decision-making the expression of genes than on sequence changes in proteins.

This article was a milestone in three respects. First, because information technology was the start comparison of a large set of proteins between closely related species, it may be considered one of the start contributions to "comparative genomics" (although no such field of study existed for another two decades). 2d, because it extrapolated from molecular data to make inferences about the evolution of form, it may also be considered a pioneering report in evolutionary developmental biology. And third, its focus on the question of human being evolution and human capabilities, relative to our closest living relative, marked the showtime of the quest to understand the genetic basis of the origins of human traits. Similar much of Wilson and his colleagues' body of work, this contribution had a nifty influence on paleoanthropologists as well as molecular biologists.

The 30th anniversary of this landmark commodity arrives at a moment when comparative genomics, evolutionary developmental biology, and evolutionary genetics are pouring forth unprecedented amounts of new data, and the entire chimpanzee genome is bachelor for report. It is therefore an opportune time to examine what has been and is being revealed near the relationship between development at the two levels of molecules and organisms, and to appraise the status of Male monarch and Wilson's hypothesis concerning the predominant role of regulatory mutations in organismal evolution.

King and Wilson used the phrase "means of life" to include both physiology and behavior (M.-C. King, personal communication) and proposed that the evolution of both anatomy and ways of life was governed by regulatory changes in the expression of genes. From the outset of this review, I brand the precipitous distinction betwixt the development of anatomy and the evolution of physiology. Changing the size, shape, number, or color patterns of physical traits is fundamentally dissimilar from changing the chemistry of physiological processes. At that place is ample bear witness from studies of the development of proteins direct involved in animal vision [2], respiration [three], digestive metabolism [iv], and host defence [5] that the evolution of coding sequences plays a key role in some (but not all) important physiological differences between species. In dissimilarity, the relative contribution of coding or regulatory sequence evolution to the evolution of anatomy stands every bit the more open question, and will be my primary focus.

The amount of direct bear witness currently in mitt is modest, and includes examples of both the evolution of coding and of not-coding, regulatory sequences contributing to morphological development. However, I will develop the statement, on the ground of theoretical considerations and a rapidly expanding body of empirical studies, that regulatory sequence development must be the major contributor to the development of form.

This conclusion poses particular challenges to comparative genomics. While we are often able to infer coding sequence function from primary sequences, we are generally unable to decipher functional properties from mere inspection of non-coding sequences. This has led to a bias in comparative genomics and evolutionary genetics toward the analysis and reporting of readily detectable events in coding regions, such every bit gene duplications and protein sequence development, while non-coding, regulatory sequences are ofttimes ignored. However, approximately two-thirds of all sequences under purifying selection in our genome are non-coding [6]. Ane consequence of the underconsideration of non-coding, regulatory sequences is unrealistic expectations near what tin can currently be learned virtually the genetic bases of morphological diverseness from comparisons of genome sequences alone. The visible variety of any group is not reflected by the nearly visible components of gene diversity—that is, the variety of gene number or of coding sequences. In order to understand the evolution of beefcake, nosotros take to written report and understand regulatory sequences, also as the proteins that connect them into the regulatory circuits that govern development. I will begin with some historical and theoretical considerations nearly regulatory and coding sequence evolution, and then delve into the insights offered by specific experimental models of anatomical evolution, and finally, I volition revisit Rex and Wilson's original focus and discuss how our emerging cognition of the development of class bears on current efforts to understand homo evolution.

A Brief History of Regulatory Thinking

Most l years ago, as the first sequences of various proteins from different species were adamant, the potential significance of macromolecules for agreement evolutionary processes was quickly recognized [7]. The great similarity among homologous proteins of different species was noted early [8] and raised the question to what degree such sequence changes were functionally significant [9]. With the appearance of the operon model of gene regulation [10], some biologists such every bit Emile Zuckerkandl began to consider the possible function of "controller genes" in evolution, including in the origin of humans from ape ancestors [11]. One of the nigh widely noted serial of theoretical contributions in this period was Roy Britten and Eric Davidson's models for gene regulation in higher organisms, which had an explicit emphasis on the importance of gene regulation in evolution [12,13].

