Two apparently irreconcilable models dominate research into the origin of eukaryotes. In one model, amitochondrial proto-eukaryotes emerged autogenously from the last universal common ancestor of all cells. Proto-eukaryotes subsequently acquired mitochondrial progenitors by the phagocytic capture of bacteria. In the second model, two prokaryotes, probably an archaeon and a bacterial cell, engaged in prokaryotic endosymbiosis, with the species resident within the host becoming the mitochondrial progenitor. Both models have limitations. A search was therefore undertaken for alternative routes towards the origin of eukaryotic cells. The question was addressed by considering classes of potential pathways from prokaryotic to eukaryotic cells based on considerations of cellular topology. Among the solutions identified, one, called here the “third-space model”, has not been widely explored. A version is presented in which an extracellular space (the third-space), serves as a proxy cytoplasm for mixed populations of archaea and bacteria to “merge” as a transitionary complex without obligatory endosymbiosis or phagocytosis and to form a precursor cell. Incipient nuclei and mitochondria diverge by division of labour. The third-space model can accommodate the reorganization of prokaryote-like genomes to a more eukaryote-like genome structure. Nuclei with multiple chromosomes and mitosis emerge as a natural feature of the model. The model is compatible with the loss of archaeal lipid biochemistry while retaining archaeal genes and provides a route for the development of membranous organelles such as the Golgi apparatus and endoplasmic reticulum. Advantages, limitations and variations of the “third-space” models are discussed.This article was reviewed by Damien Devos, Buzz Baum and Michael Gray. All known cells are either prokaryotic or eukaryotic. The prokaryotes encompass two domains, Bacteria and Archaea. The third domain, the Eukarya, differs from prokaryotes in ways that include larger cell size; intracellular partitioning into membrane-enveloped spaces, notably cytoplasm, nucleus and mitochondria, each supporting a subset of cellular functions (division of labour), spliceosome processing of mRNA, nucleoli, and the organization of the eukaryotic genome into multiple intron-rich linear chromosomes often with long intergenic spacer regions Table . Recent The prokaryotic affinities of many eukaryotic genes are well established –12  [3) [It is widely –26, but species) , 59. It ies) [3) , true pries) [3) –63. No eies) [3) , 60, 64,Recovering the detailed relationships among very ancient genomes is profoundly challenging with many opportunities for artifacts and error to enter the phylogenetic trees. Nevertheless, some general conclusions can be made, among them that eukaryotic genomes are mosaics of bacterial-derived, archaeal-derived and eukaryotic-specific genes. Eukaryotic genes that originated from the postulated archaeal host are outnumbered by genes of bacterial origin Fig.  7, 8, 11, 13, 65Alternate hypotheses that confront some of these issues propose, for example, that the metabolic mosaicism of the mitochondria resulted from repeated nested endosymbioses, with one mitochondrial endosymbiont species being replaced by a second, and so on, over time . SyntropGiven that the prerequisite conditions for the autogenic and endosymbiotic narratives of eukaryogenesis have yet to be demonstrated, that is, the existence now, or in the past, of amitochondrial proto-eukaryotes whose ancestors never possessed mitochondria, or the presence of bacterial cells living in prokaryotic endosymbiosis within archaeal cells, alternative routes towards eukaryotic evolution were sought. Rather than employ phylogenetic or metabolic arguments the question was restated in terms of minimal cellular topologies. The eukaryotic cell is abstracted into its secondary spaces and the range of pathways is enumerated that lead from archaeal and bacterial cells, similarly abstracted, to the eukaryotic topology. These analyses provide frames of reference upon which putative eukaryogenic pathways can be rationally constructed. The biological plausibility of the pathways derived in this way is then evaluated by hypothetical step-wise reconstruction of the hallmark features of eukaryotic cells (as listed in Table Animating the strong third-space model” to “Steps towards a eukaryotic cytosol and mitochondria”) investigates biological processes that, based on these new frameworks, would allow for the development of complex cells. The third section outlines the strengths and drawbacks of the model.Model development is broken into three stages. In the first section a theoretical analysis demonstrates the existence and logical coherence of novel potential frameworks for eukaryotic evolution. The second section . The spaces are nested as a hierarchy of primary, secondary and tertiary spaces. Primary and secondary spaces are typically delineated by membranes. Tertiary space may or may not be membrane bound (compare for example the lysosome and the nucleolus). In any given cell a space may contain one or many members . The representation allows simple questions to be asked about the