In a legal ‘first’, the UK removed a prohibition against modifying embryos in human reproduction, to enable mitochondrial replacement techniques (MRTs), a move the Government distanced from ‘germline genetic modification’, which it aligned with modifying the nuclear genome. This paper (1) analyzes the uses and meanings of this term in UK/US legal and policy debates; and (2) evaluates related ethical concerns about identity. It shows that, with respect to identity, MRTs and nuclear genome editing techniques such as CRISPR/Cas-9 (now a policy topic), are not as different as has been supposed. While it does not follow that the two should be treated exactly alike, one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as ‘germline genetic modification’, is thereby in doubt. Identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in law and policy. Despite this strikingly different conclusion, the report adopted a cautiously permissive approach to MRTs .When, in 2015, the UK Parliament passed regulations permitting the use of mitochondrial replacement techniques (MRTs) to prevent the transmission of serious mitochondrial disease,first, to analyze the uses and meanings of ‘germline genetic modification’ in the UK and US legal and policy debates; second, to evaluate ethical concerns about ‘identity’ raised by MRTs. The analysis has implications for policy debates regarding the reproductive use of nuclear genome editing techniques, such as CRISPR/Cas-9,Given the significance of the legal developments in the UK and the prospect of the US and other countries moving permissively to regulate MRTs,Section 2 situates the legalisation of MRTs within the UK legal framework. It also considers the position on germline genetic modification in several key international statements and conventions and how the revised UK legal position stands in relation to these.Section 3 explores possible justifications for the view that MRTs do not constitute germline genetic modification with reference to UK and US policy, regulatory, legal, scientific and academic materials. The UK classification of MRTs as something other than germline genetic modification involved construing the concept more narrowly than might otherwise be the case; two key distinctions were in play in this ‘narrowing’. First, and principally, MRTs directly concern only the mitochondrial, not the nuclear genome. Second, MRTs involve replacement , not modification. The supposed normative significance of these distinctions is that, compared to nuclear genome editing, MRTs: (a) are unlikely to alter in significant ways the identity of the person created; (b) do not introduce ‘artificial’ elements into the gene pool; and (c) are less likely to be used for human enhancement. These points played a key role in justifying the UK legalisation of MRTs. Although the US IOM report reached different conclusions about germline genetic modification, the same distinctions underpin its cautiously permissive approach.Section 4 critically assesses the argument that modifying the nuclear genome is more ethically troubling than MRTs because that could, to a greater extent, affect the ‘identities’ of future people. The section’s main conclusion is that, while there is no categorical difference (regarding identity) between MRTs and modifying the nuclear genome, a more precautionary approach to the latter may be justified because of its greater potential for non-therapeutic use.Finally, Overall, the analysis shows that the distinction between mitochondrial and nuclear and that between replacement and modification, coupled with the appeal to identity, cannot do the work typically placed on them. Rather, the permissibility of an intervention in either the mitochondrial or nuclear genome depends on the context, extent and nature of the intervention in question.We first consider recent changes to the UK legal position, the nature of the proposed MRTs, and where the UK stands in relation to the international legal position.all human embryos, no matter how created.The Human Fertilisation and Embryology (HFE) Act 1990 established a world-leading legal and regulatory framework for assisted reproduction and embryo research. Over time, however, scientific developments prompted the courts and Parliament to respond in various ways. For instance, the development of somatic cell nuclear transfer (SCNT) led to a legal scare regarding the scope of the HFE Act to govern the use of embryos created by means other than fertilisation, entailing both a legal challenge to the ActMaternal spindle transfer (MST). The ‘maternal spindle’ is the group of maternal chromosomes within the egg, which are shaped in a spindle. MST involves removing the spindle from the mother’s egg before it is fertilised by the father’s sperm. The spindle is then placed into a donor egg with healthy mitochondria .Pro-nuclear transfer (PNT). The pro-nucleus is the nucleus of a sperm or an egg cell during the process of fertilisation after the sperm enters the egg, but before they fuse. PNT involves removing the pro-nuclei  from a newly fertilised egg (which is regarded as an embryo under the Human Fertilisation and Embryology Act 1990) that has unhealthy mitochondria. The pro-nuclei are then transferred into a donated embryo, with healthy mitochondria, that has had its own, original pro-nuclei removed.The subsequently passed Regulations establish that ‘permitted’ eggs or embryos can have been the subject of particular specified processes (which detail the relevant methods) in specified circumstances (which concern the risk of disease).The HFE Act was amended in 2008 such that regulations could provide that eggs or embryos would be ‘permitted … even though the egg or embryo has had applied to it in prescribed circumstances a prescribed process designed to prevent the transmission of serious mitochondrial disease’.We next consider how these techniques should be viewed with reference to the concept of ‘germline genetic modification’, looking first at key international statements and conventions.not forms of ‘germline genetic modification’ is influenced by long-standing international opposition to such modification .The UK Government’s position that PNT and MST are By way of introduction, we note three points. First, international statements and conventions use a range of terms relevant to the discussion of germline genetic modification and these require interpretation. Second, the implications for the permissibility of particular practices are sometimes unclear. Third, at least in one early statement, there are indications of factors which might support the permissibility of germline modifications in particular circumstances, such as where the aim is to treat or eradicate disease.Genetic Engineering. Paragraph 4(a) boldly asserts that ‘the rights to life and to human dignity protected by Articles 2 and 3 of the European Convention on Human Rights [ECHR] imply the right to inherit a genetic pattern which has not been artificially changed’.therapeutic applications of genetic engineering (gene therapy), which holds great promise for the treatment and eradication of certain diseases which are genetically transmitted.[T]he explicit recognition of … [the right to inherit a genetic pattern which has not been artificially changed] must not impede development of the This strikes a more permissive note regarding ‘gene therapy’ although the meaning of that expression is unclear. It seems unlikely that it refers only to ‘somatic’ gene therapy (therapy relating to a cell in the human body that is not a germ cell)eradicate genetically inherited disease. Furthermore, the notion of ‘eradication’, together with the phrase ‘genetically transmitted’ and paragraph 4(c)’s implicit reference back to paragraph 4(a)’s statement regarding inheritance, suggests a potentially permissive (though guarded) approach towards germline modification.This early statement is the Parliamentary Assembly of the Council of Europe’s 1982 recommendation on Section 4.In this light, MRTs may be consistent with paragraph 4(c) if they treat an individual or eradicate disease, a framing prevalent in the UK policy debate.regulatory status. For example, in the context of avoiding the birth of a child with a serious genetic condition, treatment licenses have previously been granted for techniques which do not treat the future child. Under the original Act (as a matter of statutory interpretation)women to carry children’.future person.fluidity in the interpretation of ‘treatment’ in the UK policy and regulatory context, which likely has a normative purpose in the debate.It is also worth noting here that whether an intervention is a ‘treatment’ in a wider sense, or in an ethical or philosophical sense, may be (at most) only very indirectly related to its not treat and that the concept of ‘treatment’ can only be understood in this context as treatment of the prospective parents.In contrast to the UK’s characterisation of MRTs as ‘treatment’ of a future person, the US IOM report emphasizes that MRTs do treat disease, they may nonetheless contribute to the eradication of disease, and so could be compatible with the Council of Europe’s 1982 Recommendation on that basis. In this light, even if Articles 2 and 3 of the ECHR could reasonably be interpreted, as the Council recommended, to ‘imply the right to inherit a genetic pattern which has not been artificially changed’ (which is doubtful), it is questionable whether this could (or should) include the right to inherit disease. In any event, the meanings and significance of ‘genetic’ and of ‘artificially changed’ are contested, as Section 3 (below) shows.Regardless of whether MRTs Splicing Life,The vagueness of the 1982 Recommendation’s references to ‘respect for human rights’ was criticized, the same year, in a guardedly permissive US President’s Commission Report, Perhaps it is not surprising therefore that a 2003 report of the International Bioethics Committee (IBC) of the United Nations Educational, Scientific and Cultural Organization (UNESCO) found legislation in nine countries, including the UK, apparently prohibiting germline modification (using various expressions).treatment or diagnosis affecting an individual’s genome shall be undertaken only after rigorous and prior assessment of the potential risks and benefits’, as well as reference to relevant national laws.could be contrary to human dignity, such