Philosophy

HUMAN NEUROSCIENCE PERSPECTIVE ARTICLE published: 18 December 2014 doi: 10.3389/fnhum.2014.00953 Brain stimulation for treatment and enhancement in children: an ethical analysis Hannah Maslen *, Brian D. Earp, Roi Cohen Kadosh and Julian Savulescu Oxford Uehiro Centre for Practical Ethics and Oxford Martin School, University of Oxford, Oxford, UK Edited by: Peter G. Enticott, Deakin University, Australia Reviewed by: Markus Christen, University of Zurich, Switzerland Nick J. Davis, Swansea University, UK *Correspondence: Hannah Maslen, Oxford Uehiro Centre for Practical Ethics and Oxford Martin School, University of Oxford, Suite 8, Littlegate House, 16/17 St Ebbe's Street, Oxford OX1 1PT, UK e-mail: hannah.maslen@ philosophy.ox.ac.uk Davis (2014) called for "extreme caution" in the use of non-invasive brain stimulation (NIBS) to treat neurological disorders in children, due to gaps in scientific knowledge. We are sympathetic to his position. However, we must also address the ethical implications of applying this technology to minors. Compensatory trade-offs associated with NIBS present a challenge to its use in children, insofar as these trade-offs have the effect of limiting the child's future options. The distinction between treatment and enhancement has some normative force here. As the intervention moves away from being a treatment toward being an enhancement-and thus toward a more uncertain weighing of the benefits, risks, and costs-considerations of the child's best interests (as judged by the parents) diminish, and the need to protect the child's (future) autonomy looms larger. NIBS for enhancement involving trade-offs should therefore be delayed, if possible, until the child reaches a state of maturity and can make an informed, personal decision. NIBS for treatment, by contrast, is permissible insofar as it can be shown to be at least as safe and effective as currently approved treatments, which are themselves justified on a best interests standard. Keywords: brain stimulation, pediatric ethics, cogntive enhancement, functional trade-offs, autonomy Davis (2014) has called for "extreme caution" in the use of non-invasive brain stimulation (NIBS) methods to treat neurological disorders in children. His focus is on transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), which, respectively, involve passing either an electro-magnetic field or a small direct current through the skull in order to modulate neuronal activity. To justify his position, Davis calls attention to four major issues, framed as "known unknowns" in the current literature: (1) unknown effects of brain stimulation, and unknown mechanisms for producing those effects; (2) unknown side-effects of stimulation (both shortand longterm); (3) a lack of clear dosing guidelines; and (4) a lack of translational studies from adults to children. As Davis rightly points out, "children [cannot] be considered as 'small adults' when testing medical interventions" (p. 2). This is especially the case for interventions into the central nervous system, since a child's developing brain may respond differently to stimulation compared to that of an adult. Indeed, research shows that the brain continues to develop even after the age of majority (Sowell et al., 2003). Nevertheless, Davis balances his plea for caution with longer-term optimism. He argues that- when used with care-brain stimulation in children does appear to be safe and well-tolerated, and may even turn out to be "associated with fewer and less unpleasant side-effects than the neuroactive drugs [such stimulation is] intended to replace" (p. 3).1 We are sympathetic with Davis' argument (Cohen Kadosh et al., 2012). Put simply, caution and sustained clinical scrutiny are required, both for research into the effects of pediatric brain stimulation and for the application of such technology. Yet while further empirical studies into appropriate dosing, sideeffects, and so on should allow for brain stimulation in children to be made generally safer (as well as more effective therapeutically), we must also address the gaps in our understanding of the ethical implications of applying this technology to minors. In this article, we aim to contribute to such an understanding. To frame our discussion, we draw a distinction between the use of NIBS (but see Davis and van Koningsbruggen, 2013)2 as a form of treatment for a recognized neurological disorder, and its 1Consistent with this perspective, a recent review concluded that there is at least preliminary evidence of a therapeutic potential for TMS and/or tDCS in children with conditions such as depression and autism spectrum disorder; however, it should be noted that many of the studies included in this review did not have adequate control groups, and should therefore be interpreted with care (Vicario and Nitsche, 2013). 2They argue that the term "non-invasive" "is inappropriate and perhaps oxymoronic, as it obscures both the possibility of side-effects from the stimulation, and the longer-term effects (both adverse and desirable) that may result from brain stimulation. [Moreover, the] tendency for the effects of [such stimulation] to spread from the target brain area to neighboring areas is in itself contrary to the definition of non-invasiveness" (p. 1). Indeed, this ability for (intended) effects of brain stimulation to have potentially adverse Frontiers in Human Neuroscience www.frontiersin.org December 2014 | Volume 8 | Article 953 | 1 Maslen et al. Brain stimulation in children-ethics use as a form of enhancement in healthy children. Although we have argued in previous work that the treatment/enhancement distinction tends to break down in the case of adults (see Earp et al., 2014), in the case of children, we suggest, it has greater normative force. This is because, we argue, the relative weights of (parental judgements of) beneficence vs. respect for autonomy shift as the decision pertains more to "enhancement" than to "treatment".3,4 The tension between these two factors arises because some interventions may involve compensatory trade-offs or functional losses, such as potential cognitive costs in the case of brain stimulation. When these trade-offs have the effect of limiting the child's future options, they pose a threat to his or her (future) autonomy. Whilst choosing to "treat" a child will sometimes be in his or her best interests even if it precipitates cognitive tradeoffs, interventions intended to "enhance" may not be justified in this way. In the absence of a clear pathology, we suggest, greater relative weight should be placed on the child's (future) autonomy, at least in part because the certainty with which the parents can determine what would be in his or her best interests is likely to be significantly reduced. Given this, we argue that brain stimulation for "enhancement"-insofar as it involves a more controversial weighting of benefits vs. risks and costs-should be delayed until the child has reached a state of maturity. In this way, she can make an informed, personal decision about the proposed intervention. Brain stimulation for "treatment", by contrast, is permissible insofar it can be shown to be at least as safe and effective as currently approved treatments (which are themselves justified on a best interests standard). THE PERMISSIBILITY OF TREATING NEUROLOGICAL DISORDERS IN CHILDREN To begin our discussion, we ask, what makes pediatric "treatment" permissible in general? By "treatment" we intend to call to mind such interventions as surgery to correct a heart defect, or the administration of antibiotics to address an infection. In these cases, a disease or deformity is present that threatens the child's well-being, and the treatment is the best available means (or a good-enough means) to mitigate that threat. Thus, although (a) the child cannot strictly consent to the intervention, (b) the ramifications for other brain areas (and/or functions) is a key component of our ethical analysis. 3We do not suggest, of course, that there is a clear-cut, universally agreed-upon distinction between treatment and enhancement (see Maslen et al., 2014). Instead, we envision a sliding scale from interventions that are intended simply to sharpen a certain cognitive skill in a healthy child ("enhancement") to those intended to relieve a child of pain or another burden that significantly affects his or her ability to pursue the normal range of activities that children pursue ("treatment"). 4Throughout this paper we invoke three overlapping considerations: the child's "developing autonomy" (her learning to be self-governing), the child's "future autonomy" (her prospects for pursuing the life plans that she will come to value as an adult) and the child's "self-determination" (the freedom for her actions to be "up to her"). All three are relevant and closely related in our discussion. However, we use the term "future autonomy" more prominently, as this denotes best the concern with preserving options for the child to evaluate herself, once she has sufficient capacity to make such assessments. intervention may carry considerable risk, and (c) it may involve even a gross intrusion into the child's bodily sphere, it is nevertheless considered to be morally permissible. Such an intervention is permissible because, and insofar as, it is in the child's best interests-all things considered (see Hope et al., 2008). We can extend this reasoning to the case of brain stimulation. If a child is experiencing significant psychological and/or physical burdens due to a neurological disorder, the benefits of treatment with stimulation might very well be in the child's best interests in the sense just described. In fact, this could turn out to be the case even if some significant negative side-effects were generated, so long as the overall costs to the child (including the cost to autonomy) were outweighed by the benefits of performing the stimulation before an age of consent. On these grounds, it could be considered permissible, assuming that it were shown to be at least as safe and effective as other, more established treatment paradigms.5 ENHANCEMENT AND THE CHILD'S INTEREST IN AUTONOMY What about the case of "enhancement"? Ethicists are divided on the question of whether parental enhancement choices are in the child's best interests and this is often framed in terms of a