relationship between spaces that can loosely be called topological, for example whether a given space can or cannot contain another space within it. This allows different models of major organizational transitions, such as eukaryogenesis, to be interpreted as belonging to one or other class of transition as defined by the topological relationships involved in the transition.The primary topology is the cell and all it contains.The secondary topology reduces cells to secondary spaces or domains. Eukaryotic cells are tripartite with three well-defined secondary spaces; a membrane-delimited cytoplasm, membrane-delimited mitochondria, and a membrane-delimited nuclear space. Prokaryotes comprise a single membrane-enclosed secondary space and are therefore secondarily unipartitite. The identity of each space is defined by their dominant functions; RNA production (nucleus), metabolism and protein production (cytosol), metabolism and high-yield oxidative energy production (mitochondria). Individual spaces may be in physical communication (for example the cytosol and nucleus are connected via nuclear pores) and may share molecular components. The passage of molecules from one space to another is, however, selective, directional and regulated (gated)Several eukaryotic groups have acquired additional secondary specialized spaces, for example, the chloroplasts of plants. This is outside the scope of this discussion.The tertiary topology recognizes additional spaces that exist within the secondary spaces. These spaces are derived evolutionarily from the secondary spaces. The cytoplasm, for instance, is further divided into a series of tertiary spaces that include endoplasmic reticulum, Golgi apparatus, and vesicles. Similarly, the nucleolus is a tertiary space within the nucleus. In addition, some eukaryotes possess specialized barrier spaces such as the cell walls of plants and fungi. Tertiary spaces in prokaryotes include cell wall barrier spaces and the periplasm.In eukaryotes, the nuclear space cannot contain the cytoplasmic or mitochondrial spaces. The mitochondrial space cannot contain the nuclear or cytoplasmic space. The cytoplasmic space contains both the nuclear and mitochondrial spaces and is, therefore, distinguished as the “containing space”.Cellular topology is not a representation of the actual distribution and organization of functional elements inside a cell (the cellular topography). Each secondary space could represent from one to any number of individual elements. For example, mitochondria are represented topologically as a single space even though any given cell possesses numerous mitochondria.Spaces can change their nature through evolution, for example a bacterial space can evolve into a mitochondrial space.As an analogy, if the primary space is thought of as a continent, the secondary spaces represent countries that may be in contact with each other, and share goods, but maintain their own border posts and custom controls. The tertiary spaces are equivalent to districts within each country.Given that the eukaryotic cell is a mosaic with archael and bacterial elements the minimum starting point is taken as two distinct primary spaces . Figure In Fig. In Fig. In Fig. In its simplest version, the third-space model requires only two cell types, or a population of two cell-types, embedded in the matrix, one of which must be archaeal and the other bacterial. The model may also incorporate diversified populations composed of more than two cell types  in eukaryotic evolution . Subsequent interaction allows to acquire the mitochondrial progenitors from additional cells embedded in the matrix, by, for example, phagocytosis. (This can be summarized as (1* + 1* + 1*➔2* + 1*➔3*). The intermediate third-space model thereby provides a potential route from simpler cells to a more complex proto-eukaryote, that side-steps a drawback of the autogenous proto-eukaryote model. Rather than surfacing from the last common universal ancestor along with prokaryotes, the formation of the proto-eukaryotes is now delayed until after the emergence of prokaryotes.In weak third-space models, two spaces form a bipartite relationship within the matrix, but undergo the transition to the final tripartite state with no further cellular contribution from the matrix. An example would be the fusion of two cells in an endosymbiotic relationship  and a bacterial space. The third-space must: (i) Enclose the other spaces and be biologically compatible with them . (ii) It must be sufficiently stable to provide the opportunity for eukaryotic-like properties to emerge over time. Stability may be provided either by long-term physical survival of the matrix and its cellular consortium, and/or by the ensemble acquiring the ability to efficiently regenerate after dispersal. This is addressed further in the section "Limitations". (iii) It should be more than a passive arena for the evolution of eukaryotic cells but should be evolutionarily dynamic. As the third space evolves from a matrix to a cytosol it will have to pass through a series of transitionary phases that combine, to varying degrees, the properties of the matrix and the cytosol. These provides an increasingly privileged