as germ-line interventions’.somatic interventions. It certainly implies that, under the Convention, techniques such as MRTs need to be considered with reference to the concept of dignity. Taking on this challenge, the UK Government confidently stated:aimed at preventing serious hereditary conditions, would be contrary to human dignity as envisaged by Article 24 of the UNESCO declaration.In bringing forward regulations to enable mitochondrial donation we have been mindful of the UK’s obligations under international law. We do not consider that permitting mitochondrial donation, First, the 1997 UNESCO Universal Declaration on the Human Genome and Human Rights holds, in Article 1, that ‘[t]he human genome underlies the fundamental unity of all members of the human family, as well as the recognition of their inherent dignity and diversity. In a symbolic sense, it is the heritage of humanity’.ethical acceptability of [MRTs]’.The Government’s stress on the preventative purpose of MRTs highlights their positive aims and comparatively limited scope for effecting genetic change. It also suggests that treating disease does not threaten dignity, at least where issues of consent or best interests (for those who lack capacity) are addressed.not a signatory. Article 13, ‘Interventions on the human genome’, states that ‘an intervention seeking to modify the human genome may only be undertaken for preventive, diagnostic or therapeutic purposes and only if its aim is not to introduce any modification in the genome of any descendants’.also that future generations would benefit .Second, the Council of Europe’s 1997 Convention on Human Rights and Biomedicine (the Oviedo Convention) currently has 35 Member State signatories and 29 ratifications, and is described by the Council of Europe as a ‘framework Convention aiming at protecting dignity and identity of all human beings’.not ‘germline genetic modification’.Compared with the Council of Europe’s 1982 Recommendation, the 1997 UNESCO declaration and the 1997 Oviedo Convention take more overtly negative stances towards germline genetic intervention, due to concerns about human dignity and identity.are germline genetic modification, reliance on these distinctions is also central to its approach. Underlying them is a fundamental concern with not controlling or altering ‘identity’, especially by ‘artificial’ means.This section explores both interpretative and bioethical justifications for the UK Government position that MRTs are not ‘germline genetic modification’. It considers how ‘germline modification’ and ‘genetic modification’ have been defined and used in the UK and US debates and shows that the UK Government’s claim that MRTs are not ‘germline genetic modification’ relies on two distinctions: principally between the mitochondrial and nuclear genomes, and also between ‘replacement’ and ‘modification’. Although the US IOM report concludes that MRTs Government’s Response to the Consultation on the Draft Regulations in 2014:genetic modification involves the germ-line modification of nuclear DNA (in the chromosomes) that can be passed on to future generations.There is no universally agreed definition of ‘genetic modification’ in humans. … The working definition that we have adopted is that Whether MRTs are germline genetic modification was subsequently a key point in evidence taken by the House of Commons Science and Technology Committee (HCSTC). Professor Sally Davies, Chief Medical Officer (CMO), explained the rationale for the Government’s approach:clearly a germline modification because it passes through, but we needed to make the distinction between nuclear DNA, which makes us who we are and how we are – our personalities, heights, weights and whether or not we get baldness – and the 37 genes in the mitochondria which are about energy for the cell, and which we describe as the power pack. That was why we adopted that working definition.Germline is anything that is done to DNA that goes through the generations, and mitochondria go from woman to child through the generations. This is While the CMO holds that MRTs are a ‘germline modification’, she moves swiftly to contrast the mitochondrial and nuclear genomes. The reference to ‘who we are and how we are’ implies that the nuclear genome is determinative of ‘identity’ and thus highly influential and significant. By contrast, the role of mitochondria is ‘only’ energy production. Likewise, in its consultation on the draft regulations, the DH notes that ‘it is genes in our nuclear DNA, together with environmental factors, rather than mitochondrial DNA, that shape our personal characteristics and traits.’mitochondrial donation techniques do not alter personal characteristics and traits’.A key concern in the UK House of Commons debate was whether to permit MRTs would be to permit germline ‘genetic modification’. When questioned, the Under-Secretary of State for Health, Jane Ellison, maintained both that there is no internationally accepted definition of ‘genetic modification’ and that mitochondrial replacement is not an instance of it,genetic modification’ on the basis that the nuclear, rather than the mitochondrial genome, determines ‘personal characteristics and traits’. Thus, on the Government’s view, mitochondrial replacement is not ‘germline genetic modification’ because it does not alter these aspects of a person’s identity. By implication, it is therefore far less significant than nuclear intervention. We consider to what extent this is justifiable in Section 4.So although described by the UK Government as ‘germline modification’, MRTs are distinguished from ‘germline altering the genetic structure of any cell while it forms part of an embryo.’Very significantly, this association of the nuclear genome with ‘identity’ appears to have been influenced by the reasoning underlying the HFEA decision to license research into PNT at the Newcastle Fertility Centre at Life.nuclear genes that result in heritable characteristics’.alteration to the genes or the genome and the resulting heritable characteristics’.artificial creation of human beings with certain pre-determined characteristics through modification of an early embryo’s genetic structure’; it also stressed that it thought that the ‘overall’ concern of Parliament was to prohibit ‘selecting characteristics, or ensuring a predisposition as to certain characteristics’.The Committee observed that ‘genetic structure’ was ‘ambiguous’ and therefore used various ‘interpretative criteria’.physical and behavioral characteristics as well as most forms of disease’.The Committee’s reasoning relies on a distinction between the nuclear and mitochondrial genomes, supported by scientific and lay opinion. It also highlights a Parliamentary concern with ‘artificial creation’ through ‘modification’ aimed at selecting or shaping specific characteristics. The US IOM report likewise emphasizes the nuclear/mitochondrial distinction and appeals to the ‘public understanding’ of ‘genetic’, observing that ‘[w]hile mtDNA plays a central role in genetic ancestry, traits that are carried in nDNA are those that in the public understanding constitute the core of genetic relatedness in terms of [R]emoving the pronuclei from the zygote does not cause the genetic structure to be altered, nor does the depositing of the pronuclei in the cytoplasm of an enucleated egg … [and that] this does not change the genetic structure of the new cell because the nuclear material overrides any DNA in the mitochondrial DNA.It also considered the meaning of the ‘extended’ phrase ‘altering the genetic structure of any cell while it forms part of an embryo’, reasoning that, while PNT would result in a change to the ‘genetic constitution or composition’ of an embryo, it would not result in any change to its ‘genetic structure’, referring to its earlier discussion of the latter term.A second aspect of the HFEA Appeal Committee’s reasoning focused, not on the ‘target’ of alteration, but on the ‘manner’ of it. The Committee reasoned that:Subsequently, the question of the way in which MRTs involve changes to embryos or eggs became the second, and minor, theme in the legal and policy debate, to which we now turn.methods involved, methods distanced from genetic modification on the grounds that instead they amount to replacement or donation.In the run-up to the passage of the 2015 Regulations, MRTs were justified in part by reference to the nature of the germline modification – the HFEA used “germline therapy modification”’.not changing specific DNA sequences, which is generally how I as an experimental biologist would talk about genetic manipulation or modification. You are swapping an intact mitochondrial DNA genome which … happens anyway through natural reproduction. It is not as if you are engineering a specific piece of sequence … I do not see it as a form of genetic modification.You are This passage stresses replacement of a whole genome, rather than the modification of parts of one, and invokes the idea that this occurs in nature anyway. Interjecting, Dr Ted Morrow asked ‘[i]f it is germline modification, how can it not be genetic?’