The about influential single publication of this era, however, was Susumu Ohno's book Evolution by Gene Duplication [14]. Ohno focused on the importance of factor back-up in allowing "forbidden" mutations to occur that could impart new functions to proteins. His opening motto, "natural choice simply modified, while back-up created," reflected a view of natural option as a largely purifying, bourgeois process. Ohno insisted that "allelic mutations of already existing gene loci cannot account for major changes in evolution." He proposed that the duplication of regulatory genes and their control regions must have contributed greatly to the evolution of vertebrates. But the book focused exclusively on the evolution of new proteins and did not consider the creative potential of non-coding, regulatory sequences in evolutionary diversification (run across [15]).

It was against this properties that Allan Wilson and his colleagues began a series of investigations into the relationship between chromosomal evolution, protein evolution, and anatomical development in birds [16], mammals [17], frogs [18], and apes [1]. In each of four studies, the discrepancy between the evolution of proteins and the evolution of anatomy led to the conclusion that evolutionary changes in "regulatory systems" were responsible for the evolution of anatomy. Francois Jacob similarly suggested that divergence and specialization result from mutations altering "regulatory circuits" rather than chemical structures [19].

The relative contributions of dissimilar mechanisms to the evolution of beefcake depend upon both what is genetically possible, and what is permitted past natural selection. Before I delve into the data straight concerning the development of anatomy, and how well it fulfills Rex and Wilson'southward original expectations, it will be valuable to consider what mechanisms are available and what parameters volition govern their utilization in evolution, in light of what nosotros now sympathise about how genes office in multicellular organisms.

Pleiotropy and the Genetic Architecture of Multicellular Organisms

Ane critical parameter that affects the relative contribution of different genetic mechanisms to anatomical variation is the pleiotropy of mutations [20]. In general, it is expected that mutations with greater pleiotropic effects will accept more deleterious effects on organismal fitness and volition exist a less common source of variation in course than mutations with less widespread furnishings.

Over the past thirty years, several fundamental features of gene structure, function, and regulation in multicellular organisms take emerged that govern the pleiotropy of mutations and thus shape the capacity of species to generate anatomical variation and to evolve (see Box ane). Because of these features, mutations in different genes and different parts of genes (that is, non-coding and coding sequences) tin can differ dramatically in their degree of pleiotropy. For case, a mutation in the coding region of a transcription factor that functions in multiple tissues may direct touch all of the genes the protein regulates. In contrast, a mutation in a single cis-regulatory element will affect gene expression only in the domain governed by that element.

Box 1. Key Genetic Features of Multicellular Organisms

Individual regulatory proteins function in many different contexts. Signaling proteins, their receptors, signal transducers, and nigh transcription factors are deployed in multiple tissues, organs, or body parts. The function of each regulatory poly peptide is context-dependent, with different genetic targets and morphogenetic outcomes in different tissues.

The expression of private genes is regulated by multiple, modular cis-regulatory elements. The tissue-specific and temporal control of gene expression, particularly of genes encoding the regulatory proteins that shape pattern germination and cell differentiation in animals, is typically governed past arrays of detached regulatory elements embedded in regions that flank coding regions and lie within introns [23].

Many regulatory proteins are members of big families and can overlap in function. More than 20% of human genes and a much larger fraction of plant genes belong to families [75] that are the production of the duplication and evolutionary divergence of ancestral genes.

Multiple poly peptide forms may exist encoded by unmarried genetic loci. Through the employ of alternative promoters and RNA splice sites, multiple mRNAs encoding unlike protein products are often produced from a unmarried locus. Alternative poly peptide forms (isoforms) may part in unlike contexts and/or possess unlike activities.

John Gerhart and Marc Kirschner [21,22] have discussed in depth how certain features of brute genetic regulatory systems influence "evolvability"—the capacity to generate tolerable, heritable variation. For instance, redundancy reduces constraint on change by circumventing or minimizing the potentially deleterious effects of some mutations. Compartmentation besides facilitates change; by uncoupling variation in one process from variation in another, pleiotropy is decreased.