consideration of the child's interest in (future) autonomy, or self-determination. Some have argued that the enhancement of a child might lead her to feel unfree to pursue her own life-projects due to the fact that decisions about her traits and capacities have been chosen for her. In developing this argument, Habermas (2003, p. 50) has argued that, in the case of genetic enhancement (i.e., selecting for specific traits, such as intelligence), the parents' choices represent intentions and expectations relating to their child's life. Such expectations, he suggests, lead to the stifling of the child's freedom to develop in his or her own way. Others have argued that enhancement technologies would not undermine autonomy, insofar as they increase the options available in an individual's choice set. For example, Bostrom (2005) claims that an enhanced child might "enjoy significantly more choice and autonomy in her life, if the modifications were such as to expand her basic capability set. Being healthy, smarter, having a wide range of talents, or possessing greater powers of self-control are blessings that tend to open more life paths than they block" (p. 212). Such an analysis tends to assume that enhancement has the overall effect of increasing objective opportunities, even if a child might experience her freedom as being constrained by parental expectations. However, as we will now discuss, in the case of brain stimulation, the assumption of "more choice" may sometimes be mistaken. The arguments 5However, note that the permissibility of parents' choosing the intervention would depend in part on what specific "negative side effects" might be incurred by the stimulation, as well as the magnitude of the risk. As we discuss later on, one high-risk side-effect of some kinds of brain stimulation is the diminishment of a non-targeted cognitive capacity. In this case, the persistence of symptoms due to the neurological condition would have to be worse- that is, more contrary to the child's interests-than the cognitive trade-offs incurred by the stimulation (alongside any other negative side-effects and costs) for the stimulation to be considered permissible on the best interests test. Frontiers in Human Neuroscience www.frontiersin.org December 2014 | Volume 8 | Article 953 | 2 Maslen et al. Brain stimulation in children-ethics we make in what follows are about objective, not subjective, curtailment(s) of freedom. BRAIN STIMULATION AND COGNITIVE TRADE-OFFS While early research into brain stimulation in healthy adults has focused on its potential to enhance cognitive functions, the cognitive costs that might be associated with such enhancement have largely been neglected. However, as Davis points out, no brain region exists in isolation. Indeed, there is evidence that enhancing one aspect of cognition may be detrimental to other cognitive faculties, making neuromodulation "a zero-sum proposition" (Brem et al., 2014; but see Luber, 2014). For example, it has been shown that enhancing cognitive performance on one task can be associated with poorer performance on a different cognitive task (Iuculano and Cohen Kadosh, 2013; Sarkar et al., in press). It must be acknowledged that the evidence for such enhancement tradeoffs has thus far been obtained only from well-controlled laboratory experiments that have poor ecological validity. However, this preliminary evidence should alert us to the possibility of similar trade-offs that might occur in more ecologically valid settings. Laboratory experiments can help to demonstrate what would be theoretically expected, based on the cognitive function that is targeted and the brain regions that are stimulated. Crucially, such experiments suggest that it is theoretically likely that enhancement of one domain of cognition will sometimes come at the cost of impairment in another. Thus, any decision to enhance could be also a decision to impair. When this is coupled with the emerging probability of long lasting effects on the brain (see Snowball et al., 2013), a situation arises in which parents might inadvertently or even knowingly limit (at least some) future options for their children when they choose to enhance particular capacities at the expense of others. For example, imagine a parent who has aspirations for her child to be the star of the school's quiz team. The parent encourages the child to memorize facts whilst her brain is stimulated to enhance long-term memory. However, as a result, the child's visuospatial working memory is impaired and her ability to quickly solve mental arithmetic problems suffers (see de Jongh et al., 2008 for a review of such trade offs with respect to pharmacological enhancements). Although the child performs well on general knowledge tests, she performs less well in mental arithmetic: mathematics-related pursuits are, to a certain degree, limited as a result of the intervention. In this example, by choosing to enhance the child's long-term memory and, correspondingly, the ease with which activities employing this particular cognitive capacity can be pursued, the parent is also choosing to impair a different capacity, making the pursuit of activities involving visuospatial working memory more difficult. It is our contention that making these opportunity-limiting choices on behalf of the child may not be permissible. This is the case even if opportunities associated with the enhanced cognitive domain are increased. Parents cannot know what the child will grow up to value and so should not restrict opportunities based on what they want their child to pursue. Whilst there are many decisions that parents can make in the best interests of their child, which cognitive capacities are more valuable, we contend, is usually not one. This argument applies most strongly to cases in which there is (roughly) a one-to-one trade-off. However, if a given enhancement intervention substantially increased function in one domain, while only slightly reducing function in another (as judged by a reasonable observer), then the decision would turn more heavily on considerations of what would be in the best interests of the child, overall. Such valuations are hard to make, and are likely to be highly subjective in many cases. The more subjective they are (that is, the less clear an "objective" observer would be about the relative weights to assign to the enhanced vs. diminished capacities), the more the decision about intervening should be left to the individual who must live with the consequences. AT WHAT AGE CAN CHILDREN DECIDE TO BE "ENHANCED"? Let us summarize our argument so far. First, when "enhancement" interventions involve a functional trade-off, the agent whose relevant capacities will be altered should usually be the one to make a decision about whether the intervention is desirable, all-things-considered. However, young children are unlikely to know which capacities they will value later in life, since their self-knowledge and ability to make and pursue long-term goals is yet to develop. Therefore, there is a problem in terms of a child's capacity to consent-that is, to fully understand what an intervention involves and what the material consequences will be. Moreover, there is a problem in terms of the child's limited insight into what she will value over time. At what point, then, can children make meaningful decisions regarding self-enhancement, taking into consideration the apparent risk of cognitive trade-offs? To begin with, we should point out that a child's inability to provide informed consent does not make pediatric interventions impermissible per se. As we have already suggested, when it comes to treatment, at least, parents (or legal guardians) can legitimately make decisions in the best interests of the child. Similarly, when an intervention is carried out for purposes of medical research, a child's lack of capacity to consent is not necessarily prohibitive either. In these cases, clinicians or researchers must seek (and obtain) the child's assent to participate in the study (as well meet all other ethical requirements, see Caldwell et al., 2004).6 For minor interventions, then-such as venipuncture for the purposes of a study-a child's assent may be all that is needed. This is because the risks that are associated with such a 6What is assent? Although there are several different theories of assent, at its most basic, it involves agreement to or acceptance of the intervention. It is often argued that the requirements for assent are less cognitively demanding than for consent (see John et al., 2008; Waligora et al., 2014), such that individuals whose capacities to make informed judgments are still developing may nevertheless be able to meet them. In relation to assent for pediatric research, for example, Roth-Cline and Nelson suggest that the child should "understand why he or she is being asked to participate and what will be his or her experience if he or she decides to participate" (Roth-Cline and Nelson, 2013, p. 296). Precise age ranges vary, but a child's assent is thought to be (ethically) obtainable by approximately age five or six, depending upon the specific intervention being proposed (including its risk profile, etc.), and also adjusting for the child's individual stage of development. Frontiers in Human Neuroscience www.frontiersin.org December 2014 | Volume 8 | Article 953 | 3 Maslen et al. Brain stimulation in children-ethics procedure are either immediate and transitory (e.g., pain, stress, or discomfort) or rare (e.g., hemorrhage or infection), assuming that the intervention is properly performed. By contrast, the effects of brain stimulation for "enhancement" may have consequences that reach far into the child's future. Therefore, in order to evaluate the reasons one might have for refusing such an "enhancement" (such as a desire to leave one's cognitive functions intact), one must be capable of meaningful temporal self-projection. Yet such projection is usually not possible for very young children.7 It may be possible, however, for older children and/or adolescents. Accordingly, some scholars have suggested that genuine consent may be possible before an age of legal majority (typically 18), at least for certain kinds of "medical" interventions (see, e.g., Levy et al., 2003). For simple procedures with minimal risks, children as young as 10 may be capable of giving age-appropriate