.germline modification; we have always said that’.germline modification that would require a change in primary legislation’.not modifying the actual genome of the mother and father; you are simply moving it into another bag.’This distinction was a focus in various pieces of evidence to the HCSTC in late 2014. For instance, Professor Robin Lovell-Badge, a member of the HFEA’s Review Panel, stated ‘you could call [MRTs] swapping one person’s mtDNA for another’s. This is in contrast to techniques for modifying nDNA which involve snipping gene sequences from one cell and splicing them into another’.Differences between replacement and alteration were stressed elsewhere, for instance by the Wellcome Trust.modifying or altering a genome (the nuclear one) from replacing one entire ‘unhealthy’ genome  with another ‘healthy’ one. The use of terms such as ‘transferring’, ‘swapping’, ‘moving’ or ‘replacing’ a whole genome, as opposed to ‘modifying’ or ‘altering’ parts of one, is striking. It is also noteworthy given that, in the amended HFE Act, the legality of PNT in particular has been established as an exception to the prohibition in Schedule 2, paragraph 1(4), which states (in part) that ‘[a] licence under this paragraph cannot authorise altering the nuclear or mitochondrial DNA of a cell while it forms part of an embryo, except for the purpose of creating something that will by virtue of regulations under section 3ZA(5) be a permitted embryo’.altered mitochondrial DNA to be classified as ‘permitted’ eggs or embryos …’.change to an embryo’s DNA prior to implantation’, and that ‘[a]s such, they have been the subject of some opposition and controversy.’These statements all distinguish, in various ways, composition of the egg or embryo, rather than alteration of mitochondrial DNA per se. This would be consistent with the HFEA Appeal Committee’s reasoning: that mitochondrial replacement does involve ‘interventions’ in eggs or embryos (through a ‘replacement’ process), ones that change their genetic ‘composition’, but no alteration to or modification of the mitochondrial DNA itself. The emphasis on this last point seems motivated by a wish to distance MRTs from modification of the nuclear genome, no doubt in the light of the history of the HFE Act and the international background. Thus, the contrast between the genomes  is accompanied by an emphasis on the nature of the intervention: that is, on the substitution of whole ‘natural’ elements, rather than ‘engineered’ changes, seen as ‘artificial’.Given what is actually involved in MRTs, the only way to understand the Act, the Explanatory Notes and the House of Commons Library statement is to interpret the Act as referring to, and permitting as an exception, ‘alteration’ of the genetic replacement of whole, intact, and naturally occurring mitochondrial genomes’ with ‘any approach for modifying nDNA, which would likely involve editing rather than en bloc replacement of chromosomes’.no direct modification or editing of the mtDNA sequence itself, the novel combination of mtDNA from one woman and nDNA from another would not occur in unassisted sexual reproduction or in other ARTs.’To some degree, the same move is made by the US IOM, which contrasts ‘[t]he genetic modification” of the oocyte or zygote.’genetic modification’, not because they involve genome editing, but because they entail a ‘novel combination’ of genetic material. Moreover, noting that ‘germline modification’ is defined by the FDA as ‘human inheritable genetic modification’, the Committee also finds that:genetic modification of germ cells, but … it constitutes heritable genetic modification (germline modification) only if used to produce female offspring because mtDNA is solely maternally inherited, and therefore MRT to produce male offspring would not constitute heritable genetic modification (germline modification).MRT results in the Hence, in a significant departure from the UK Government and the HFEA Appeal Committee, the IOM finds that MRTs would amount to germline genetic modification if female offspring were born.From here on, however, the UK and US part company. Thus, working with the terms of reference and definitions established by the FDA, the report views ‘genetic modification’ as ‘changes to the genetic material within a cell’.not involve ‘genetic modification’ , research into PNT could not have been licensed without a change to the original HFE Act. Thus, defining (by implication) ‘germline genetic modification’ to exclude interventions relating only to the mitochondrial genome was essential for research to progress at that time. This move undoubtedly influenced the terms of the UK debate. Significantly however, despite differing from the UK Government position that MRTs are not germline genetic modification, the IOM report is nonetheless consistent with the Government’s emphasis on the mitochondrial/nuclear distinction , and on that between ‘wholesale replacement’ and ‘modification’.Of course, while the IOM had to work within the framework established by the FDA, this did not include a legislative background that might bar certain kinds of intervention. By contrast, if the UK HFEA Appeal Committee had not held, as a matter of statutory interpretation, that MRTs do replacement, MRTs are aligned with genetic selection – long established in prenatal diagnosis (PND) and PGD. They are thereby distanced from genetic modification and nuclear genome ‘editing’. In this way, and given the aim of disease prevention, children born through MRTs are positioned far from the hypothetical one discussed in the President’s Council on Bioethics Report, Reproduction and Responsibility, in 2004, ‘who … designed to certain specifications might be viewed as more of an artefact – or more answerable to the will of his or her parents – than a child who is merely selected for his or her existing characteristics’.Seen as centrally involving mitochondrial and the nuclear, and between replacement/selection and modification.In the next section, we examine the (supposed) normative significance of the distinctions between the This section focuses on what was earlier shown to be central to the legal and policy debate: the claim that nuclear genetic modification is more ethically problematic than mitochondrial replacement because it is more likely significantly to affect the resultant child’s identity.We start by drawing a distinction between numerical, qualitative, and narrative identity.Numerical Identity. A and B are identical in this sense if and only if A and B are the very same object or person. So, for example, Theresa May is numerically identical with the woman who became British Prime Minister in July 2016 and what this means is that ‘they’ (May and the woman who became Prime Minister) are one and the same woman, not merely two similar women.Qualitative Identity. On a strict definition, A and B are ‘qualitatively identical’ if and only if they share all of the same properties or qualities. One obvious response to this is to point out that it is rarely, if ever, the case that two distinct objects are exactly the same, because there will always be small differences. If we start also to consider objects’ relational properties (how they are related to other objects in the universe) and their spatiotemporal properties (where and when they are) it is tempting to conclude that, for A and B to be qualitatively identical, they must also be numerically identical, a thesis known as the Identity of Indiscernibles.extreme similarity; many pairs of so-called ‘identical twins’ would meet this less exacting standard.Narrative Identity, Self-Conception, or ‘Sense of Self’ are psychological phenomena, but ones with potential ethical significance:Another relevant sense of identity or self is a psychological, not numerical, sense. It consists of the properties or qualities that an individual considers important to who he is, to what kind of person he is, to what properties of himself he identifies with.Finally, Is the Distinction between Identity-affecting and Non-identity-affecting Interventions Ethically Significant?(i) If the distinction between identity-affecting and non-identity-affecting interventions is ethically significant, its significance is neither obvious nor straightforward, and identity-affecting interventions are certainly not always wrong or problematic. Imagine that Amelia is planning to use Bobby as a sperm donor. Shortly before donation occurs she discovers that he carries a serious genetic condition; his offspring will suffer from painful and life-shortening disease. Amelia therefore uses another sperm donor instead: Callum, who has no known heritable diseases. In this case, Amelia has changed the  identity of her future child. She was going to have a child with Bobby’s sperm but instead used Callum’s. A different child ensues, one with a much lower risk of genetic disorder. Many would not think of Amelia’s action as wrong, nor that it is made worse by her child’s  identity being altered.If numerical identity is used to distinguish nuclear genetic modification from MRTs, the underpinning claim must be that reproductive technologies which alter the nuclear genome (often) result in a numerically different person coming into being, whereas MRTs  do not. However, this argument is flawed. For it is not clear that the distinction between identity-affecting and non-identity-affecting interventions maps onto a clear ethical line. Nor is it obvious that nuclear modification is identity-affecting while MRTs are not. In what follows we look at each of these points in turn.selective reproduction rather than therapy.There may however be more subtle ethical differences. One is that whereas non-identity-affecting interventions can directly benefit, cure, or harm determinate future individuals, identity-affecting ones cannot. When a reproductive technology is not identity-affecting and yet has some effect on the future person  that person (once she exists) can look back upon the intervention and see that she has directly benefitted. It is not so clear however that the same can be said of identity-affecting interventions because the alternative to life with a disorder in such cases is non-existence, not life without a disorder.could be used to differentiate some reproductive technologies from others: in this case, nuclear genetic modification from MRTs.Whether this distinction between therapy and selective reproduction is ultimately ethically significant is beyond the scope of this work. For the present, we merely note that it does open up an interesting line of argument which (ii) Is Mitochondrial Replacement Therapy or Selective Reproduction?The argument that MRTs are less ethically problematic than modifying the nuclear genome, because only the latter is seriously identity-affecting, could only work however if  genetic modifications of nuclei were identity-affecting while  instances of mitochondrial replacement were not. Is this plausible? There are two reasons for thinking not. First, some practices termed ‘mitochondrial replacement’ may themselves be identity-affecting. Second, it may be possible to modify a cell nucleus without altering the resultant person’s  identity. Therefore, the distinction between identity-affecting and non-identity-affecting does not line up in a neat or systematic way with the nuclear/mitochondrial distinction.Furthermore, concerns about eugenics and human dignity may be thought to engage more powerfully when interventions are selective rather than therapeutic.decision to use PNT (the other kind of MRT) is identity-affecting. Imagine, for example, the choice between having a child through sexual reproduction versus having a child