Several genetic features contribute to redundancy and compartmentation. For example, factor duplication creates initially redundant paralogs. Mutations that may have been deleterious in the ancestral gene may be tolerated and permit for the "exploration" of new variation, which can occur in coding or regulatory sequences, or both (Figure 1A). Likewise, the expanded number and diversity of cis-regulatory elements establishes compartmentation past enabling the independent control of gene transcription in different torso parts (Effigy 1B). The use of alternative promoters and RNA splice sites as well contributes to compartmentation by enabling tissue- or cell-typespecific production of alternative forms of a poly peptide (Figure 1C). Variation may arise either in regulatory sequences governing promoter apply or splice site choice, or in coding sequences of exons. The iii mechanisms factor duplication, regulatory sequence expansion and diversification, and alternative protein isoform expression attain essentially the same full general result—they increase the sources of variation and minimize the pleiotropy associated with the development of coding sequences. The global question of the genetic basis of the development of form then boils downward to the relative contribution of gene duplication, regulatory sequence development, and the evolution of coding sequences, over evolutionary time. I volition first examine what is known most the role of regulatory sequences and then discuss the contributions of coding sequences and gene duplication to the evolution of anatomy.

Figure ane. Different Modes of Cistron Evolution Increment the Multifariousness of Factor Function and Minimize Pleiotropy

The office of a progenitor factor with the simple structure of 1 cis-regulatory element (red circle) and a pair of exons (blackness rectangles) can exist expanded and diversified in several ways.

(A) Gene duplication followed by mutations (asterisks) in either coding or regulatory sequences of the initially identical paralogs will produce genes that may be expressed in different ways or proteins with distinct functions, while the original function tin be maintained.

(B) An expansion in the number of cis-regulatory elements by any of a number of means (transposition, rearrangement, duplication, bespeak mutation) can expand the number of tissues in which the gene is active, just preserves the original function.

(C) The evolution of a new exon and splicing sites creates the potential for alternative forms of a protein to be made. Mutations in alternative exons (asterisks) demand not affect the original function of the protein.

https://doi.org/10.1371/journal.pbio.0030245.g001

Regulatory Sequences and the Evolution of Anatomy

Over the past decade or so, comparative studies of gene expression in diverse animals and plants, across all taxonomic levels, have revealed a general clan betwixt the gain, loss, or modification of morphological traits and changes in gene regulation during development [23,24]. Changes in the expression of an individual cistron may evolve through alterations in cis-regulatory sequences or in the deployment and action of the transcription factors that command gene expression, or both.

Progress toward elucidating the mechanisms governing the evolution of specific traits and genes has required the study of models in which genetic and molecular methods enable the identification and autopsy of functional differences amid populations or species. The traits and species for which such detailed analysis has been possible include the trichome [25–27], bristle [28], and pigmentation patterns [29] in fruit flies; blossom coloration [30], compages [31], and branch patterns [32] in plants; and limb [33] and axial diversity in vertebrates [34].

A scattering of studies have genetically demonstrated that evolution at item loci has affected the gain [32], loss [26,27,33], or modification of morphological traits [25]. These studies—highlighted below—have firmly eliminated coding sequences as a possible cause and thereby implicated regulatory sequence evolution at loci encoding pleiotropic transcription factors. In a few cases, direct testify of functional changes in cis-regulatory elements has been obtained [34–36].

Fruit flies display all sorts of conspicuous patterns of black pigmentation on their head, thorax, abdomen, and wings. These patterns are regulated by a diverseness of well-conserved signaling pathways and transcription factors that control the spatial expression of the enzymes that promote or inhibit the formation of the pigment melanin [37]. In Drosophila melanogaster and other members of the genus, structural genes, such as yellowish, are regulated by an assortment of cis-regulatory elements that govern their expression in different torso parts, such as the wing and abdomen [36] and the bristles and larval mouthparts. This modular arrangement of cis-regulatory elements had suggested that gene expression and pigment patterns evolve independently in different torso parts through changes in individual cis-regulatory elements. Recent studies have demonstrated this to exist exactly the case [35,36] (Effigy 2A).