consent. As the risks increase, however, and as the need for temporal projection becomes more central to the decision-making process, a higher threshold for consent is required. In the case of "enhancement" decisions involving potential trade-offs, such as the impairment of a cognitive capacity, the threshold should be higher still. This is for two reasons: first, as we have discussed, a child's brain is still developing, and in numerous ways that are not yet understood. Indeed, even adolescent and adult brains continue to develop. Nevertheless, and second, adolescents (and adults) have much greater insight-compared to very young children- into their own future values. It is this forward-looking capacity, we contend, that is especially important when making decisions about how to weigh the relative value of different cognitive functions; and younger children seem to lack this capacity. Therefore, in the case of pediatric enhancement involving long-term cognitive tradeoffs, we suggest that consent may be (ethically) obtainable by later adolescence, perhaps around the age of 16, but usually not earlier than this. PARENTS' TRADITIONAL INFLUENCE ON CHILDREN'S SKILL DEVELOPMENT A first response to our argument might be to point out that parents already make many (relatively unproblematic) decisions when, for example, they allow their children to take part in certain extra-curricular activities but not others. A parent might encourage her child to go to drama club instead of French lessons or to practice football rather than sing. However, there are important differences between these decisions and the sorts of cognitive trade-offs under discussion.8 7Therefore, a simple understanding of the immediate experience of the intervention would not be sufficient to make the enhancement intervention morally permissible. Indeed, the acceptance that is characteristic of child assent must be supplemented with a strong, considered preference in cases in which long-term trade-offs are under consideration. 8There will, of course, be examples of "hyperparenting" (see Sandel, 2009), which may in fact be as problematic as electing opportunity-limiting interventions involving direct intervention into a child's brain-and for similar reasons. This will be the case particularly when the child is made to spend a considerable amount of time developing a certain skill despite her sustained dissent. Whilst parents should encourage children to try different activities, First, developing a skill through participation in an extracurricular activity does not directly impair the skills that would have been developed had a different activity been selected: practicing music, for example, does not directly impair the ability to speak French. The significance of this disanalogy with cognitive trade-offs will depend upon two things. First, the extent to which the failure to develop a capacity is comparable (in an opportunity-limiting sense) to directly impairing it: if, later in life, non-developed skills can more easily be developed than impaired skills, then the child retains more options. Second, the permanency of the impairment will be highly relevant: temporary enhancement may only result in temporary impairment. If impairment to a capacity subsides, or is compensated for, then it becomes equivalent to a non-developed capacity and the (moral) distance between traditional intervention and neuro-intervention decreases. Thus, neuroscientific evidence regarding the permanency- and extent-of cognitive costs associated with brain stimulation will be essential to determining the permissibility of parental "enhancement" decisions. It will also be crucial to know how these effects differ between one-off vs. repeated interventions, as well as whether the sought-after benefit can be achieved later in life, when the (future) adult can decide for himself or herself. Such knowledge is currently lacking. Accordingly, we highlight the need for careful consideration of these variables, and conclude that "enhancements" involving significant long-term cognitive tradeoffs should be delayed until the individual to be affected can express a considered preference (i.e., adolescence). CONCLUSION Whilst adults are in a position to decide whether effect X is valuable enough (to them) to justify incurring impairment Y, children do not yet have the capacity or the life experience to make such trade-off decisions. They do not know what they will value when they grow up and nor do their parents. Whilst an intervention that improves X may count as an enhancement for the individual who does not care much about Y, another individual, valuing Y over X, will view the very same outcome as an impairment. In such cases-that is, cases in which the very status of an intervention's being an (overall) enhancement vs. an impairment is controversial-the weight of considerations should shift toward delaying the intervention until the individual who will actually be affected by it has sufficient capacity to decide. The more permanent and substantial the trade-off, the more this argument has force. The gaps Davis identifies in the literature on brain stimulation suggest that we do not currently have enough evidence to properly assess the magnitude and permanency of any trade-offs and, consequently, that the caution he recommends is indeed warranted. However, we have suggested that even when science and sometimes override dissent when a child is less-than-enthusiastic on a particular occasion, a child's long-term resistance to an extra-curricular activity renders parental force morally questionable at best, and morally impermissible at worst. This is due to the failure of such parental pressure to nurture the child's developing autonomy and its prevention of the child's pursuit of alternative extra-curricular options, which may become increasingly difficult to master as time goes on. Frontiers in Human Neuroscience www.frontiersin.org December 2014 | Volume 8 | Article 953 | 4 Maslen et al. Brain stimulation in children-ethics can tell us about the effects of brain stimulation in more detail, the permissibility of parental decision-making may remain limited in some cases in which the aim is only to "enhance" an intact cognitive capacity. In contrast, the treatment of atypical cognitive abilities using brain stimulation will be permissible insofar as the stimulation is (at least) as safe and effective as existing treatments in providing an overall benefit to the child. ACKNOWLEDGMENTS This work was supported by the Wellcome Trust [086041/Z/08/Z]; the Oxford Martin School; and the Uehiro Foundation on Ethics and Education. REFERENCES Bostrom, N. (2005). In defence of posthuman dignity. Bioethics 19, 202–214. doi: 10.1111/j.1467-8519.2005.00437.x Brem, A. K., Fried, P. J., Horvath, J. C., Robertson, E. M., and Pascual-Leone, A. (2014). Is neuroenhancement by noninvasive brain stimulation a net zerosum proposition? Neuroimage 85, 1058–1068. doi: 10.1016/j.neuroimage.2013. 07.038 Caldwell, P. H., Murphy, S. B., Butow, P. N., and Craig, J. C. (2004). Clinical trials in children. Lancet 364, 803–811. doi: 10.1016/S0140-6736(04)16942-0 Cohen Kadosh, R., Levy, N., O'Shea, J., Shea, N., and Savulescu, J. (2012). The neuroethics of non-invasive brain stimulation. Curr. Biol. 22, R108–R111. doi: 10.1016/j.cub.2012.01.013 Davis, N. J. (2014). Transcranial stimulation of the developing brain: a plea for extreme caution. Front. Hum. Neurosci. 8:600. doi: 10.3389/fnhum.2014.00600 Davis, N. J., and van Koningsbruggen, M. G. (2013). "Non-invasive" brain stimulation is not non-invasive. Front. Syst. Neurosci. 7:76. doi: 10.3389/fnsys.2013. 00076 de Jongh, R., Bolt, I., Schermer, M., and Olivier, B. (2008). Botox for the brain: enhancement of cognition, mood and pro-social behavior and blunting of unwanted memories. Neurosci. Biobehav. Rev. 32, 760–776. doi: 10.1016/j. neubiorev.2007.12.001 Earp, B. D., Sandberg, A., Kahane, G., and Savulescu, J. (2014). When is diminishment a form of enhancement? Rethinking the enhancement debate in biomedical ethics. Front. Syst. Neurosci. 8:12. doi: 10.3389/fnsys.2014.00012 Habermas, J. (2003). The Future of Human Nature. Cambridge: Polity Press. Hope, R. A., Savulescu, J., and Hendrick, J. (2008). Medical Ethics and Law: The Core Curriculum. London: Elsevier Health Sciences. Iuculano, T., and Cohen Kadosh, R. (2013). The mental cost of cognitive enhancement. J. Neurosci. 33, 4482–4486. doi: 10.1523/jneurosci.4927-12.2013 John, T., Hope, T., Savulescu, J., Stein, A., and Pollard, A. J. (2008). Children's consent and paediatric research: is it appropriate for healthy children to be the decision-makers in clinical research?. Arch. Dis. Child. 93, 379–383. doi: 10. 1136/adc.2007.118299 Levy, M. D. L., Larcher, V., and Kurz, R. (2003). Informed consent/assent in children. Statement of the ethics working group of the Confederation of European Specialists in Paediatrics (CESP). Eur. J. Pediatr. 162, 629–633. doi: 10. 1007/s00431-003-1193-z Luber, B. (2014). Neuroenhancement by noninvasive brain stimulation is not a net zero-sum proposition. Front. Syst. 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M., Welcome, S. E., Henkenius, A. L., and Toga, A. W. (2003). Mapping cortical change across the human life span. Nat. Neurosci. 6, 309–315. doi: 10.1038/nn1008 Vicario, C. M., and Nitsche, M. A. (2013). Non-invasive brain stimulation for the treatment of brain diseases in childhood and adolescence: state of the art, current limits and future challenges. Front. Syst. Neurosci. 7:94. doi: 10. 3389/fnsys.2013.00094 Waligora, M., Dranseika, V., and Piasecki, J. (2014). Child's assent in research: age threshold or personalisation? BMC Med. Ethics 15:44. doi: 10.1186/1472-693915-44 Conflict of Interest Statement: Roi Cohen Kadosh has filed a patent entitled "Apparatus for Improving and/or Maintaining Numerical Ability" (International Application PCT/GB2011/050211). Received: 25 September 2014; accepted: 09 November 2014; published online: 18 December 2014. Citation: Maslen H, Earp BD, Cohen Kadosh R and Savulescu J (2014) Brain stimulation for treatment and enhancement in children: an ethical analysis. Front. Hum. Neurosci. 8:953. doi: 10.3389/fnhum.2014.00953 This article was submitted to the journal Frontiers in Human Neuroscience. Copyright © 2014 Maslen, Earp, Cohen Kadosh and Savulescu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Human Neuroscience www.frontiersin.org December 2014 | Volume 8 | Article 953 |