via IVF and PNT. The chances of the very same egg and spermatozoon coming together at fertilisation in both scenarios are miniscule and even the decision to use IVF could mean that a different person comes to exist, because of that decision’s effects on the timing of conception. But this should come as no surprise for, as has been noted, reproductive decisions affecting numerical identity are ubiquitous. So the attempt to defend MRTs by arguing that they do not (while nuclear genetic modification does) alter  identity fails. For at least some forms of mitochondrial replacement are identity-affecting; indeed, possibly they all are if we take a wider-context view of the matter and consider not just the technique itself but the position of people who are choosing whether to use IVF and MRTs in the first place.As noted earlier, Wrigley and others have plausibly argued that, at least in practice, MST (one kind of MRT) will be identity-affecting because it causes a different sperm to be used at fertilisation.ex hypothesi) affect important personal characteristics. Second, intuitively we would want to say that an embryo can survive this small degree of change – that the embryo is altered, not destroyed-and-replaced. The embryo can carry on being  the same embryo as it was before the intervention – just as an adult might carry on being the same person (and the same organism) despite having an organ removed or replaced.This conclusion is bolstered by the fact that some instances of nuclear genetic modification may not be identity-affecting. The leading examples here are ones in which nuclear genetic modification makes only a trivial difference.[M]odification of the mtDNA is likely to change the  identity of the future person … a person without a mtDNA disease will have a different life experience, a different biography and perhaps also a different character.However, given the meaning of ‘qualitative identity’, this is hardly a surprising claim. If ‘qualitative identity’ is defined stringently, as Bredenoord and others do  then pretty much anything we ever do to a person will affect qualitative identity. As such, the claim that modification of the mitochondrial genome affects qualitative identity is almost trivially true. But even if one takes a broader view of qualitative identity and sees it as merely extreme similarity, MRTs will still affect qualitative identity. For the difference between having and not having a mitochondrial disorder is often huge.MRTs will undoubtedly affect ‘qualitative identity’:So MRTs alter qualitative identity, but this is not surprising. For this reason, we question whether ‘qualitative identity’ is a very helpful expression. It might be clearer and more informative to talk instead just about altering characteristics or properties. The problem with labelling such changes ‘identity-affecting’ is that it encourages people to think that something more is being claimed than is really the case. If all that is being claimed is that the characteristics of the person are affected, then why not just use the language of altering characteristics or properties?Having noted these concerns about language, what about the substantive ethical issue? Does the fact that MRTs ‘affect qualitative identity’ have any ethical or legal implications?whether it has been altered but how:Many medical treatments and interventions are intended to improve a person’s health  compared to their identity had the treatment or intervention not been used. The important ethical question is whether changing the person’s qualitative identity is likely to adversely affect them.Bredenoord and others suggest that one implication of all this is that it is hard to draw an ethical distinction between nuclear and mitochondrial interventions and they are clearly right that there is no categorical difference (as far as identity is concerned) between modifying nuclear DNA and modifying mtDNA; both alter qualitative identity.There is not in general anything morally troubling about altering qualitative identity. What matters, as the NCoB pointed out in 2012, is not so much with considerable precision important personal characteristics, including significant aspects of behaviour, mind, and personality. But this is not true of MRTs. The latter may have a huge bearing on people’s lives – but crucially this impact is not controlled and targeted to anything like the same degree as that of some conceivable forms of nuclear genetic modification.possible with mitochondrial replacement, but seems much less probable because of the relative lack of fine-grained control that MRTs provide.One is this. Nuclear ‘genome editing’ potentially allows people to manipulate A further difference between nuclear genome editing and MRTs is that the former has the potential to introduce into future people characteristics that are fully ‘designed’ or ‘artificial’, unlikely ever to occur naturally. However, this is not true of current MRTs, which involve importing a naturally occurring mitochondrial genome ‘lock, stock and barrel’. We might therefore think that while nuclear genome editing is genetic modification, mitochondrial replacement is selective reproduction. We alluded to this distinction during the earlier discussion of the Non-Identity Problem, but here it does different work. The idea is that selective reproduction is liable to be safer and less ethically troubling because it involves merely choosing between gametes, embryos, or mitochondrial genomes, all of which already exist ‘in nature’ and could easily have gone on to play a role in ‘natural’ reproduction in any event.selecting between naturally occurring materials is less dangerous than modifying things at a genetic level. Clearly there is no necessary connection between the selection/modification distinction and levels of risk; we can imagine selection technologies with dire effects, and conversely benign forms of genetic modification. But perhaps there is some contingent link between the two inasmuch as most known forms of selection  have quite limited and slow effects on the ‘gene pool’, whereas the genetic modification of embryos could have more dramatic effects. So there may be grounds for taking a more cautious approach to nuclear genetic modification, given that the scale and scope of the risks are greater.What ethical significance this has is far from clear, although it may have some. One way of understanding it is as an appeal to the intrinsic superiority of ‘the natural’ over ‘the artificial’ but, for reasons that have been extensively rehearsed elsewhere, any such appeal is doomed to fail.Most people, most of the time, do not think much about numerical identity. People think more often about their narrative identity. This involves an individual’s self-conception: her most central values, implicit autobiography, and identifications with particular people, activities, and roles.So what ethical issues does this generate and how do these apply when considering interventions (such as MRTs) which impact directly – not on existing people – but only on the eggs and embryos which may go on to become people?Identity in this third sense is intuitively more closely bound up with ethical issues than either numerical or qualitative identity. As DeGrazia puts it:valid consent; that changes cause or constitute inauthenticity; that changes are harmful. Only the last of these applies directly to human reproduction.Narrative identity is easiest to understand when applied to the core case: existing adults with mental capacity. We start therefore with this and work back to reproduction. If we think about what might be wrong with modifying an existing adult’s narrative identity, three main kinds of worry emerge: that changes take place without Concerns about consent cannot apply because the relevant person does not exist yet and so does not have an opinion. In addition, as Gyngell and others note, it is not clear ‘why the consent of future generations should be seen as vital for decisions involving GGE [germline gene editing] but not for other major decisions with long term effects’, such as environmental policy or the decision to allow (or not) the development of new communications technologies.hypothetical consent. On this view, the position of the future person is like that of the existing child in the following scenario:will endorse what the parent has done and will consent to comparable measures that might be needed to maintain or enhance the immunity this established.A parent is concerned to have her beloved child vaccinated against a deadly and/or debilitating disease. As the child unhappily resists, the parent comforts herself (and perhaps even the child) with the thought that later on, if and when he is more mature, more thoughtful, and more adequately informed about health matters, the adult that the child becomes This may be a useful heuristic perhaps but, in reproductive cases, appealing to hypothetical consent does not add much to what we would (or should) do in any event: that is, have regard for the welfare of the child.Some may argue that we can appeal instead to there is no true self and there are no values to be altered in ‘inauthentic’ ways. This is not to say that there are no ethical objections to controlling or ‘designing’ future people’s characteristics.Much the same goes for inauthenticity. The main concern here is not that people are literally caused to become someone else, but rather that they are changed in ways which are inconsistent with their ‘true selves’ or ‘authentic values’. While there is much to be said about what ‘authenticity’ is, we do not address this here, since there are some general and fundamental reasons why inauthenticity arguments do not engage.harmful effects on narrative identity. The question of how MRTs will affect future people’s narrative identities is a complex empirical matter with any claims made being highly speculative. That said, and crucially for the concerns of this paper, the case for assuming any kind of systematic connection between – (a) the extent to which future narrative identity will be adversely affected and (b) whether a modification is nuclear or merely mitochondrial – appears weak. This is because, as noted earlier, one can imagine relatively trivial forms of nuclear genetic modification, or ones which precisely target a specific disease, not having a significant effect on narrative identity. In such