Figure 2. The Modular Architecture of the cis-Regulatory Regions of Pleiotropic Genes Enables the Contained Development of Gene Expression in Unlike Trunk Parts

(A) Expression of the yellow pigmentation gene of Drosophila is controlled by several dissimilar cis-regulatory elements (red circles). Differences in the activeness of selected elements (fly and wing spot) underlie differences in paint patterns between species (Figure based on [35].)

(B) Similarly, the expression of the Pitx1 factor of vertebrates is inferred to exist controlled by multiple elements (red circles). In pelvic-reduced stickleback fish, Pitx1 expression is absent from the pelvic region. This is proposed to occur through of a selective loss of activity of the hindlimb regulatory chemical element (cantankerous through the reddish circle) (Effigy based on [33].)

https://doi.org/x.1371/journal.pbio.0030245.g002

There are several salient general features of the evolution of pigment patterns in fruit flies. Many or all of the structural genes involved are pleiotropic; they have roles in multiple parts of the body and in other physiological processes (for instance, neurotransmitter synthesis and behavior). Furthermore, they are regulated, at least in role, by widely deployed, highly conserved pleiotropic regulatory proteins, some of which are themselves regulated by deeply conserved and evolutionarily stable global regulators of body pattern germination [29]. Thus, while the coding sequences of the structural and regulatory proteins are constrained past pleiotropy, modular cis-regulatory regions enable a great diversity of patterns to arise from alterations in regulatory circuits through the evolution of novel combinations of sites for regulatory proteins in cis-regulatory elements [35]. This diverseness is produced by the sort of "tinkering" with existing components envisaged by Jacob [nineteen].

Is what is truthful of coloration, truthful of more circuitous traits? It is possible that because torso colour patterns are so disquisitional to organismal adaptation, the genetic systems that affect them might exist more flexible than those governing more than complex traits such as body organization, appendage formation, and other, more slowly evolving characters. The bachelor bear witness suggests, notwithstanding, that the diversification of other traits that are governed past highly pleiotropic and well-conserved proteins can also exist accounted for past regulatory sequence evolution.

For case, shifts in the rostrocaudal boundaries of Hox cistron expression are associated with large-scale differences in axial patterning in vertebrates, arthropods, and annelids [24]. Once, the Hoxc8 cistron of the chicken and mouse, differences in the function of one cis-regulatory chemical element have been demonstrated to govern differences in gene expression boundaries in the neural tube and paraxial mesoderm [34].

While such differences in centric morphology are thought to evolve slowly and relatively infrequently, some features of the vertebrate skeleton, such as the pelvic skeleton of stickleback fish, evolve rapidly [38] and repeatedly [33]. Reduction of the pelvic fin, the homolog of the tetrapod hindlimb, is due to changes at the Pitx1 locus [33]. The Pitx1 poly peptide is a pleiotropic transcription factor that affects the development of multiple tissues in fish and mice, including the hindlimb. Pelvic-reduced sticklebacks have lost Pitx1 expression in pelvic fin precursors, but possess a perfectly intact Pitx1 coding region with no sequence changes relative to populations with fully formed pelvic structures. The only explanation consistent with these observations is that regulatory mutations in a cis-chemical element governing expression in the pelvic fin precursors has selectively abolished Pitx1 expression in this one part of the developing animal, while gene expression elsewhere is not affected (Effigy 2B).

The crucial insight from the development of Pitx1, yellow, and Hoxc8 is that regulatory mutations provide a mechanism for change in one trait while preserving the function of pleiotropic genes in other processes. This is peradventure the about important, most fundamental insight from evolutionary developmental biology. While functional mutations in a coding region are ordinarily poorly tolerated and eliminated past purifying selection, even complete loss-of-role mutations in regulatory elements are possible considering the compartmentation created by the modularity of cis-regulatory elements limits the effects of mutations to individual body parts.