Philosophy

Hofmann Evo Edu Outreach (2017) 10:1 DOI 10.1186/s12052-017-0064-4 HISTORY AND PHILOSOPHY Rate variation during molecular evolution: creationism and the cytochrome c molecular clock James R. Hofmann* Abstract Molecular clocks based upon amino acid sequences in proteins have played a major role in the clarification of evolutionary phylogenies. Creationist criticisms of these methods sometimes rely upon data that might initially seem to be paradoxical. For example, human cytochrome c differs from that of an alligator by 13 amino acids but differs by 14 amino acids from a much more closely related primate, Otolemur garnettii. The apparent anomaly is resolved by taking into consideration the variable substitution rate of cytochrome c, particularly among primates. This paper traces some of the history of extensive research into the topic of rate heterogeneity in cytochrome c including data from cytochrome c pseudogenes. Keywords: Molecular evolution, Cytochrome c, Molecular phylogenetics, Mutation rates, Molecular clocks, Creationism © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Background The most egregious and widespread creationist misuse of cytochrome c sequence data is surely the spurious "equidistance" anomaly generated when amino acid sequences taken from several members of a large clade are compared to the sequence for a single member of an outgroup to that clade. Far from being anomalous, data of this sort were both predicted and confirmed by Emanuel Margoliash as early as 1963 (Margoliash 1963, p. 677). In spite of Margoliash's trenchant discussion of this point and numerous subsequent readily available confirmations in prominent scientific and popular sources, the claim that the cited types of equidistance are anomalies for evolutionary theory continues to circulate in creationist venues.1 The topic of molecular clocks thus provides unfortunate examples of how misguided creationist arguments can proliferate by means of uncritical repetition. More subtle molecular clock issues arise from the fact that mutations can result in variable amino acid replacement rates in proteins, especially among primates. For example, in a 30 September 2014 video entry for his blog, The New Creationist, Eugene Gateley pointed out that the cytochrome c of the American alligator, Alligator mississippiensis, has an amino acid sequence slightly closer to that of humans than is the corresponding sequence in the cytochrome c of a primate, the bush baby Otolemur garnettii (Gateley 2014). To put the paradoxical aspect of this fact in context, Fig. 1 illustrates the evolutionary consensus that among early primates two major taxa, strepsirrhines and haplorhines, diverged from each other at least 70 million years ago. Haplorhines subsequently diversified into the tarsiers, the new world monkeys, the old world monkeys, the apes, and eventually Homo sapiens. 1 This case is discussed in detail in Hofmann (2014). Open Access *Correspondence: firstname.lastname@example.com; Liberal Studies Department, California State University Fullerton, Fullerton, CA, USA Page 2 of 17Hofmann Evo Edu Outreach (2017) 10:1 Strepsirrhines also diverged into two main subgroups, lemurs and Lorisiformes. Lorisiformes in turn diversified into the lorises and the galagos, commonly referred to as bush babies. The bush baby O. garnettii thus is a strepsirrhine primate. Consequently, among the primates O. garnettii is quite distantly related to humans but is of course much more closely related to humans than is any non-primate. The data cited by Gateley thus do not appear to agree with the evolutionary consensus that the strepsirrhine primate O. garnettii is much more closely related to humans than alligators are. The following excerpt is a transcript of the conclusion of Gateley's recorded comments; allowance should be made for the fact that these are Gateley's spoken remarks rather than writing intended for publication. ... the question then is, why in the world would an alligator be more similar, at 87.62%, would an alligator be more similar to human than another primate at 86.67%, rounded up, you know. So, the evidence here is drastically and ridiculously contradicting the theory. And I have, this just boggles my mind how anyone could present cytochrome c as evidence for evolution in light of this evidence (Gateley 2014). Most biochemists or molecular phylogeneticists familiar with molecular clocks would probably respond more or less flippantly that it has been known since the 1960s that cytochrome c has a variable substitution rate. Although amino acid sequence data sets for cytochrome c do have phylogenetic implications over long time periods, its relatively short amino acid sequence cannot be expected to provide precise divergence times and phylogenetic relationships in all cases. This is especially true for relatively recent and rapid processes such as the diversification of primates. Gateley is implicitly assuming that simply counting and comparing amino acid differences for three sequences is sufficient to determine the correct phylogeny for their respective species. By doing so he ignores fifty years of progress in molecular clock techniques in general and the study of mutation rates for cytochrome c in particular.2 While this response is accurate, it does not address the specific data set that Gateley cites. Why, in particular, are there fewer amino acid differences when human cytochrome c is compared to that of alligators than there 2 For some of the many available summaries of these methods, see Welch and Bromham (2005), Rutschmann (2006), Lanfear et al. (2010), Bromham (2016). Fig. 1 A simplified phylogeny of primate evolution Page 3 of 17Hofmann Evo Edu Outreach (2017) 10:1 are when humans are compared to the much more closely related primate O. garnettii? It turns out that the example Gateley poses as if it were a new discovery of anomaly actually falls within the intersecting purviews of research areas that now are in their fifth decade. The following historical summary highlights some relevant stages in these investigations including recent analysis of cytochrome c pseudogenes. Although the stochastic nature of mutations always has to be acknowledged, a great deal of molecular evolution can be clarified, especially for a protein as thoroughly studied as cytochrome c. Early applications and analyses of the cytochrome c molecular clock The general idea of a molecular clock was developed by Linus Pauling and Emile Zuckerkandl shortly after the prerequisite developments in protein chemistry during the late 1950s.3 At that point it was known that each protein is constructed from a sequence of amino acids that fold into a distinctive shape required by the protein's function. Each of the 20 possible amino acid molecules consists of a carboxyl group (–COOH) opposite an amino group (–NH2) at the other end of the molecule. In between these extremities is a so-called alpha carbon atom from which an additional side chain is attached that gives each amino acid its distinctive structure. These side chains vary considerably in size and complexity and thus can be expected to be a factor in explaining why only certain amino acids are found at crucial locations in the operative protein. When linked together in the polypeptide chain that constitutes a protein, the carboxyl group of one amino acid binds to the amino group of another with the release of a molecule of water. After this binding process, the remaining "residue" of each amino acid takes a distinctive location in the resulting polypeptide chain. Based upon this understanding of protein structure, the initial idea of a protein molecular clock was that the number of amino acid differences found when sequencing the same protein for two different species could be used to measure the time that has elapsed since their divergence from a common ancestor. A large number of amino acid differences between two sequences would be expected to be due to a larger period of elapsed time than the time corresponding to a relatively small number of differences. Translation of a specific number of differences into an absolute measurement of time rather than a relative one requires a calibration of the clock. That is, one or more well dated events in the fossil or geological 3 For historical commentary, see Dietrich (1998), Morgan (1998), Hagen (1999), Dietrich and Skipper (2007), Sommer (2008), Suarez-Diaz and Anaya-Munoz (2008), Hagen (2009, 2011), Suarez-Diaz (2014), O'Malley (2016). record are used to determine the number of amino acid changes per unit of time, the rate at which a molecular clock is "ticking". Amino acid sequence comparisons for a specific protein can only be used as a molecular clock due to mutations in the gene coding for that protein. These mutations take place in the three-lettered DNA codons that code for the amino acids that make up the protein. The phrase "mutation rate" typically and most accurately refers to the rate at which these mutations occur. Due to redundancies in the genetic code, many of these mutations do not result in a change in amino acid. For example, codons GGT and GGC both code for the same amino acid, glycine. A "synonymous" mutation of this type from GGT to GGC would not result in one of the amino acid changes that are counted in the application of a protein molecular clock. Other mutations of course do result in a change in amino acid. For example, codon AGC codes for amino acid serine while AGA codes for arginine. The result of a "non-synonymous" mutation from AGC to AGA would be a change in amino acid that potentially would be counted in a protein molecular clock analysis. For this to be the case the relevant non-synonymous mutation must first become fixed throughout a population. Once this happens a new amino acid has been substituted in a specific location within the amino acid sequence that constitutes the protein. The rate at which these amino acid substitutions take place for a particular protein is typically referred to as the "substitution rate" or "replacement rate" for that protein. By the late 1970s researchers were also comparing DNA sequences and they often cited either mutation rates or replacement rates for the nucleotides that make up the genes that code