cases, the resultant person either may not care at all about their having been ‘modified’ or may understand this as on a par with childhood vaccination. Conversely, being one of the first MRT babies in the world could have a psychological impact on self-conception that is just as significant as being one of the first ‘genome-edited’ children.That just leaves the suggestion that some changes may have Perhaps the child’s emerging sense of self could in some way become confused through knowing that a third person was involved in their conception, unlike their peers; children might not be able to understand why the mitochondrial donor is not included in family events and communications; tensions may develop, if the child wants more information about the mitochondrial donor than parents are able or willing to provide.For these reasons, MRTs are not necessarily in a better position than nuclear genetic modification. Yet neither is the opposite something that we can take for granted because one can also imagine specific narrative identity problems emerging in the case of ‘genetically modified children’: fears about them being ‘designed’, ‘artificial’, or even ‘polluting the gene pool’. So it is possible to imagine problems regarding narrative identity for both MRTs and nuclear genetic modification. This suggests that there is as yet  no strong narrative identity argument for treating MRTs more favourably than nuclear genetic modification.In a recent paper on this topic, Jackie Leach Scully asks how MRTs might impact upon narrative identity. She discusses various challenges for MRT children. As a new and unusual social group, they may lack a clear origin-narrative, or their sense of self may be adversely affected by media coverage.Are MRTs less troubling than interventions altering the nuclear genome, because they influence to a lesser extent the identities of future people?Regarding numerical identity, no clear difference between MRTs and nuclear genome modification was found. Each has the capacity to be identity-affecting, depending on the context, extent, and nature of the procedure; conversely, each could conceivably be used in ways that do not alter numerical identity.with considerable precision important characteristics. Another is that nuclear genome editing has greater potential to introduce characteristics that are fully ‘designed’ or ‘artificial’, and unlikely to occur naturally. These differences might serve partially to justify the suggestion that nuclear genome editing is more dangerous than MRTs and that therefore a more precautionary approach towards nuclear genetic modification is warranted. Nevertheless, two caveats must be stressed. First, it is only a pragmatic argument, not a categorical difference between nuclear and mitochondrial. Second, whether this argument should be classified as a concern about identity is doubtful. Really the worry here is about the technology generating adverse outcomes and about its going beyond therapy towards enhancement and ‘designer babies’.Something similar goes for qualitative identity; both mitochondrial and nuclear interventions would affect this. We did however find that nuclear genome editing may be different from MRTs in some relevant ways. One is that, compared to MRTs, nuclear genome editing provides an ability to manipulate Finally, we concluded that, regarding narrative identity, there is no clear difference between the mitochondrial and nuclear genomes. Interventions in either may or may not be problematic.This paper has considered the uses and meanings of the term ‘germline genetic modification’ in the UK and US legal and policy debates and has evaluated the underlying ethical concerns about ‘identity’.nuclear genome and that the concerns in this context relate principally to worries regarding ‘artificial’ changes to ‘identity’, as well as about ‘dignity’, consent, human enhancement, and safety. A central move in the UK and US debates has been to demarcate a clear boundary between the mitochondrial genome and the nuclear genome and to emphasize the latter’s more significant role in determining personal characteristics and traits. Similarly, replacing one mitochondrial genome with another ‘naturally occurring’, ‘donated’ one has been contrasted with, and presented as more innocuous than, ‘altering’, ‘editing’, or ‘modifying’ the nuclear genome .Its analysis has shown that the broadly prohibitive international position regarding germline genetic modification is best understood as relating to the nuclear genome in reproduction should be permitted. Our analysis has shown that, as far as identity is concerned, MRTs and nuclear genome ‘editing’ are not as different as has been supposed. It does not follow from this that they should be treated alike, since there may be other reasons that justify different legal and policy stances; these include the latter’s greater capacity for non-therapeutic application and its greater potential to effect rapid and radical change in the human ‘gene pool’. It does however mean that one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as ‘germline genetic modification’, is in doubt. The concept of identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in policy debates, international statements and conventions and legal positions.The question of how much normative significance these distinctions have, therefore, is important both to MRTs as a development in reproductive medicine and to the now current question of whether modifications of the


DNA analyses indicate that the Bay of Fundy population of the intertidal tellinid bivalve Macoma petalum is genetically divergent from coastal populations in the Gulf of Maine and Nova Scotia. To further examine the evolutionary forces driving this genetic break, we performed double digest genotype by sequencing (GBS) to survey the nuclear genome for evidence of both neutral and selective processes shaping this pattern. The resulting reads were mapped to a partial transcriptome of its sister species, M. balthica, to identify single nucleotide polymorphisms (SNPs) in protein‐coding genes. Population assignment tests, principle components analyses, analysis of molecular variance, and outlier tests all support differentiation between the Bay of Fundy genotype and the genotypes of the Gulf of Maine, Gulf of St. Lawrence, and Nova Scotia. Although both neutral and non‐neutral patterns of genetic subdivision were significant, genetic structure among the regions was nearly 20 times higher for loci putatively under selection, suggesting a strong role for natural selection as a driver of genetic diversity in this species. Genetic differences were the greatest between the Bay of Fundy and all other population samples, and some outlier proteins were involved in immunity‐related processes. Our results suggest that in combination with limited gene flow across the mouth of the Bay of Fundy, local adaptation is an important driver of intraspecific genetic variation in this marine species with high dispersal potential.Mitochondrial  They found relatively high levels of gene flow, likely due to rafting in this species, but also genetic splits between the East and West Gulf of Maine Coastal Currents and Nova Scotia. Semibalanus balanoides, an intertidal barnacle with pelagic larvae, was found to exhibit genetic structure both between the Gulf of Maine and the Gulf of Saint Lawrence (GSL) and within the GSL, and this differentiation within the GSL was attributed to the potential effects of selection due to temperature differences , the Gulf of Saint Lawrence (GSL), the Bay of Fundy (BF), and Nova Scotia (NS) to study the roles of selection and genetic drift driving patterns of genetic differentiation between these regions. We aimed to build on previous works that used mtDNA and limited nuclear markers to investigate the phylogeographic break at the Bay of Fundy  protocol. We added 10 μl RNase A to the extraction homogenate and incubated overnight at 56°C. Genomic DNA from each individual was eluted in 100 μl of EB buffer. We checked the quality of extracted DNA by running an aliquot on a 1% agarose gel for UV imaging and measuring concentration with the QUIBIT 2.0 Fluorometer Broad Range assay (Invitrogen). Mitochondrial sequences from previous analysis of the same individuals were used to confirm that all individuals were indeed 2.3We digested 100 ng of genomic DNA from each individual with two restriction enzymes, 5 U/μl of Sau96I and 10 U/μl of MluCI (BioLabs) with 1× buffer in a 20 μl reaction. Digestion was completed in a thermocycler at 37°C (2 hr), 80°C (20 min), and 4°C (hold). Barcoded Illumina adapters were ligated to digested DNA at 22°C (2 hr), 65°C (30 min), and 4°C (hold). We used 60 unique barcodes (0.0125 μmol/L) of 4–10 bp to avoid sequencing bias during Illumina calibration, along with four barcodes on the Y‐adapter (1.25 μmol/L), one barcode per 15 individuals, between 5 and 8 bp in length. Reaction components additionally included 1× buffer, 1 μmol/L of ATP, and 400 U of T4 ligase in a 40 μl reaction. We multiplexed individual DNA into two libraries to maximize PCR amplification using 10 μl of ligated DNA from each individual, with 32 individuals in the first library and 28 individuals in the second. The pooled ligates were purified using the QIAquick PCR Purification Kit (QIAGEN), with 50 μl eluate, prior to amplification to remove adapter and enzyme contamination. We followed the NEBNext High‐Fidelity 2× PCR Master Mix (Biolabs) protocol for PCR in 5 × 40 μl reactions per pool. Thermal cycling conditions were 98°C (30 s) initial denaturation, 98°C (10 s), 65°C (30 s), and 72°C (30 s) repeated for 16 cycles, 72°C (5 min) final extension, and 4°C (hold).Four of the five 40 μl PCRs were pooled for each library prior to an additional purification using the QIAquick PCR Purification Kit (QIAGEN), with a final elution volume of 25 μl per library. The remaining PCR product for each pool was used to verify success of PCR in a 1.5% agarose gel and to determine the concentration of DNA after purification.We ran the two pooled libraries of PCR products in a 1.5% agarose gel  for a gel extraction. We used the section of the gel between 200 and 700 bp to ensure removal of nonspecific bands <200 bp in size. The QIAEX II Gel Extraction Kit was used to purify the gel sections, finally eluting 30 μl of each pool and combining for a total of 60 μl for the