Does this mean that coding sequences cannot contribute to morphological evolution? Not at all. There are several clear examples of functional sequence changes in proteins that impact form, and I volition highlight them next. The key questions to keep in mind are, how often and under what circumstances do coding sequences of regulatory molecules functionally evolve?

Coding Sequences and the Development of Beefcake

The body plans of arthropods and tetrapods have evolved around the use of a fairly stable complement of Hox genes in each phylum [24,39]. The stability of Hox gene number, and the conservation of Hox ortholog sequences and role, led to the initial impression that Hox proteins take not significantly diverged in function. However, it is now understood that several arthropod Hox proteins have changed in ways that are associated with shifts in form or developmental mechanisms, including the Hox3, Fushi tarazu, Ultrabithorax (Ubx), and Antennapedia [twoscore] proteins. In the case of Hox3 and Fushi tarazu, Hox-type role has been lost in particular lineages while new functions have been gained. The Fushi tarazu protein of sure insects lost sequence motifs involved in Hox functions, and gained a motif for a new activity involved in segmentation [41,42]. Similarly, the Hox3 protein lost Hox function in insects and gained a novel dorsoventral centrality patterning part. It subsequently underwent a duplication that produced two divergent genes involved in early patterning of the two body axes in one clade of flies [43–45].

In the Ubx poly peptide, functional motifs evolved while the protein retained Hox role. Comparative and functional studies have shown that the carboxy terminus of the Ubx protein was extended in the crustacean lineage and serves as an activeness-modulating domain [46]. In the insect lineage, this domain was replaced past a brusque glutamine/alanine-rich motif that has been well preserved throughout the course of more than 300 million years of insect evolution [47].

These arthropod Hox proteins demonstrate that some of the virtually conserved proteins can, nether certain circumstances, evolve new and unlike activities. In these examples, choice against coding changes might have been relaxed considering of functional back-up amongst Hox paralogs. Even so, these events are, in the long span of the history of these lineages, rare relative to the all-encompassing diversification of body forms. It must as well be stressed that both ftz and Hox3 (and its derivatives zen and bcd) acquired entirely novel regulatory elements that governed their expression in new domains and patterns. Furthermore, Ubx regulation has been extensively diversified amongst arthropods [24], including inside the insects [48–fifty]. Thus, even in the infrequent instances of overt coding sequence evolution in regulatory proteins, regulatory sequence evolution is a critical component of functional evolution, and further diversification of factor part.

Are at that place more common and rapid means of evolving morphological variety via coding mutations? Definitely. One prominent instance is the melanocortin-1 receptor (MC1R), which modulates the quantity and blazon of melanin synthesis in melanocytes. Mutations in the MC1R factor are associated with scale, fur, or feather color variation and divergence in a broad range of species [51]. The ecological significance of alternative phenotypes suggests that the MC1R gene has evolved nether natural and sexual selection. The clear-cut case of MC1R evolution raises the question, why is coding sequence evolution so prevalent in the diversification of vertebrate pigmentation, while the evolution of gene regulation plays a central role in flower and fruit fly pigmentation?

There may exist particular backdrop of MC1R that take enabled it to play this starring role. MC1R is a fellow member of a family of five related receptors and is the only member involved in pigment synthesis regulation [52]. Thus, the structural and regulatory diversification of this receptor family unit (that is, the evolution of MC1R expression in melanocytes) has produced a protein that has a much greater degree of evolutionary freedom than more pleiotropic receptors. It should exist noted that MC1R coding mutations result in body-broad effects on pigmentation, and do not create or alter spots, stripes, or other patterns. The evolution of spatial patterns of pigmentation in vertebrates is even so probable to involve regulatory evolution in the expression of pigmentation proteins, or regulators of receptor activity [53], via mechanisms like to those underlying the development of insect color patterns.