for proteins. These rates for DNA nucleotide changes in specific genes are of course the basis for resulting amino acid substitution rates in the corresponding proteins. Pauling and Zuckerkandl began their investigations of molecular evolution with the reasonable expectation that species with a relatively recent common ancestor should have relatively few differences when their amino acid sequences for a particular protein are compared. For example, the 104 amino acid sequence for mammalian cytochrome c is identical in humans, chimpanzees, gorillas, orangutans and gibbons. The common ancestor for these species existed so recently in the past that no cytochrome c substitutions have become fixed in any of its descendants. We are still waiting for the first "tick" of the cytochrome c clock since divergence from that common ancestor. On the other hand, species that are relatively distantly related would generally be expected to have more differences between their amino acid sequences for a particular protein. Exceptions to this general expectation can plausibly be attributed to variations Page 4 of 17Hofmann Evo Edu Outreach (2017) 10:1 in amino acid substitution rates either across species at a particular time or during an elapsed time span for particular lineages. For example, suppose a molecular clock for a particular protein has been calibrated using well established events in the fossil record. If the clock is used to study a poorly understood clade, the results will not be accurate if the protein temporarily experienced an accelerated substitution rate within that clade. Uncorrected application of this clock to two species in the clade would make them appear to be more distantly related than they actually are. That is, naïve reliance upon a temporarily accelerated molecular clock could place the common ancestor of two species farther in the past than it actually is. Developing an explanation of an anomaly such as Gateley's by attributing it to a variable substitution rate becomes particularly apt when the relevant phylogenetic relationships can be determined with high precision independently of the molecular clock in question. In contrast to the situation during the 1960s when cytochrome c analyses were first carried out, the large number of protein and whole genome analyses and accurate fossil calibrations now at hand mean that the relevant phylogenies are sufficiently trustworthy to pinpoint the timing and nature of variable substitution rates. One reason cytochrome c is such an extensively researched protein is due to its important function in the mitochondrial electron transport system. Eukaryotic respiratory transport systems are made up of approximately 90 proteins that collectively accomplish oxidative phosphorylation, the primary source of aerobic energy stored in ATP. Electrons are transported through four protein complexes, three of which use energy to pump protons into the intermembrane space of the mitochondrion. The potential energy in the resulting proton gradient across the membrane then drives protons back through the fifth complex of the system, ATP synthase, yielding ATP. Cytochrome c contributes to this respiratory chain by acting as an electron shuttle between Complex III (ubiquinol cytochrome c reductase) and Complex IV (cytochrome c oxidase). The initial form of this chemiosmotic theory of oxidative phosphorylation was developed by Peter Mitchell during the 1960s, the same decade in which the genetic code linking DNA codons to amino acids was deciphered.4 Early investigations of cytochrome c substitution rates were linked both to its molecular structure and to the role of specific amino acids in cytochrome c function. For example, throughout 1963, as amino acid sequences for cytochrome c became available for an increasing number 4 For detailed historical analyses of Mitchell's research, see Prebble and Weber (2003), Weber and Prebble (2006). of species, comparisons showed that some residues vary far less frequently than others.5 Meanwhile, work had also begun on the tentative construction of phylogenetic trees using cytochrome c amino acid sequences. Richard Eck and Margaret Dayhoff published some of the earliest of these trees in their 1966 edition of the Atlas of Protein Sequence and Structure (Eck and Dayhoff 1966). Their cytochrome c based phylogeny relied upon an estimate of what the tree would be for a minimum number of amino acid substitutions. At this point they did not attempt to incorporate complications resulting from substitution rate variability. That step was taken by Margoliash and Walter Fitch who used cytochrome c data for a 1967 publication that Francisco Ayala would later refer to as "the founding document of molecular phylogenetics".6 Variations in the cytochrome c substitution rate were now estimated quantitatively. Fitch and Margoliash constructed phylogenetic trees based upon "mutation distances" between the cytochrome c genes for any two species. These were calculated by determining the minimum number of nucleotide replacements that would result in the transformation of the cytochrome c amino acid sequence for one species into that of another. They then argued that the most likely phylogenetic tree would be the one that minimized the composite mutation distances consistent with the amino acid sequence data.7 They realized that their results called attention to some lineages as particularly prone to substitution rate variation. Thus the method indicates those lines in which the gene has undergone the more rapid changes. For example, from the point at which the primates separate from the other mammals, there are, on the average, 7.5 mutations in the descent of the former and 5.8 in that of the latter, indicating that the change in the cytochrome c gene has been much more rapid in the descent of the primates than in that of the other mammals. (Fitch and Margoliash 1967, p. 283). Fitch and Margolis made similar comments in later publications in 1968.8 Further exploration of the topic 5 See Margoliash (1963), Smith and Margoliash (1964, p. 1244). For all seven sequences available in 1964, cysteine was found to be an invariant amino acid residue at positions 14 and 17 where bonds form to the central heme complex of the molecule. On the other hand, at position 89 six different residues were found. By 1999 113 eukaryotic cytochrome c protein sequences had been catalogued; see Banci et al. (1999). In 2013 a total of 285 sequences were catalogued and only cysteine at position 17 was known to be entirely invariant in all these species; see Zaidi et al. (2014, p. 232). 6 Fitch and Margoliash (1967). For Ayala's comment, see Ayala (2011, p. 5). 7 For historical commentary, see Felsenstein (2001) and (2004, pp. 130–133). 8 For examples, see Margoliash and Fitch (1968, p. 370), Margoliash et al. (1968, p. 271). Page 5 of 17Hofmann Evo Edu Outreach (2017) 10:1 appealed to Richard Dickerson who was intrigued by the possibility that amino acid sequence comparisons might inform his primary interest in cytochrome c molecular structure and function. Structural studies of cytochrome c Following his initial x-ray crystallographic analysis of cytochrome c structure during the 1960s, Dickerson collaborated with illustrator Irving Geis to produce several valuable popularizations of new developments in protein biochemistry. A 1972 essay for Scientific American included illustrations in which Geis provided schematic representations of how the amino acid sequence of cytochrome c is coiled around the heme complex with its central iron atom (Dickerson 1972). Each amino acid residue was shown schematically as a single ball representing the alpha carbon atom from which an additional side chain would be attached in each actual amino acid structure. A point of emphasis for Dickerson was that although genetic mutation is a stochastic process, the resulting changes in amino acids are not all equally acceptable if the protein is to function properly. For example, the glycine amino acid residues at positions 6, 29, 34, 41, and 84 are located in tight corners of the cytochrome c structure where there is no room for a long side chain. Since glycine is unique in having only a single hydrogen atom as its side chain, it makes good structural sense that it is usually found in these locations. In Geis's 1972 illustration shown in Fig. 2, all 104 mammalian cytochrome c amino acids are enumerated and the 35 invariant residues known at that time are labelled using their abbreviations. The only side chains shown are for those residues that attach to the heme, residues 14, 17, 18 and 80. The invariance of these residues for all the sequences available during the 1970s thus could plausibly be attributed to their crucial role in binding to the heme via their distinctive side chains. On the other hand, other residues such as 89 were noted to be highly variable. Residues 44 and 89 in fact will turn out to be relevant to the example raised by creationist Eugene Gateley. Although Dickerson himself emphasized very longterm averages in substitution rates, his structural studies of cytochrome c during the 1970s coincided with much more extensive research that indicated rate variation. As Walter Fitch and Margoliash had done during the 1960s, but now with access to much more advanced statistical methods, Fitch and Charles Langley, as well as Morris Goodman and his colleagues at Wayne State, constructed increasingly detailed phylogenetic trees and then used the nodes of these trees to compare substitution rates along particular evolutionary branches. In general, the central method here was to construct the phylogenetic tree that minimized the number of mutations compatible with the sequence data. Additional statistical factors were then introduced to compensate for gene duplications and multiple mutations at the same site, including backmutations. Once such a tree was constructed, the number of substitutions along various branches leading to extant species could be compared. Langley and Fitch also published a series of studies in which they used expanded maximum likelihood procedures to argue for variation in the cytochrome c substitution rate.9 A typical conclusion drawn from their research was that "It is quite clear that the hypothesis of overall constant evolutionary rate for each protein or even overall constancy for this group of proteins as a