final library. We sent 50 μl of the library to the McGill University and Génome Québec Innovation Centre for 100‐bp paired‐end sequencing in a single lane of the Illumina HiSeq 2000 PE100 platform.2.4M. balthica transcriptome reference using the CLC Assembly Cell 4.3 algorithm with default parameters, a length fraction of 0.9, and similarity fraction of 0.8. A consensus sequence was extracted from this mapping, with any unmapped regions filled in by the transcriptome reference assembly, and used as the reference for all future bioinformatics analyses . Variants were detected for each individual and for the reference as a whole using CLC's Basic Variant detection tool. All variants except single nucleotide variants (SNVs) were excluded, and only those SNVs that had at least two reads of coverage for each of the 60 individuals were kept. Therefore, only biallelic SNPs with coverage on the transcriptome reference as well as in all individuals were kept for future use. SNPs were filtered for a minor allele frequency of ≥0.05 to obtain a final SNP dataset for use in future analysis.Raw Illumina reads were downloaded from the Nanuq portal and imported into CLC 8.0.3. We demultiplexed the reads based on Y‐adapter and unique barcodes, discarding reads lacking barcodes. Reads were trimmed for a minimum PHRED score of 30, a limit of two ambiguous nucleotides, and for a minimum length of 30 bp. Reads were then mapped to our partial 2.5P), expected heterozygosity (He), and observed heterozygosity (Ho) at all sampling sites as well as pairwise FST values between each sampling site. We calculated locus‐specific FST values in Genepop 4.5.1  of 0.1. We ran 1 million simulations with the infinite allele mutation model. Analyses were repeated 10 times and only outliers present in all 10 runs were retained. For both LOSITAN and BayeScan, runs were performed with data divided into the two sampling regions based on the assignment tests using STRUCTURE  in Arlequin 3.5.2.2 with 10,000 permutations using the full SNP dataset, the outlier dataset, and the neutral dataset to partition variance among regions, among populations within regions, and within populations. Populations were assigned to one of the two regions indicated by the structure analysis.R  to visualize population structure at a greater resolution. PCA reduces the genetic variation of multivariate data into principle components, which are then visualized in a plot to infer genetic relationships between individuals. We used the adegenet 2.0.0 package Jombart,  in R , 32.90% of the processed reads, to the M. balthica reference to extract a consensus sequence. Finally, 36.85% of processed Illumina reads  were mapped to the consensus reference, resulting in 24,163 contigs and singletons with at least two contributing reads from each individual. Only variants that mapped to the M. balthica reference transcriptome were included in further analysis.Illumina sequencing of We detected a total of 1.199 million variable sites in the reference sequence, 454,264 of which had at least two contributing reads, including indels, replacements, and multinucleotide variants. We filtered this down to 14,310 SNPs with a minimum of two reads of coverage per individual, and the average depth of the heterozygous SNPs  due a high proportion homozygous sites among individuals.Summary statistics for each sampling site and region consisted of the proportion of polymorphic loci, observed heterozygosity, and expected heterozygosity averaged across all loci Table . The pro3.3Using the results of the STRUCTURE analysis as a guide, we detected a total of 531 outliers from LOSITAN, and nine from BayeScan, between the Bay of Fundy and populations sampled elsewhere. Of these outliers, 92 from LOSITAN and all from BayeScan were putatively under directional selection. One BayeScan outlier was not found by LOSITAN, yielding a cumulative 93 putatively directionally selected outlier loci. A combination of all detected outliers from both datasets was used to filter the 2,583 SNPs into a conservatively neutral dataset consisting of 1,089 SNPs, each from a single contig to minimize bias caused by linkage disequilibrium.3.4k clusters. The analysis of the LnP(D) for all three datasets indicated that k = 2 was the most likely number of clusters , neutral , and outlier (93 loci) SNP datasets in STRUCTURE and used the method described by Evanno et al.  to deter,583 loci Figures . For allp < .05). The full SNP dataset  yielded a ΦCT of 0.0122 between the two regions, indicating a small but significant differentiation between BF and elsewhere  was attributed to within population differentiation, with very little variance among populations within groups (ΦSC = 0.0062). For the neutral SNP dataset, AMOVA results showed a small but significant amount of variation among groups (ΦCT = 0.0057) or among populations within groups (ΦSC = 0.0114). In contrast, results for the outlier SNP dataset showed a much greater amount of variation among regions (ΦCT = 0.2144) and among populations within regions . Previous analysis of mtDNA in M. petalum showed hierarchical structure among four broad geographic regions  for this species as well as the presence of a phylogeographic break at the Bay of Fundy  permit the analysis of nuclear single nucleotide polymorphisms (SNPs) throughout the genome, providing both neutral and putatively selected markers  than those sampled in the Bay of Fundy and Gulf of Maine  and 1,2‐dihydroxy‐3‐keto‐5‐methylthiopentene dioxygenase . Two top BLAST hits represented predicted uncharacterized proteins, and two were related to transposable elements. Frequencies of the alternative and the reference alleles are available in Table ASGPR2 had the highest frequency of the alternative allele in the GSL. The frequency of the alternative allele in BF was 0, although only 12 individuals were sampled from BF so it is possible that this allele occurs at low frequencies in BF as the alternative allele was found in both GOM and NS. ASGPR, formed by subunits ASGPR1 and ASGPR2, has been associated with immune response in shrimp against virions and bacterial infections , which are widespread throughout eukaryotic genomes (Ivanov, Melnikov, Siunov, Fodor, & Ilyin, jockey‐like retrotransposons account for the formation of many pseudogenes (Bebikhov, Postnoc, & Nikinenko, jockey‐like mobile element are not themselves experiencing selective pressures, but that they are linked to loci that are under putative selection in M. petalum.The final two outliers were both related to transposable elements. The transposable element Tcb1 transposase was a strong outlier with locus‐specific M. petalum in the Atlantic and propose some correlations between observed phylogeographic patterns and putative selection, not claim outright the causation of these patterns or to rigorously determine all genes putatively under selection.A major limitation of using reduced representation genomic methods with restriction enzymes is exclusion of some variable sites. Mutations in the enzyme cut sites themselves would result in an alternate allele being unknowingly discarded (Arnold et al., Mya arenaria (Metzger, Reinisch, Sherry, & Goff, M. petalum in the Bay of Fundy could be caused by selection for immunity to such pollutants and their immunological effects, or other disease causing agents, as well as limited gene flow from the EMCC.The outliers detected and described above do not show a clear, single‐factor differentially affecting sample sites in the BF compared to the GSL, GOM, or NS regions. However, many outlier sequences did not have BLAST results, and may represent important proteins that have not been adequately described in bivalves to date. Additionally, no alternative alleles were differentially fixed between regions. Regardless, the differentiation between the BF and the other regions is clear, and is most likely caused by a mix of neutral factors, such as the EMCC limiting gene flow and facilitating genetic drift, and natural selection. Two of the outliers with BLAST results had functions related to immunity. Selection for immunity could be in response to parasites, infectious bacteria, viruses, or cancerous diseases as in 5Macoma petalum shows a distinct genotypic difference between the Bay of Fundy and the other three regions examined, Nova Scotia, the Gulf of Maine, and the Gulf of Saint Lawrence. While previous work on Macoma petalum identified a genetic discontinuity at or near the mouth of the Bay of Fundy (Einfeldt et al., M. petalum in the Bay of Fundy.None declared.SLM, JHK, and JAA conceived and designed the study; SLM and JHK performed the experiments and collected the data; SLM, JHK, and JAA analyzed and interpreted the data; JAA contributed resources; SLM and JAA drafted the manuscript; and SLM, JHK, and JAA edited and revised the final draft. Click here for additional data file.