The widespread interest of MC1R coding variation in the visible variety of vertebrates may then be a relatively special example, enabled by the dedication of MC1R to pigmentation and its minimal pleiotropy. It would be expected that other, more than pleiotropic proteins would exist constrained in their sequence variation and, hence, their contribution to morphological variation. However, it has recently been shown that morphological variation in dog breeds is associated with variation in the length of repeated amino acid sequences in the coding regions of a diversity of developmentally important transcription factors [54]. These repeats are encoded by microsatellite sequences that expand or contract at very high rates, and spontaneous or induced mutations of these sites touch on visible traits. The extraordinary variation in repeat lengths, and their potential effects on morphology, raises the possibility that these repeats are a source of variation in natural populations. All the same, this variation may take accompanying deleterious, pleiotropic effects that, while manageable under domestication, would limit its contribution to evolution under natural selection.

Gene Duplication and the Development of Beefcake

The history of Hox genes and the MC1R gene reflects that 1 condition contributing to the potential evolution of coding sequences is the generation of new genes by duplication. E'er since Ohno [xiv], and indeed well earlier [55], in that location has been widespread belief and expectation that gene duplication has been a major driving force in development. Empirical evidence suggests, all the same, that while gene duplication has contributed to the evolution of form, the frequency of duplication events is not at all sufficient to account for the continuous diversification of lineages. This conclusion is based primarily upon two sets of observations.

First, the estimated rate of cistron duplication is about once per factor per 100 meg years [56]. This figure suggests that gene duplication can contribute to genome development over longer spans of evolutionary time (for example, greater than 50 one thousand thousand years), but this charge per unit is not sufficient to account for variation in populations (for example, quantitative trait differences) or for difference amongst related species such as the 300,000 known species of beetles, or 10,000 species of birds.

Second, the relative infrequency of gene duplication is documented past the bodily histories of key developmental regulatory factor families. For example, while it is very clear that during the early development of animals, there was an expansion in the number of Hox genes, and that during the early evolution of the vertebrates, there was an expansion in the number of Hox gene clusters, the number and multifariousness of Hox genes in highly diversified phyla, such every bit the arthropods and tetrapods, appears to have remained fairly stable for very long periods (perhaps approximately 500 million years). Other cistron families, such as the Wnt family unit of signaling ligands, likewise exhibit deep ancestral complication. Of 12 Wnt subfamilies known in vertebrates, 11 have been identified in a cnidarian [57]. Such deep bequeathed complexity is much greater than would exist expected under the hypothesis that variety evolves primarily through the development of new genes [39,58]. Similarly, despite widespread speculation that the human genome would comprise many more than genes than other species, it does not, and the keen majority of human genes take syntenic orthologs in the mouse [6].

Furthermore, the contribution of gene duplication to the evolution of class may exist governed primarily by the difference of the regulation of newly duplicated genes, rather than novel functions acquired by coding mutations. Both theoretical considerations and empirical information have suggested that the partitioning of the progenitor gene'southward functions may occur about often through regulatory mutations, or the partitioning of regulatory sequences in the original duplication event [59].

The Relative Contribution of Regulatory and Coding Sequences to Anatomical Evolution

The examples I have described demonstrate that both regulatory sequences and coding regions of the genome tin and do contribute to the evolution of class. The more subjective result is whether, from the modest sample of case studies mentioned here and in the literature, one can make (and defend) statements about the relative contribution of regulatory and coding sequence evolution to the evolution of anatomy. We are, subsequently all, in much better position at present to do so than King and Wilson were xxx years ago.

While the doubter, "wait and come across" position would appear safer, that would not at all be in keeping with the bold spirit of the pioneers who offset wrestled with the question. Moreover, I debate that a trend is evident, and that that tendency should, of course, inform ongoing and future piece of work. Based upon (i) empirical studies of the evolution of traits and of cistron regulation in development, (2) the rate of gene duplication and the specific histories of of import developmental cistron families, (iii) the fact that regulatory proteins are the most slowly evolving of all classes of proteins, and (iv) theoretical considerations concerning the pleiotropy of mutations, I argue that there is adequate footing to conclude that the development of anatomy occurs primarily through changes in regulatory sequences.