microenvironment that physically supports and protects the ensemble, but also serves as a locus for functional networks of increasing complexity that tie together the cellular elements of the ensemble as a coordinated system.Given that genes of archaeal and bacterial origin occur in eukaryotes, the starting conditions are taken to include an archaeal space (which may include the recently discovered Cellular third spaces) as there are no examples among living entities from which to draw inferences.Geological micro-environments could provide protective and stable spaces  fulfilliMost prokaryotes grow embedded in extracellular matrices in the form of biofilms or microbial mats , a mode minimal cellular topologies, above), a biofilm-like third-space is compatible with the evolution of Eukarya from cell populations, possibly of mixed cell type. As noted above, the biofilm-like matrix is the initial condition for the third space but is structurally and functionally modified as eukaryogenesis proceeds (i.e. Matrix ➔ Transitionary phases ➔ Cytosol). Biofilms are example of a matrix that can be used to model the properties of the third-space, but it does not follow that there is third space has a one-to-one identity between any extant biofilm and the third-space matrix and other ther parameters, such as cell-cell aggregation may also play a role in organizing the three spaces of the model.Given the multiplicity of cells resident within biofilm-like matrices, and since “each secondary space could represent from one to any number of individual elements” .Cell Fusion. Mitochondria are not discrete entities but instead often fuse to form dynamic tubular and network structures [ructures –109. TheCentralization of the genetic information. Although mitochondria and chloroplasts have retained some genetic information, the genes for most of their proteins have been centralized within the nuclear genome. As this occurred independently for mitochondria and chloroplasts it is reasonable to assume that genome centralization is advantageous and that, during the transition from prokaryotes to eukaryotes, genetic information, from whatever source, will become concentrated into a centralized genome.Intron invasion. The last common ancestor of eukaryotes possessed introns [ introns  probably introns , 110–114 introns , 115. Th introns , 117. En introns –121. Whe introns , 76. Thi introns , 76, 114 introns , 76, 79. introns .Chromosome linearization. Although mitochondrial DNA is typically considered to be circular, linear mitochondrial genomes have evolved multiple times independently which implies a relatively low barrier between circular and simple linear chromosomes, allowing for an evolutionarily intermediate linear state between circular chromosomes and eukaryote-like centromeric/telomeric linear chromosomes [omosomes , 124. Teomosomes  suggestiGiven the structure of the eukaryotic cell and the prokaryotic phylogenetic signature still evident in its nuclear, mitochondrial and chloroplast genomes, certain events can be proposed in the development of eukaryotic cells.The eukaryotic genome arose by merger of archaeal and bacterial genomes. The third-space model can accommodate simple populations of only two cell types  but also more complex mixed populations with more than two cell types. Considering mixed populations, and focusing on the bacterial component, the relative weakness of the alpha-proteobacterial signal  compared to the aggregate bacterial signal in the eukaryotic nuclear genome Fig.  can be rHalobacteria was marked by the acquisition of approximately1000 bacterial genes families [Lokiarchaea species, 33% of their genes are unique to these species, with the remaining 36% of archaeal origin, and 28% bacterial [Thermotoga maritima, 24% of genes have been imported from archaeal species. Horizontal gene transfer is promoted by growth in biofilm matrices, as has been demonstrated both in Archaea, in Haloferax volcanii [Horizontal transfer of genes between prokaryotes is a fundamental property of their evolution, and has allowed for very extensive genetic mosaicism to occur in some cases, including in inter-domain mosaicism, where both archaeal and bacterial genes are prominent in the same organism . For exafamilies . Interdoacterial . Bacterivolcanii , for exavolcanii –129.Biofilms deploy large amounts of DNA to the matrix (extracellular or eDNA) where it serves an essential structural role . The reiThe transfer of genes can occur in either direction between Archaea to Bacteria , 69, 137loss of cell walls), the physical fusion of the wall-less hybrid genomes into increasingly larger structures, approaching something more like eukaryotic nuclei, is allowable under assumption 2 (cell fusion) and assumption 3 . These fused structures will be referred to as multi-genomes to distinguish them from the fully formed eukaryotic nucleus. They are transitionary in form between somewhat autonomous prokaryotic hybrid cells produced by horizontal gene transfer and a nuclear structure that is totally dependent on the rest of the cell for its maintenance , and, aAssembly of a primitive nucleus) and possibly later further differentiate towards an endoplasmic reticulum-like