Glossina fuscipes fuscipes is the main vector of the Trypanosoma parasites responsible for these diseases as well for the animal African Trypanosomiasis (AAT), or Nagana. We used highly polymorphic microsatellite loci and a mitochondrial DNA (mtDNA) marker to provide fine scale spatial resolution of genetic structure of G. f. fuscipes from 42 sampling sites from the northern region of Uganda where a merger of the two disease belts is feared. Based on microsatellite analyses, we found that G. f. fuscipes in northern Uganda are structured into three distinct genetic clusters with varying degrees of interconnectivity among them. Based on genetic assignment and spatial location, we grouped the sampling sites into four genetic units corresponding to northwestern Uganda in the Albert Nile drainage, northeastern Uganda in the Lake Kyoga drainage, western Uganda in the Victoria Nile drainage, and a transition zone between the two northern genetic clusters characterized by high level of genetic admixture. An analysis using HYBRIDLAB supported a hybrid swarm model as most consistent with tsetse genotypes in these admixed samples. Results of mtDNA analyses revealed the presence of 30 haplotypes representing three main haplogroups, whose location broadly overlaps with the microsatellite defined clusters. Migration analyses based on microsatellites point to moderate migration among the northern units located in the Albert Nile, Achwa River, Okole River, and Lake Kyoga drainages, but not between the northern units and the Victoria Nile drainage in the west. Effective population size estimates were variable with low to moderate sizes in most populations and with evidence of recent population bottlenecks, especially in the northeast unit of the Lake Kyoga drainage. Our microsatellite and mtDNA based analyses indicate that G. f. fuscipes movement along the Achwa and Okole rivers may facilitate northwest expansion of the Rhodesiense disease belt in Uganda. We identified tsetse migration corridors and recommend a rolling carpet approach from south of Lake Kyoga northward to minimize disease dispersal and prevent vector re-colonization. Additionally, our findings highlight the need for continuing tsetse monitoring efforts during and after control.Uganda is the only country where the chronic and acute forms of human African Trypanosomiasis (HAT) or sleeping sickness both occur and are separated by < 100 km in areas north of Lake Kyoga. In Uganda,  T. b. gambiense and T. b. rhodesiense). The geographic location of this region creates the risk that these distinct foci could merge, which would complicate diagnosis and treatment, and may result in recombination between the two parasite strains with as yet unknown consequences. Both strains require a tsetse fly vector for transmission, and in Uganda, G. f. fuscipes is the major vector of HAT. Controlling the vector remains one of the most effective strategies for controlling trypanosome parasites. However, vector control efforts may not be sustainable in terms of long term reduction in G. f. fuscipes populations due to population rebounds. Population genetics data can allow us to determine the likely source of population rebounds and to establish a more robust control strategy. In this study, we build on a previous broad spatial survey of G. f. fuscipes genetic structure in Uganda by adding more than 30 novel sampling sites that are strategically spaced across a region of northern Uganda that, for historical and political reasons, was severely understudied and faces particularly high disease risk. We identify natural population breaks, migration corridors and a hybrid zone with evidence of free interbreeding of G. f. fuscipes across the geographic region that spans the two HAT disease foci. We also find evidence of low effective population sizes and population bottlenecks in some areas that have been subjects of past control but remain regions of high tsetse density, which stresses the risk of population rebounds if monitoring is not explicitly incorporated into the control strategy. We use these results to make suggestions that will enhance the design and implementation of control activities in northern Uganda.Northern Uganda is an epidemiologically important region affected by human African trypanosomiasis (HAT) because it harbors both forms of the HAT disease ( Glossina) is the major vector of human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). The diseases occur throughout sub-Saharan Africa, causing extensive morbidity and mortality in humans and livestock  and  and G. fc events 35]. We . We G. fG. f. fuscipes in Uganda [The map in n Uganda . At eachn Uganda 29] were wereG. fn Uganda , justifyT. b. gambiense sleeping sickness . The. TheT. br bodies . Habitatr bodies ], our saase belt . Some diase belt , which mase belt , we coll2 and 0.1 μl of 5 units/μl GoTaq , 0.1 μl of 100X BSA , 0.5 μl of 10 μM fluorescently-labeled M13 primer, 0.5 of μl 10 μM reverse primer, and 0.3 μl of 2 μM M13-tailed forward primer. For loci C7b and GmL11, 0.5 units of Taq Gold polymerase  were used instead of Promega GoTaq. PCR products were multiplexed in groups of two or three and genotyped on an ABI 3730xL Automated Sequencer  at the DNA Analysis Facility on Science Hill at Yale University . Alleles were scored using the program GENEMARKER v2.4.0  with manual editing of the automatically scored peaks.DNA was extracted from two to three legs per sample, using PrepGEM Insect DNA Extraction kit , following the manufacturer’s protocols and stored at -20°C. We collected genotypic data from 16 microsatellite loci . Sequence chromatograms were inspected by eye and sequences trimmed to remove poor quality data using GENEIOUS v6.1.8 . The forward and reverse strands were used to create a consensus sequence for each sample, and the sequences trimmed to a length of 490 bp. Only a subset of the samples screened for microsatellite variation was also sequenced at the mtDNA locus  with the "adegenet" package v1.4–2  in the RO), expected heterozygosity (HE), number of alleles, allelic richness (AR) and , whe, wheO), DEMEtics  implemenDEMEtics . GeograpDEMEtics . The sigDEMEtics .EESTIMATOR v2.01 . The. TheG. fG. f. fuscipes sampled from northern Uganda  and  and G. f species . However species . This suG. f. fuscipes, depending on the exact Ne and generation time of the population in question 31][[31][G. f[[31][G. question . Signals of 2009 41]. Sim. SimG. f of 2009 [25]. On  of 2009  in the d of 2009 ,31, whilClustering  and multG. f. fuscipes nuclear lineages found at the Victoria Nile, which harbor a mix of mtDNA haplotypes of both northern and southern associated lineages . This portheast  and 0.53vs 0–2% on either side. The pattern of mismatch in the North/South contact zone suggest differential introgression of mtDNA and nuclear loci, which could be due to Wolbachia infections . We . We 81].G. f. fuscipes from the southeast into the northeast . [25. [25G. fortheast  109]. N. NG. f. ortheast , shiftinThe observations from this study have important implications on the epidemiology of the two HAT diseases, as well as on future vector control and monitoring efforts in this region. A dense sampling scheme across a relatively small geographic area allowed an unprecedented spatial resolution of genetic structure in this region. Our results point to the presence of four genetic units, three of which have high levels of gene flow among them. The genetic distinctiveness of the West from the other three units suggests that this unit could be treated as a separate entity from the Northern ones. However, when planning control and monitoring strategies, it is opportune to look at the patterns and levels of genetic discontinuities between West vs. South and West vs. North genetic backgrounds in more detail to more precisely define the boundaries of each genetic unit at a country-wide scale. Given the results of this work, for sampling sites North of Lake Kyoga, control efforts undertaken at the unit level are unlikely to produce long-lasting results due to re-invasion from adjacent units, unless physical barriers are incorporated to avoid re-invasion from adjacent units. The best strategy would be a “rolling-carpet” initiative where control is initiated from the Northeast through the Transition Zone into the Northwest followed by intense monitoring and additional control to manage fly migration from previously cleared sites due to population recrudescence after control.G. f. fuscipes populations connected–a fact that should be taken advantage of when designing control. The genetic connectivity we found along waterways provides further support for a vector control strategy that incorporates targets along waterways and barriers to recolonization from adjacent stretches of riverbanks. This idea is also supported by a recent study that comprehensively evaluated a “tiny targets” vector control strategy along riverine savannah and found that a target density of 20 per linear km can achieve >90% tsetse control , it , it 31],T. b. gambiense and T. b. rhodesiense) is unlikely to be prevented by an incompatibility between vector populations in the region of contact. Given the extent of connectivity in the three northern genetic units and the apparent genetic stability of G. f. fuscipes populations in the region [Of major relevance for disease control is the finding of high levels of genetic intermixing and gene flow in the Transition Zone, which implies that a fusion of the two diseases  were used, following selection of principal components using a-score optimization, to plot (PDF)Click here for additional data file.S2 FigTriangles, diamonds, and squares identify sampling sites within the Northwest, Transition Zone, Northeast genetic units, respectively.(PDF)Click here for additional data file.S1 TableG. f. fuscipes. ** Indicates samples collected prior to 2014, N = number of individuals analyzed, AR = Allelic richness, Ho = Observed heterozygosity, He-Expected heterozygosity, Fis = inbreeding coefficient.Sample geographic locations, sample sizes, and genetic diversity statistics for 16 microsatellite loci and for a 490bp mtDNA COI-COII gene fragment in 42 populations of (XLSX)Click here for additional data file.S2 TableThe table reports loci names followed by the forward (F) and reverse (R) primers names and sequences. The last column reports its source. M13 tails are marked with an asterisk (*).(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableST and ɸST comparisons for microsatellites (A) and mtDNA (B) respectively. FST values are reported in the lower diagonal. Since most values are significant, we highlight those that are non-significant in bold. All computations were done in ARLEQUIN. Significance was calculated based on a P<0.05.Pairwise F(XLSX)Click here for additional data file.S6 TableEST values and the Benjamini-Hochberg corrected significance p-values, respectively. Estimates were made in the R package DEMEtics [The first two columns show the sampling site pairs, while the third and fourth columns report their mean DDEMEtics .(XLSX)Click here for additional data file.S7 Table2), p-value for the Mantel test (p), and slope and intercept of the linear regressions are shown. Significant correlations are indicated in bold (p<0.05).Results for the Northwest, Transition Zone, Northeast, West units, and all samples combined  are shown separately. Genetic group, root mean square values (R(DOCX)Click here for additional data file.S8 TableEstimates of effective population size (Ne) were computed for each of the 42 sampling sites across the geographic regions in northern Uganda using three methods: LD, modified temporal method of Waples  based on(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 TableGeneral information is displayed . Total, female, and male migrants are shown as counts from each unit based on both analyses.(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.