This conclusion comes as no surprise, given the hypotheses of Male monarch and Wilson and others framed decades ago. Indeed, nearly aficionados of evolutionary developmental biology would find no news here. However, I am not convinced that what we have learned about the evolution of form is existence adequately considered in comparative genomics and population genetics, where the potential office of regulatory sequence evolution appears to be a secondary consideration, or ignored altogether. This neglect has fundamental bearing on the issue that kickoff drew King and Wilson'south interest—the origins of differences between chimps and humans.

Chimps and Humans Redux

The morphological differences between modern humans, human ancestors, and the great apes are the product of evolutionary changes in development. I take argued elsewhere [lx] that the development of circuitous traits such as brain size, craniofacial morphology, cortical voice communication and language areas, hand and digit form, dentition, and body skeletal morphology must accept a highly polygenic and largely regulatory basis. The keen and difficult challenge, with the genome sequences of humans, chimps, and other mammals now bachelor, is to map changes in genes to changes in traits. Many approaches are being taken, and a few intriguing associations of candidate genes and the development of item traits take been discovered, such as the FOXP2 gene and the evolution of spoken language [61], and the MYH16 muscle-specific myosin pseudogene and the evolutionary reduction of the masticatory apparatus [62]. My concern here is not whether these specific associations did or did not play a part in human evolution; rather, my concern is the exclusive focus, past choice or by necessity, on the development of coding sequences in these and more genome-wide population genetic surveys of chimp–human differences [63].

At that place exists some disconnect between what studies in model species have underscored—the ability or sufficiency of regulatory sequences to account for the evolution of physical traits—and which models of evolution are implicitly or explicitly being tested when only coding sequence departure is considered. Two stories concerning the FOXP2 gene illustrate the dramatically different conclusions i might draw, depending upon the methodologies and assumptions practical.

The human FOXP2 gene encodes a transcription factor, and mutations at the locus were discovered to be associated with a spoken communication and linguistic communication disorder [64]. The human being FOXP2 protein differs from the gorilla and chimp protein at simply two residues, raising the possibility that the two replacements that occurred in the human lineage might be significant to the development of speech communication and linguistic communication. Furthermore, population genetic analysis indicates that the FOXP2 locus has undergone a selective sweep within the last 200,000 years of homo evolution [61]. While it would certainly be convenient if the two changes in the FOXP2 protein were functional, the additional hypothesis must be considered that functional regulatory changes might have occurred at the FOXP2 locus. In weighing alternative hypotheses of FOXP2 or any gene'southward potential involvement in the evolution of class (or neural circuitry), we should ask the following questions. (i) Is the gene product used in multiple tissues? (two) Are mutations in the coding sequence known or likely to exist pleiotropic? (3) Does the locus incorporate multiple cis-regulatory elements?

If the answers are yes to all of these questions, and then regulatory sequence evolution is the more likely style of evolution than coding sequence evolution. For FOXP2, this appears to be the case. FOXP2 is expressed at multiple sites, non merely in the brain, but in the lungs, heart, and gut as well [64,65]. Patients with the FOXP2 mutation do have multiple neural deficits [66]. And, because FOXP2 is expressed in different organs and different regions of the brain, information technology is certain to possess multiple regulatory elements. Furthermore, it is an enormous, complex locus, spanning some 267 kb. Based upon a simple average base pair difference of one.two%, there should be over 2,000 nucleotide differences between chimps and humans in this span. Because there is much more than potential for functional divergence in non-coding sequences, at that place is no specific reason to favor coding sequence difference over regulatory sequence divergence at FOXP2.

The discovery of FOXP2 and its association with homo speech has inspired consideration of the potential role of FOXP2 in the development of vocalization in other animals, and here is where strikingly different conclusions were reached depending upon the hypothesis tested and the methodology used. Song learning has evolved in three orders of birds. There are some behavioral and neural similarities betwixt bird vocal and human speech in terms of their being learned at critical periods and the involvement of auditory and motor centers and specialized encephalon centers. A standard comparative assay of the FOXP2 coding sequences of humans and song-learning and non-learning birds did not reveal any amino acrid substitutions that were shared between song-learning birds and humans, nor whatever stock-still differences between vocal-learning and not-learning birds. The study ended there was "no prove for its [FOXP2] role during the evolution of song learning in nonhuman animals" [67].