unit must be rejected" (Langley and Fitch 1974, p. 169). Similarly, in 1976, when Goodman published a study of vertebrates with G. William Moore, Richard Holmquist, and several other coauthors, he and his colleagues could bluntly state that "Non-uniform rather than uniform rates characterize cytochrome c evolution".10 Margoliash was thoroughly convinced by these arguments, as he made clear in 1976. Suffice it to point out that the much more precise recent study of statistical phylogenetic trees based on amino acid sequences show that the rate of evolutionary change in cytochrome c is not constant either in a single line of descent during different evolutionary intervals, or in separate lines of descent in the same evolutionary interval... (Margoliash et al. 1976, pp. 146–147). The conclusion that the substitution rate for cytochrome c varies significantly over time thus was firmly in place by 1976. Furthermore, the molecular structure of the cytochrome c molecule was well enough understood to pick out some residues as particularly prone to substitution. It also had become clear that some of the most interesting periods of rate variation took place during the diversification of primates. Cytochrome c among the primates One of the primary reasons for Morris Goodman's research with primate cytochrome c was his interest in the relationship between molecular evolution and 9 For examples, see Langley and Fitch (1974), Fitch (1976), Fitch and Langley (1976a, b). Francisco Ayala summarized Fitch's work on this topic in his obituary tribute. "Walter demonstrated that the variance in the rate of molecular evolution was statistically larger than expected under the theory of the molecular clock and, thus, the underlying assumption of "neutral" replacements in DNA or proteins was not correct. However, he demonstrated that by combining the data from several genes or proteins, the average number of differences observed converged to the expected time since the divergence of the species investigated." Ayala (2011, p. 9). 10 Moore et al. (1976, p. 33). Page 6 of 17Hofmann Evo Edu Outreach (2017) 10:1 Fig. 2 A simplified folding diagram for cytochrome c. Illustration by Irving Geis in Dickerson (1972, p. 59). Highly variable residue 89 is in a peripheral location at the top of the diagram, far from the central heme. Relatively invariant residues, such as glycine 84, are labelled with their abbreviated names preceding their residue number Page 7 of 17Hofmann Evo Edu Outreach (2017) 10:1 morphological change. During the 1970s and early 1980s Goodman and his colleagues emphasized the variability of the cytochrome c substitution rate and tried to determine whether this variability could be correlated with specific stages in primate evolution. In some particularly influential 1981–1982 publications they applied maximum parsimony methods to cytochrome c data for 87 species to construct a phylogeny from which they could compare substitution rates for a specific time period along various branches.11 They expressed their results in units of "nucleotide replacements per 100 codons per 100 million years".12 That is, they compiled and compared data for nucleotide substitution rates rather than the resulting amino acid substitution rates. The rate of nucleotide replacements was found to peak during the period between 90 and 40 million years ago, reaching an average rate of 17.3 nucleotide replacements per 100 codons per 100 million years during that period.13 Since the number of amino acids in cytochrome c is 104 and thus requires 104 codons, the 17.3 replacement rate per 100 codons also corresponds to a rate of change of approximately 17.3%. This period of maximum nucleotide replacement rate and associated amino acid substitution rate stretched from the approximate date for the origins of placental mammals through the point of divergence of new world monkeys.14 Between 40 and 25 million years ago the nucleotide substitution rate dropped slightly to 12.6% and then plunged sharply to 1.9% after the 25 million year point when apes had diverged from Old World monkeys. The substitution rate thus was highest during the eras crucial for early primate radiation and then fell abruptly after 25 million years ago, a phenomenon Goodman referred to as the "hominoid slowdown".15 Due to the inevitable incompleteness of the fossil record, particularly for primates, molecular analyses can generally be expected to give earlier divergence times than is directly supported by fossil evidence.16 The actual time of divergence of a new species from an ancestral population necessarily precedes the date assigned to the earliest relevant fossil evidence. Accurate molecular clock analysis thus can be expected to give an earlier divergence time than the date of the earliest relevant fossil. Even at present there still is some uncertainty in the 11 Moore et al. (1976); Baba et al. (1981), (1982); Goodman (1981). 12 Baba et al. (1981, p. 204). 13 Baba et al. (1981, p. 204); and (Baba et al. 1982, pp. 20–21). 14 In the early 1980s the divergence of platyrrhines from other primates was thought to be approximately 40 million years ago; some recent estimates place it at 47 million years ago. 15 See Goodman (1985) for a summary. Goodman had argued for this idea throughout the 1960s and 1970s. 16 For a summary and many examples, see Hedges and Kumar (2003). precise dating of some of the nodes in the primate phylogeny summarized in Fig. 1.17 Nevertheless, there is full agreement that the time interval Goodman highlighted between 90 and 25 million years ago includes the origin of primates, the diversification of strepsirrhines into lemurs, lorises and bush babies, and the haplorhine diversification into tarsiers, monkeys and apes. More particularly, it includes the origin of tarsiers at approximately 60–70 million years ago and the common ancestor of lorises and bush babies at approximately 40 million years ago. Because tarsiers diverged from other haplorhines relatively early, it is customary to refer to the haplorhines other than tarsiers as anthropoids. Central to Goodman's research agenda was his argument for a link between accelerated mutation rates and functional innovations in the anthropoid molecular structure of cytochrome c. Dickerson's work was helpful in this respect since the functions of most of the 104 vertebrate cytochrome c amino acids now were at least approximately understood. Goodman analyzed the distribution of mutations over the span of amino acids in the cytochrome c sequence by distinguishing several different functional groups. His fourth group, the oxidase-reductase area of the protein, was expected to be of primary importance for phosphorylation. During the preceding decade Dickerson and Margoliash and many others had focused on 16 amino acids as probably crucial for this function; these were in positions 7, 8, 11, 12, 13, 15, 16, 19, 21, 25, 27, 72, 81, 83, 86, and 87. Five of these residues are substitution sites that distinguish human cytochrome c from that of O. garnettii: 11, 12, 15, 21, and 83. By 1981 Goodman and his colleagues thus had not only confirmed that cytochrome c has a variable substitution rate, but also determined that the time period for the fastest pace of change was during the early stages of primate evolution and was concentrated in residues crucial to the interaction between cytochrome c and cytochrome c oxidase during phosphorylation. In an extensive 1990 study of cytochrome c, Geoffrey R. Moore and Graham Pettigrew summarized Goodman's results and included an illustration shown in Fig. 3 based upon one used by Goodman in 1981.18 The same two relatively recent periods of high genetic replacement rates again stand out, 25–40 million years ago and especially 40–90 million years ago, time periods that span the 17 Pozzi et al. (2014) is a recent analysis that includes comparisons to several other studies carried out between 2008 and 2013. 18 Figure 6.10 from Moore and Pettigrew (1990, p. 278), based upon Figure 4 from Baba et al. (1981, p. 205). By 1986 cytochrome c had been sequenced for 92 different eukaryotic species; see Hampsey et al. (1986). Baba et al. (1982) mentions 94 species. Page 8 of 17Hofmann Evo Edu Outreach (2017) 10:1 major primate divergences, including the separation of strepsirrhines from haplorhines. For Goodman, the recognition of mutation rate variation in cytochrome c was a preliminary motivation for further study of the causes of variation. His research thus stands in sharp contrast to more recent creationist reactions. Creationist critics typically focus on what they interpret to be an unexpected set of data and then attempt to highlight it as a conclusive falsification of common descent. Goodman set out to see what could be learned from rate variation to understand primate evolution. In the creationist example under consideration, when a strepsirrhine primate such as the bush baby O. garnettii is found to have more cytochrome c amino Fig. 3 The rate of change of cytochrome c. From Moore and Pettigrew (1990, p. 278) Page 9 of 17Hofmann Evo Edu Outreach (2017) 10:1 acid differences when compared to humans than an alligator does, this might suggest several questions for further research. Does other evidence exist that implies an increased or decreased mutation rate or substitution rate in one of the relevant lineages? Is this rate variation linked with similar variations in other proteins that share a function with cytochrome c in the respiratory sequence of electron transport? Have any of the relevant cytochrome c amino acids been found to be more subject to substitution than others, and if so, are there functional or adaptational reasons? Have episodes of relatively rapid protein evolution been correlated with morphological changes? All of these questions generated productive research in the case of cytochrome c. Goodman's group found that variable nucleotide substitution rates in the cytochrome c gene are correlated with similarly variable rates for other components of the electron transport chain, especially subunits of cytochrome c oxidase that come into direct interaction with cytochrome c during oxidative phosphorylation.19 In a 2004 review article they emphasized how the increased substitution rate in COX4-1, a sub-unit in cytochrome c oxidase, was correlated with that of cytochrome c during the same two time periods, 25–40 19 