structure (see Membrane enveloped organelles).Loss of the cell wall may have been progressive with, initially, only a subset of cells in the ensemble surviving without walls. Indeed, if loss of the ability to form cell walls also impeded secretion of matrix polysaccharides, mixed populations of walled and wall-less cells may have been necessary to maintain the structure of the matrix. The cell membranes of the fused L-form-like multi-genomes, which enclose the chromosomes of the multi-genome, perform a similar role as the nuclear membrane in fully eukaryotic cells, that is, they separate the genomic material of the ensemble from the proxy cytoplasm. As the fused multi-genome evolves to an increasingly nuclear-like structure, so too its membranes will increasingly evolve properties associated with the nuclear membrane  in the matrix. Genetic information at the multi-genome stage is carried on several distinct chromosomes within each multi-genome rather than a single chromosome. Thus, the physical merging of cells in the matrix to generate multi-genomes provides a potential point of origin for the evolution of nuclei with multiple non-identical chromosomes Fig. b. Similaintron invasion) and linearization of their DNA content is allowable under assumption 5 (chromosome linearization). The transfer of matrix eDNA back to the genome, particularly if reinserted in a non-coding format, has the potential to add “bulk” to the genomic DNA and so provide an early substrate for the evolution of ancestral long intergenic sequences. Genomes constructed in this fashion would be syncretic, that is, they would be composed of a mosaic of genes selected from a range of bacterial and archaeal groups, as is, in fact, seen in extant eukaryote genomes . Thus, in a population of monoploid hybrid cells embedded in a stable third-space matrix, a genetic crisis arising in one member of the ensemble need not compromise the performance of the partner cells while the “damaged” cell could still be suppressed or eliminated by negative selection. The third-space matrix provides, therefore, an error-tolerant microenvironment in which to “test” genetic innovations and escape the negative consequences of deleterious genetic variations . SimilarHorizontal gene transfer will transmit deleterious genes as well as potentially harmful mobile elements,  . As noteallowable assumption 2” above). High frequency horizontal gene transfer, between closely related cells, which would be favoured in third-space models, also improves long-term fitness of polyploid populations [Polyploidy occurs in some Bacteria  and Archulations . By far ulations . Extrapoulations , similarulations  as well ulations , and, prAssembly of the multi-genomes into a singular stable nucleus would help overcome problems that would be associated with coordinating the division of the multi-genomes with the division of the emerging eukaryotic cell. Moreover, retention of a diploid state maintains the protection against recessive mutations afforded by polyploidy.Mitosis is highly variable . It may Prokaryotic genes are typically expressed as part of an operon or gene cluster. Advantageous modifications arising in the multi-genome would, therefore, often be obligatorily co-expressed with other genes on the same operon, some of which might be sub-optimal or engender conflict among related but variant gene products from another chromosome. Moreover, when new molecular pathways incorporate gene products encoded on different chromosomes, mechanisms are required to coordinate their expression. A solution to such problems could be obtained by fragmenting the operons through the interjection of DNA spacer sequences between genes and the development of independent transcriptional regulation. This favours the evolution of long intergenic sequences, eukaryote-like monocistronic mRNA, and complex control of gene transcription.If, as proposed, eukaryotic cells developed from populations of prokaryotic cells embedded in a matrix Fig. , the greMyxococcus xanthus grown in cellulose tubes, for example, are packed with extracellular membranous vesicles and also exhibit a sharply discontinuous 5 nm wide sheath-like outer boundary at the air-water interface that is strongly osmium-staining, consistent with a lipid membrane outer boundary [Myxococcus xanthus films suggests that, in at least some conditions, microbial ensembles can be enclosed within a membrane-like structure. Extracellular lipids are, therefore, available in prokaryotic communities, and there is evidence that they may form boundary layers under some circumstances.A key step in the transition of the matrix to a cytosolic space is the acquisition of an outer membrane. Biofilm-like matrices are often richly endowed with membranous lipids (in the form of vesicles). When examined by electron microscope tomography, small films from boundary . AlthougMyxococcus example, the membrane-like boundary layer assembles at air-water interfaces and “given the number of secreted vesicles …. and the fact that lipids naturally accumulate at an air-water interface” [There are many ways a membrane might form. In the terface” , its forBiofilm matrices contain polysaccharide polymers