In cracking contrast, when FOXP2 mRNA and protein expression in the developing and developed brains of a diverseness of song-learners and non-learners were examined, a hit increase in FOXP2 expression was observed in Area 10, a centre necessary for vocal learning that is absent from non-learners [68] (Figure 3A–3C). This increase occurs in zebra finches over the developmental menstruation when song learning occurs. Furthermore, in adult canaries, seasonal changes in FOXP2 expression were observed in Area X, associated with changes in the stability of the bird's song (Effigy 3D–3F). Thus, remarkable changes in the regulation of FOXP2, simply not the protein sequence, are correlated with the evolution and evolution of vocal learning in birds. These changes could ascend through the development of FOXP2 cis-regulatory sequences, or of the regulatory or coding sequences of transcription factors that control FOXP2.

Figure 3. The Regulatory Development of FOXP2 and the Origins of Vocal Learning

(A–F) The patterns of FOXP2 expression in sections of bird brains are depicted The dark-green area is the striatum. FOXP2 is upregulated in the vocalism heart known as Area X (pink spots) in song-learning species such as the zebra finch (A) and black-capped chickadee (B) only not in non-learning species such every bit the ringdove (C).

(D–F) In the canary, FOXP2 expression in Surface area Ten varies over seasons; elevated expression is associated with periods during which the vocal is plastic (pink spot).

(Figure based on [68].)

https://doi.org/ten.1371/journal.pbio.0030245.g003

The contrast betwixt the negative conclusions drawn from the analysis of coding sequences and the fascinating correlation revealed by the comparative study of gene regulation in vivo highlights the general inadequacies of, and potential error in, the sectional analysis of coding regions when considering the development of beefcake. But that inadequacy applies more broadly than just to the evolution of form. While standard population genetic tests have been used to search human protein sequences for statistical evidence of positive choice [63,69], several examples of positive pick on cis-regulatory sequences of physiological genes are documented [seventy–72]. This includes the very articulate case of the erythroid-specific loss of expression of the Duffy antigen chemokine receptor in populations resistant to Plasmodium vivax malaria [73]. This loss is due to a regulatory mutation that affects an erythroid cis-regulatory sequence simply has no outcome on receptor expression elsewhere in the body [74].

Any statements or claims, then, about the genetic changes that "make us human" must be weighed critically in lite of the ability and limitations of the methodology employed, and the scope of the hypotheses being tested. While it is understandable that some biologists have reached for the "low-hanging fruit" of coding sequence changes, the task of unraveling the regulatory puzzle is nevertheless to come up.

Conclusion

The hypothesis of regulatory evolution put forrad by King and Wilson 30 years ago was founded entirely on negative information, that is, the apparent insufficiency of coding sequence divergence to account for gross organismal differences. Information technology has required several decades to obtain evidence that regulatory sequences are and so often the basis for the evolution of form that, when considering the evolution of anatomy (including neural circuitry), regulatory sequence development should be the primary hypothesis considered. The analysis of regulatory sequence evolution poses item challenges in that it is impossible to distinguish meaningless from functional changes by mere inspection. But, in nonhuman models where extensive experimental tools are available, there is crusade for optimism that the contribution of regulatory sequences to development volition be increasingly well understood in the nigh term. In order to arroyo the origins of human traits, much greater accent has to exist placed on comparative studies of gene expression, regulation, and development in apes and other primates. This is precisely the requirement forecast past King and Wilson 30 years agone [one], only at present we take the means to meet information technology.

Acknowledgments

I thank Thousand.-C. Male monarch for correspondence regarding her 1975 paper with A. C. Wilson, 50. Olds for the artwork, and A. Rokas, B. Williams, C. Hittinger, B. Hersh, P. Carroll, and South. Paddock for helpful comments. Work in my laboratory is supported by the Howard Hughes Medical Institute.

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Source: https://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.0030245

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