See Andrews and Easteal (2000), Grossman et al. (2001), (2004), Wildman et al. (2002), Doan et al. (2004), Pierron et al. (2011). million years ago and 40–90 million years ago (Grossman et al. 2004). The substitution rates thus increase in multiple proteins in the electron transport chain following the divergence of anthropoid primates from tarsiers. In this analysis there was no particular reason to call attention to a specific strepsirrhine primate such as O. garnettii. However, if we do look at the relevant data for that species the results are quite in keeping with Goodman's more general conclusions. Bush babies, Homo sapiens, and alligators Table 1 shows a correlated comparison of the 14 human or ape cytochrome c amino acid residues that differ from those of the bushbaby O. garnettii. Recall that the entire 104 amino acid sequence for cytochrome c is identical in Homo sapiens and all the apes. In Table 1 the residues at the 14 locations that distinguish humans and apes from O. garnettii are also compared to those of two lemur species, a tarsier, and several non-primate vertebrates: gray whale, rat, alligator, and bullfrog. The rat cytochrome c comes in two forms, one found in somatic cells, rat(s), and the other found exclusively in sperm cells, rat(t), and only expressed during spermatogenesis. The top row of the table shows the total number of residue differences for each species when compared to the sequence shared by humans and apes. Throughout the table, differences in specific O. garnettii residues when compared to the human and ape sequence are highlighted in yellow. Table 1 Cytochrome c amino acid differences between human or ape and the bushbaby Otolemur garnettii # DIFS→ 0 14 9 10 10 10 9 17 13 16 AA# Human or ape Bushbaby Mouse lemur Lemur caa Tarsier Grey whale Rat(s) Rat(t) Alligator Bull frog 1 G S G G G G G G G G 3 V I V A V V V A V V 11 I V V V V V V I V V 12 M Q Q Q Q Q Q Q Q Q 15 S A A A A A A A A A 21 E D E E E E E E E E 44 P A A A A V A P P A 46 Y F F F F F F F F F 50 A D D D D D D D E D 58 I T T T T T T I T T 83 V A A A A A A A A A 85 I V I I I I I I I I 89 E G G G G G G S P G 96 A D A A A A A Q A A Page 10 of 17Hofmann Evo Edu Outreach (2017) 10:1 Locations where species have an amino acid differing from both humans and from O. garnetti are shown in green. What is the most straightforward explanation for the 14 differences between human and O. garnettii? First of all, it is striking that so many of these amino acids are identical in most of the species listed except for humans and apes. Seven of the 14 differences between humans and O. garnettii, residues 11, 12, 15, 46, 50, 58, and 83, apparently involve mutations in the relatively recent anthropoid lineage that leads to monkeys, apes and humans after their divergence from tarsiers and long after their earlier divergence from strepsirrhines such as O. garnettii and the lemurs.20 Five of the remaining amino acid differences appear to have happened along the divergent branch leading to the strepsirrhine bush baby O. garnettii (residues 1, 3, 21, 85, and 96). Residues 44 and 89 have apparently undergone multiple substitutions resulting in differences not only between humans and O. garnettii but with the other listed species as well. This is not surprising since residues 44 and 89 were discovered by Dickerson to be located far from the heme 20 One exception for residue position 58 is an old world monkey, the hamadryas baboon, which also has T at position 58. This is one of three differences it has when compared to humans. The others are at positions 4 and 33. core of the cytochrome c molecule and thus allow a high degree of variability. These data are in keeping with the temporarily accelerated substitution rate thoroughly documented since the early 1980s. Goodman and his colleagues had in fact included a cytochrome c analysis in one of their 2001 studies of the evolution of the electron transfer complex (Grossman et al. 2001). Figure 4 highlights some details from their illustration of the extensive amino acid replacements occurring along the Catarrhine stem after the divergence of both Rattus norvegicus (brown rat) and Oryctolagus cuniculus (European rabbit) and prior to the divergence of Old World monkeys such as Ateles sp. (spider monkey) 25 million years ago. Along with highly variable residue 89, the figure labels precisely those seven amino acid changes that stand out from a straightforward perusal of the data (amino acid #s 11, 12, 15, 46, 50, 58, and 83). Additional changes in the highly variable residues 44 and 89 are also indicated. Although data for O. garnettii are not shown in this diagram, as a strepsirrhine primate it diverged from the other primates prior to the highlighted changes in the Catarrhine stem leading to monkeys and apes as well as Homo sapiens. Seven of the 14 amino acid differences between human and O. garnettii thus are accounted for by recent changes in the anthropoid lineage, five can be attributed to the Fig. 4 Amino acid replacements during primate evolution. Labelled detail from Grossman et al. (2001, p. 31) Page 11 of 17Hofmann Evo Edu Outreach (2017) 10:1 strepsirrhine lineage leading to O. garnettii, and the remaining two, 44 and 89, have been subject to multiple substitutions. Substitution rates among some strepsirrhine lineages have more recently been found to be generally very high, even compared to other primates. These conclusions are of course based on much more thorough sequencing techniques than earlier ones that relied simply upon individual proteins (Eizirik et al. 2004, pp. 54–55). The upshot of these and many other studies is that instead of using cytochrome c simplistically as a molecular clock assumed to have a fixed substitution rate, other more reliable timing mechanisms have been used to link the variable substitution rate of cytochrome c to its structure and function and to particular episodes in primate evolution. Similar conclusions can be drawn from the data for a comparison of alligators and humans shown in Table 2. Of the thirteen amino acid differences between alligators and Homo sapiens, six can be assigned solely to the anthropoid lineage (11, 12, 15, 46, 58, and 83). Two others apparently involve substitutions in both anthropoids and crocodylians (50 and 89), and five are found only in the crocodylian lineage (36, 62, 100, 103, and 104). One question that these data prompt is why only a total of seven amino acid replacements have taken place along the long crocodylian lineage in contrast to the eight assigned to a much shorter time period within the primate lineage. A reasonable place to look for an explanation would be to see what the mutation rate is in the crocodylian lineage. It turns out that in contrast to primates, the crocodylian lineage has an unusually low genetic substitution rate. In their 2014 study using whole genome-alignments, Richard Green and colleagues found that alligators and crocodiles have "exceptionally low rates of evolution relative to mammals" (Green et al. 2014, 1254449-3). As a result, it is not surprising that only seven crocodylian cytochrome c amino acid replacements have contributed to the difference between human and alligator cytochrome c. Eight O. garnettii substitutions took place during a much shorter time period. One more aspect of the alligator, O. garnettii, and human cytochrome c data is worth mentioning. As shown in Table 3, human and O. garnettii cytochrome c sequences both differ from alligator cytochrome c by 13 amino acids. Of the 13 differences between human and alligator sequences, six are at amino acids where all the other primates listed have the same amino acid as alligator (amino acid #s 11, 12, 15, 46, 58, 83). As we have Table 2 Amino acid differences between human or ape and alligator cytochrome c AA# Human or ape Alligator Mouse lemur Lemur caa Tarsier Grey whale Kangaro o Hipp o Horse Bushbaby Bull frog Tuna 11 I V V V V V V V V V V V 12 M Q Q Q Q Q Q Q Q Q Q Q 15 S A A A A A A A A A A A 36 F I F F F F F F F F I F 46 Y F F F F F F F F F F Y 50 A E D D D D D D D D D D 58 I T T T T T I T T T T V 62 D E D D D E D E E D D N 83 V A A A A A A A A A A A 89 E P G G G G G G T G G G 100 K E K K K K K Q K K S S 103 N S N N N N N N N N S S 104 E N E E E E E E E E K Table 3 Amino acid differences for alligator and primates Alligator Microcebus coquereli (mouse lemur) tarsius bancanus (Western tarsier) Lemur catta (ring-tailed lemur) Otolemur garnettii (bush-baby) Human and ape AA DIFS→ 0 8 9 9 13 13 Page 12 of 17Hofmann Evo Edu Outreach (2017) 10:1 seen, all of these six differences have arisen in the anthropoid lineage. Five of the remaining seven differences are shared by humans and the other primates listed (amino acid #s 36, 62, 100, 103, and 104). The data thus are very much as would be expected from accelerated mutation and substitution rates within primate lineages. In sharp contrast to the multi-faceted investigation of molecular evolution by the scientific community, creationist responses to cytochrome c data demonstrate quite a different attitude. A common reaction is to simply use intuitively unexpected cytochrome c sequence data for specific primates as a reason to categorically reject amino acid sequence data as evidence for common descent. Eugene Gateley presents his examples as if they are recent discoveries, even giving the impression that he might be the first to have noticed them. Data sets of this type have in fact been subject to interesting research for decades, research that securely explains them as consequences of variation in the rate of amino acid replacement. The apparent anomaly generated by the 14 differences between human and O. garnettii cytochrome c amino acid sequences thus is resolved as part of a more general analysis of the variable rates of molecular evolution in cytochrome c. Human cytochrome c pseudogenes Interesting additional confirmation of the variable substitution rate in the evolutionary history of cytochrome c comes from its numerous pseudogenes. In general, pseudogenes are versions of a gene that no longer carry out that gene's initial function. In some cases unitary pseudogenes are the direct remains of a gene that has become dysfunctional due to mutations. In other cases a gene has undergone duplication and one copy has mutated and become a pseudogene. In still other cases a processed pseudogene is the result of transcription and retrotransposition, that is, reinsertion of a nucleotide sequence back into the genome after being transcribed, stripped of introns, and then left without a promoter to generate subsequent transcription. Processed pseudogenes thus are relatively easy to identify due to their lack of introns.21 The fact that human cytochrome c has a large number of processed pseudogenes attracted research interest during the 1980s.22 In humans the functioning gene for cytochrome c is located on chromosome 7 and has two 21 See Zhang and Zheng (2014). 