During the last few decades, extensive fragmentation has caused severe population decline and local extinction of golden langur from several fragments. However, no studies are available on the impact of habitat fragmentation and the genetic diversity of golden langur in the fragmented habitats. The present study aimed to estimate the genetic diversity in the Indian population of golden langur. We sequenced and analyzed around 500 bases of the mitochondrial DNA (mtDNA) hypervariable region-I from 59 fecal samples of wild langur collected from nine forest fragments. Overall, genetic diversity was high  and comparable with other colobines. Populations in smaller fragments showed lower nucleotide diversity compared to the larger forest fragments. The median-joining network of haplotypes revealed a genetic structure that corresponded with the geographical distribution. The Aie and Champabati Rivers were found to be a barrier to gene flow between golden langur populations. In addition, it also established that T. geei is monophyletic but revealed possible hybridization with capped langur, T. pileatus, in the wild. It is hoped that these findings would result in a more scientific approach towards managing the fragmented populations of this enigmatic species.Gee’s golden langur  However, its distribution range rarely overlaps with that of the capped langur (Trachypithecus pileatus) but hybrids between golden and capped langur have been reported in Bhutan [Gee’s golden langur ileatus) . It has ileatus)  and Scheileatus) –6. Its dileatus) –11. The 2), Royal Manas NP  and Phibsoo Wildlife Sanctuary (WLS) (266 km2) [2) and Chakrashila WLS (45.58 Km2), the major populations are found in Reserve Forests (RFs), Proposed Reserve Forests (PRFs) and Unclassified State Forests with a little or no protection. In the last few decades due to forest fragmentation and degradation, its habitat in India has been reduced by more than 30% leading to severe population fragmentation [In Bhutan, the golden langur is mostly found in three Protected Areas viz. Black Mountain National Park (NP) 1,730 km266 km2) , whereashila WLS 5.58 Km2,entation –17. Rece6 km2 , wHabitat fragmentation consistently has large, negative effects on measures of biodiversity such as population size, distribution and genetic diversity, especially in habitat specialist species –22. Low Semnopithecus and Trachypithecus meet. Furthermore, no information is available on the genetic diversity of fragmented populations of golden langur. The present study aims to estimate the genetic diversity in the Indian populations of golden langur using HVRI. It also aims to examine the phylogenetic position of Indian golden langur with respect to the Bhutanese population and closely related Trachypithecus spp. using cytb. It is hoped that this study would serve as a platform for a more scientific approach towards managing the wild population of this enigmatic species.Genetic data generated so far on golden langur was intended to examine its taxonomic placement within the langur group in the subfamily Colobinae using mitochondrial cytochrome b gene (cytb) ,42,43. I2 (NY) to 592.5 km2 (CR) and are inhabited by as few as 25 golden langur individuals (BG) to more than 1450 individuals (CR) [Eight forest fragments in Assam that vary in size, time since isolation and disturbance level  were parals (CR) –15. Aparals (CR) ,44.th March, 2013) and Assam State Biodiversity Board . Fresh fecal samples were collected between 29/06/2014 and 16/12/2015 from wild langurs after watching them defecate. Care was taken not to collect multiple samples from the same individual. A total of 71 samples were collected from CR (N = 15), CS (N = 12), KJ (N = 10), ND (N = 9), NY (N = 6), BH (N = 6), UM (N = 5), MN (N = 4) and BG (N = 4). Approximately 5g of the samples were stored in small plastic bottles using the two-step method [Sample collection was performed without direct interaction with the golden langurs and without causing any disturbance to their habitat. Samples were collected only from government-owned lands. Permission to collect fecal samples from Assam state were obtained from the Principal Chief Conservator of Forests (Wildlife), Assam Forest Department , a PCR was performed with universal vertebrate mitochondrial cytochrome b (cytb) forward primer  and 16022, 0.25 μM each of forward and reverse primers and 0.75 units of Taq polymerase . Utmost precautions were taken while performing PCR to avoid contamination from an external source of DNA. All PCR reactions were carried out with a negative control. Pre- and post-PCR works were carried out at separate places and using separate micropipettes. The PCR products were visualised on a 2% agarose gel. DNA was eluted from the gel using Gel Elution Kit (Bioserve India).PCRs were carried out with initial denaturation at 95°C for 5 min, 35 cycles each with initial denaturation at 95°C for 20 sec, annealing at 52°C for 30 sec and extension at 72°C for 30 sec followed by final extension at 72°C for 10 min. The total reaction volume was 15 μl with 1X BSA, 1X PCR buffer, 0.25 mM of each DNTP, 2.5 mM MgClDNA sequencing was carried out using BigDye Terminator cycle sequencing Kit (Applied Biosystems) in ABI 3730XL sequencer. Sequencing was repeated twice in both the directions. A maximum 639 bases of a mitochondrial DNA fragment containing the partial tRNA-Thr sequence, complete tRNA-Pro sequence and 543 bases of HVRI were generated. Cytb sequences of a maximum 521 bases were generated from eight samples.http://www.codoncode.com/). The primer sequences along with low quality bases at the ends which were removed. CLUSTAL W with default settings as implemented in MEGA5 was used to realign the edited sequences.The sequences were assembled and manually edited to check for base-calling errors using CodonCode Aligner v.2.0.6 (h) and nucleotide diversity (π) for each fragment. Population pairwise FST were calculated from pairwise nucleotide differences between fragments and their statistical significance was checked using 10,000 permutations as implemented in Arlequin v.3.5.1.2 [http://www.fluxus-engineering.com) was used to construct a median-joining haplotype network [Diversity estimations were done using DnaSP v.5.10 . Genetic.3.5.1.2 . To repr network .T. geei were used. Four orthologous sequences of other Trachypithecus spp. truncated from mitogenome sequences from GenBank nucleotide database were part of this 529 base dataset: two sequences of T. shortridgei (accession numbers HQ149048.1 and KP834334.1) and one sequence each of T. pileatus (acc. no. KF680163.1), and T. obscurus (acc. no. EU004477.1). Two sequences of Nasalis larvatus (acc. nos. DQ355298.1 and KM889667.1) were used as the outgroup. In addition to HVRI sequences, cytb sequences were used for ascertaining the phylogenetic position of Indian T. geei sequences with respect to T. geei from Bhutan, T. pileatus and T. shortridgei. For this purpose, apart from the aforementioned mitochondrial sequences, three partial cytb sequences of T. geei  and one of T. pileatus (acc. no. EU526386.1) of Bhutanese origin were also included in the analysis.For determination of intra-specific genetic structure, all unique HVRI haplotype sequences of The software jModelTest v.2.1.3  was usedTrachypithecus sequences in GenBank, our sequences clustered with authentic mtDNA sequences instead of confirmed pseudogenes. No stop codons were detected in the partial cytb sequences.The possibility of amplifying numts was greatly reduced by the fact the all our DNA was extracted from fecal samples, which are naturally enriched with mtDNA molecules due to their small size, high copy number and lower vulnerability to degradation compared to nuclear DNA . MoreoveOut of 71 samples collected, 59 samples gave successful amplification for HVRI and 518 bases were sequenced and aligned. Nineteen haplotypes were revealed based on 47 mutations (45 transitions and two transversions) spread over 46 segregating sites, out of which 43 were parsimony-informative. Haplotype sequences were deposited to GenBank (accession numbers KX189494-KX189512).h = 0.938) but the nucleotide diversity was low (π = 0.02443). The haplotypes were assigned an alphabetical code (from GL-A to GL-S) based on the chronology of discovery. Haplotype GL-C was the most frequent, represented by eight individuals from two fragments, followed by GL-A represented by seven individuals. More than half of the haplotypes represented fewer than three individuals.The overall haplotype diversity was high (π = 0.000483).Haplotype and nucleotide diversities were calculated for each population . CS and A median-joining haplotype network was drawn based on the HVRI sequence data . The 19 ST were calculated between each fragment and statistical significance assessed with 10,000 permutation steps  and GL-M (ND) were basal to the rest of the 15 haplotypes of which 14 fell into two clades of high statistical support, and one (GL-O from CR) remained unresolved. The BH-KJ clade showed a typical stepping stone-like progression from BH haplotypes to the KJ haplotypes, with high support for all nodes but one. On the other hand, the CR-CS clade also showed a similar progression from CR haplotypes to the rest. However, the basal node had low support and the derived sequences fanned out instead of being strictly dichotomous, but the nodes had high support.The 19 HVRI haplotypes of t clades . In bothT. geei formed four haplotypes . Haplotype sequences were deposited to GenBank (accession numbers KX189513-KX189516). To check for the taxonomic placement of T. geei, a dataset containing 392 bases of partial cytb sequences was used in phylogenetic tree reconstruction. T. geei, T. pileatus and T. shortridgei clustered together with very high support, as expected, but the relationship among the three remained unresolved , whereas the other T. pileatus sequence [We also found that the cytb sequences obtained from resolved . Our seql., 2008 ) also clsequence  separateh = 0.938) and a moderately high nucleotide diversity (π = 0.0244) compared to other Asian colobines. For example, the critically endangered white-headed langur, T. leucocephalus  and Guizhou snub-nosed monkey, Rhinopithecus brelichi  showed much less genetic diversity even when a much higher percentage of their population was sampled [R. roxellana  [R. bieti  [Nasalis larvatus  [Rhinopithecus spp. [While all previous genetic work carried out on golden langur were aimed at addressing questions regarding its taxonomic placement among congeneric species ,42,43, t sampled ,40. Thes= 0.034) , Yunnan = 0.034) ,56 and p= 0.022) . Notablycus spp. ,43.2), whereas lowest was in Kakoijana (10 km2). Interestingly two of the largest (>350 km2) fragments showed lower haplotype diversities. This might be due to over-sampling of major haplotypes. Nucleotide diversities showed greater correspondence to the fragment areas with larger fragments showing higher nucleotide diversities compared to smaller fragments, with the exception of Nayekgaon, thus reflecting the greater gene flow expected in large, continuous forests. Nayekgaon’s unusually high nucleotide diversity may be due to the presence of individuals from divergent populations which originated from adjoining fragments which it was connected with until the 1990s. Kakoijana, although not the smallest fragment, has the lowest haplotype and nucleotide diversities, which could be due to relatively large geographical and temporal isolation from adjoining forest  seems to be founded by individuals from or near Chakrashila. This has to be ascertained to prevent any inadvertent genetic intermixing during future repopulations. Most forest fragments lost their connectivity between 1970s and 1990s. These fragments should be protected from further degradation and steps may be taken to connect them to maintain genetic diversity.Golden langurs are monophyletic, and it appears that they are capable of hybridizing with capped langurs in the wild and Hanuman langurs in captivity. In fact, some of the individuals kept in Indian zoos may be hybrids. Therefore, the genetic affiliation of captive golden langurs has to be ascertained before they are used in captive breeding programs and reintroductions.