22 See Wu et al. (1986), Evans and Scarpulla (1988), Virbasius and Scarpulla (1988), Zhang and Gerstein (2003, 2004), Mills (1991), Zhang et al. (2003). Due to the relatively small data sets available during the 1980s, early studies were carried out in conjunction with comparisons to genes and pseudogenes in rodents. Recent analysis based upon both fossil evidence and molecular clocks place the origin of rodents at approximately 60 million years ago during the period of very high cytochrome c substitution rate. See Wu et al. (2012). introns. By 2003 Zhaolei Zhang and Mark Gerstein had identified 49 cytochrome c pseudogenes distributed over 18 different human chromosomes (Zhang and Gerstein 2003). They called particular attention to nine highly variable residues (11, 12, 15, 44, 46, 50, 58, 83, 89), all of which are among the 14 residues that distinguish human cytochrome c from that of O. garnettii. As we have seen, all of these substitutions, except for the highly variable sites 44 and 89 have been attributed to mutations that took place solely in the anthropoid lineage long after anthropoid divergence from strepsirrhines such as O. garnettii. Zhang and Gerstein followed a prior protocol in distinguishing between two sets of cytochrome c pseudogenes. The four pseudogenes in class 1 (ψ15, ψ21, ψ45 and ψ46) all code for sequences that have a high degree of similarity to the functional human cytochrome c. This implies that the cytochrome c gene experienced a period of significant mutation relatively recently in the anthropoid lineage that gave rise to the four pseudogenes in class 1 only after these mutations. Table 4 shows class 1 pseudogene data for all 14 of the amino acid differences for O. garnettii compared to humans or apes along with the somatically expressed rat cytochrome c, rat(s). Differences from human cytochrome c are shown in yellow. These data contributed to the 2001 conclusion by Goodman's research group that the class 1 pseudogenes originated during a period of accelerated cytochrome c substitution rate between 40 and 25 million years ago.23 As we have seen, they assigned substitutions in amino acid #s 11, 12, 15, 46, 50, 58, and 83 solely to the anthropoid lineage. The pseudogenes in class 1 all came about after these substitutions and preserved them in all but a very few residues. Secondly, Zhang and Gerstein placed the remaining 45 of the total 49 pseudogenes in a set labelled class 2. They noted that the amino acid sequences coded for by these pseudogenes bear very few identities to human cytochrome c at highly variable locations such as 11, 12, 15, 44, 46, 50, 58, 83, and 89. The most straightforward interpretation of the data is that the 45 members of class 2 are relatively old pseudogenes compared to class 1. Zhang and Gerstein used the known age of retrotransposon insertions to estimate the age of the oldest class 2 pseudogene to be at least 80 million years. As a result of their age, and in contrast to the pseudogenes in class 1, pseudogenes in class 2 should have a relatively high degree of correlated similarities or dissimilarities to both the O. garnettii gene and the human gene at amino acid positions that distinguish O. garnettii from humans and apes. 23 Grossman et al. (2001, p. 31). Page 13 of 17Hofmann Evo Edu Outreach (2017) 10:1 For example, as illustrated in Fig. 5, at positions 1, 3, 21, 85, and 96 we would expect to see differences between class 2 pseudogenes and O. garnettii but similarities to humans. This is because mutations took place for these residues only in the strepsirrhine lineage leading to O. garnettii but not in the anthropoid lineage. On the other hand, at positions 11, 12, 15, 46, 50, 58, and 83 we should see just the opposite, namely, a similarity to O. garnettii and dissimilarities to humans. Mutations at these locations took place only relatively late in the anthropoid lineage leading to humans and apes but not in the strepsirrhine lineage leading to O. garnettii. These expectations are summarized in Fig. 6. The highly variable sites 44 and 89 can be expected to differ from both human and O. garnettii due to mutations in both the strepsirrhine and anthropoid lineages. Of course since pseudogenes generally sustain arbitrary mutations to a higher degree than do functioning genes, Table 4 Class 1 human cytochrome c pseudogenesAmino acid # 1 3 11 12 15 21 44 46 50 58 83 85 89 96 Human/ape G V I M S E P Y A I V I E A ψ15 G V I M S E P H A I V I E A ψ21 G V I M S E P Y A I V I E A ψ45 G V I M S E A Y V T V I E A ψ46 P Y A I V I E A Rat(s) G V V Q A E A F D T A I G A Otolemur garnettii S I V Q A D A F D T A V G D Fig. 5 A simplified primate phylogeny with some events in cytochrome c evolution Page 14 of 17Hofmann Evo Edu Outreach (2017) 10:1 we should not expect these correlations to be without exceptions. Nevertheless, the data do generate quite striking patterns. Figure 7 shows the data with colors coordinated for class 2 pseudogene residues that match either O. garnettii or human cytochrome c. Residues that match neither species are shown in red. These data thus once again confirm the conclusion that mutations at positions 11, 12, 15, 46, 50, 58, and 83 all took place in the anthropoid lineage leading to humans and that mutations in residues 1, 3, 21, 85, and 96 came about within the strepsirrhine lineage leading to O. garnettii. The highly variable sites 44 and 89 have undergone multiple substitutions with the result that neither human nor O. garnettii has very many similarities to any of the ancient class 2 pseudogenes at these locations. As Zhang and Gerstein concluded, "our findings strongly support the hypothesis that this gene has evolved at a very rapid rate in the recent human lineage" (Zhang and Gerstein 2003, p. 71). More specifically, the pseudogene data support detailed phylogenetic assignments for all the amino acid residues that distinguish human cytochrome c from that of O. garnettii. The cytochrome c mutation and substitution rates certainly are not claimed to be arbitrarily variable. On the contrary, specific amino acid changes can plausibly be assigned to either the anthropoid or the O. garnettii lineage in such a way as to be compatible with all the protein and pseudogene data. Conclusion The history of cytochrome c research shows that the present understanding of its mutation rate heterogeneity has progressed in conjunction with study of its molecular structure and its associated pseudogenes. The initial correlation of cytochrome c substitution rate changes with specific time intervals was gradually supplemented by sequence data from entire genomes and a multitude of other molecular clocks. Rather than simplistically using the irregular cytochrome c clock to determine precise primate phylogenies, much broader data sets have clarified interesting episodes in the variation of the cytochrome c substitution rate. Although cytochrome c no longer plays a cutting edge role in the determination of divergence dates among primates, it has played an important role in the historical development of the field. The contrast between scientific inquiry into this topic and creationist commentary is severe. When calling attention to an apparent molecular clock anomaly, it is not illuminating to simply count and compare amino acid residue differences without asking further questions. This is particularly true when many of the relevant questions have received increasingly detailed answers over several decades. Zuckerkandl and Pauling made this point over 50 years ago. Counting numbers of differences in amino acid sequence is only one stage of the analysis, and recordFig. 6 Class 1 and Class 2 pseudogene substitutions Page 15 of 17Hofmann Evo Edu Outreach (2017) 10:1 Amino acid 11 12 15 46 50 58 83 Amino acid 1 3 21 85 96 Amino acid 44 89 Human/ape I M S Y A I V Human/ape G V E I A Human/ape P E Bushbaby V Q A F D T A Bushbaby S I D V D Bushbaby A G ψ1 V Q A F D I A ψ1 G V E I A ψ1 V A ψ2 V Q A F D T A ψ2 G I E I D ψ2 T _ ψ3 F R S F D I T ψ3 G V _ I A ψ3 /P T ψ4 A /Y D T V ψ4 V T A ψ4 T A ψ5 V X V F D I T ψ5 G V E V P ψ5 A A ψ6 V Q A F D T A ψ6 G V _ I A ψ6 I A ψ7 V Q A F D T A ψ7 S V E I A ψ7 V A ψ8 F T T S D T T ψ8 S V E I T ψ8 I V ψ9 I Q A F E V S ψ9 G A E I Q ψ9 P S ψ10 V Q A F D T A ψ10 S V E I A ψ10 V A ψ11 V Q A F D T A ψ11 G A E I A ψ11 T A ψ12 V Q A F D T A ψ12 G A E I A ψ12 I A ψ13 V Q A F D T A ψ13 G A E I A ψ13 I A ψ14 F L V F D I I ψ14 A E I Q ψ14 P _ ψ16 V Q S P _ X ψ16 V E ψ16 R ψ17 I Q V F D T F ψ17 G V E /V T ψ17 I A ψ18 V /M A F V S ψ18 V K ψ18 P ψ19 I Q A L D T A ψ19 D V E I A ψ19 V _ ψ20 V Q A F D T G ψ20 G V E I A ψ20 I G ψ22 V Q V F D T A ψ22 G V E I A ψ22 P V ψ23 V Q A F E S A ψ23 G V E M A ψ23 V A ψ24 V Q A F D T A ψ24 G V E I A ψ24 V A ψ25 V Q A F D T A ψ25 G V E I A ψ25 I A ψ26 V Q A F D T A ψ26 G V E I A ψ26 V A ψ27 T E A F D T A ψ27 G I E I A ψ27 I _ ψ28 V E V F D T T ψ28 G A E I A ψ28 A /A ψ29 V Q A Y D T A ψ29 G A Q I A ψ29 V A ψ30 V Q A _ D T A ψ30 G V E I A ψ30 _ T ψ31 V Q A F D T A ψ31 G V E I A ψ31 V A ψ32 V Q A L E T A ψ32 G V E I A ψ32 I A ψ33 V Q A L D T S ψ33 R V E I T ψ33 I T ψ34 V Q A F D T A ψ34 G V /E I A ψ34 I A ψ35 K K T S E T A ψ35 E I A ψ35 A S ψ36 V Q A F D T A ψ36 S A E I A ψ36 A T ψ37 A Q A F D A A ψ37 G V E I A ψ37 V V ψ38 V X A F D T Y ψ38 G V G K ψ38 F ψ39 V Q A F D T A ψ39 G V E I A ψ39 I A ψ40 V P A F D T T ψ40 D V E ψ40 A ψ41 V Q A L E T ψ41 G A E ψ41 I ψ42 V Q S F D A A ψ42 E V ψ42 P ψ43 A Q A F D N T ψ43 V V E I A ψ43 V A ψ44 V Q A F D T S ψ44 G V E I A ψ44 V T ψ47 S ψ47 E ψ47 ψ48 A /Y D T A ψ48 V T A ψ48 T A ψ49 L Q A L E T A ψ49 A S E I A ψ49 I A Fig. 7 Class 2 pseudogenes Page 16 of 17Hofmann Evo Edu Outreach (2017) 10:1 ing the nature of the differences is a necessary further step in the establishment of a molecular phylogeny. 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