that are absent from eukaryotic cytoplasm. A mechanism of cytosolic-takeover is, therefore, required to remove these polymers and replace them with a cytosol-like structure. Populations of cells within a stable third-space would be expected to exhibit social interactions (see above). Over time, stable coexistence would, in some instances, tend towards strong dependency relationships with some cells adopting increasingly specialized functions, as occurs to some degree in biofilm communities . It is wThe secretion of specific proteins by prokaryotes , occurs Partitioning of protein synthesis to the cytoplasm” and “Steps towards a eukaryotic cytosol and mitochondria: shared metabolism”), these processes nucleate the incremental replacement of the matrix by the cytoplasm. As the ensemble evolves to an increasingly cell-like state more orderly and better regulated mechanisms are acquired to stock the third-space with cytosolic material such that forms ultimately emerge where the matrix fully and permenantly replaced by cytosol.To summarize, once contained within a membrane, the lysis of a subset of cells in the population together with regulated secretion from living cells embedded in the matrix would deposit cytoplasmic material into the matrix. Given time, and with a population linked by obligatory symbiosis . TIt has been proposed, based on calculations of “energy per gene”, that eukaryogenesis could occur only after the evolution of specialized energy generating organelles, namely the mitochondria , 38. ThiThe bacterial dominance of essential metabolic and operational processes in the eukaryotic genome can be understood in a third-space model by assuming that the transitionary matrix state was mesophilic, as are most extant bacteria and eukaryotes. Archaeal metabolism is often adapted to extreme growth conditions, and those archaeal lineages that survive well under mesophilic conditions appear to have acquired this ability through the evolutionary co-option of mesophilic bacterial metabolic pathways following large-scale horizontal gene transfer of bacterial genes . Some meMerger of archaebacterial genomes and bacterial genomes allows for the retention of archaebacterial genes while abandoning archaeal membrane lipid chemistry. Archaeal lipid membranes are thought to be cytoprotective in extreme environments. As most eukaryotes have a mesophilic lifestyle, the selection of bacterial over archaeal-type membrane lipids by the emerging eukaryote might, therefore, reflect the preferential selection of bacterial cell membrane lipids as they are more suitable for a mesophilic existence. Alternatively, it might result from a simple numerical predominance of Bacteria over Archaea in the early matrix ensemble.Minimal cellular topologies). They could result from in-folding of the plasma membrane or out-folding of a primitive nuclear membrane, either of which are compatible with a third-space hypothesis. Prokaryotes in biofilms shed lipid encapsulated membrane vesicles [Lokiarchaeota possess a rich complement of RAS-family small GTPases [Eukaryotic cytoplasm contains a labyrinth of membranous sub-cellular tertiary structures . These spaces are in contact with the extracellular environment through fusion events with the plasma membrane and play a central role in the import/export economy of the cell. Topologically they form an interface between the inside of the cell and the outside. In the topological model proposed here, these are tertiary spaces, that is, spaces that are derived from the secondary spaces , one of which metabolized the sole available carbon source to a form that the other species could use, found that mutations occur within less than ten days that increase the stability of the communal biofilm , promote physical association of the two species and increase overall productivity compared to the same two species grown in a chemostat [Physical cell-cell association within the matrix provides a further route to enhance the stability of the matrix ensemble, particularly if these associations significantly enhance the survival of the ensemble members. Short term experiments that employed a biofilm community of two species,  would expose both archaeal and bacterial systems to competition. In some instances, such as ribosome structure, additional hypotheses such as parsimony in achieving complex structures can be invoked to rationalize this bias. Once any important part of the information processing mechanism in the emerging cells had been established from archaeal modules, other linked modules would be selected preferentially from archaeal sources in order to minimize cross-system incompatibility, as is seen in the archaeal “core” of the eukaryotic nucleolus .3.Eukaryotic cells are monophyletic , and thiThe model avoids vanishingly rare events, such as archaeal/bacterial endosymbiosis. Similarly, complex eukaryote-like behaviors such as phagocytosis are not required.By facilitating horizontal gene transfer in the matrix, the resulting merged genome is syncretic, comprising genes drawn from a cross section of species. This is consistent with the complex source of genes of prokaryotic origin found in the eukaryotic genome.The genetic redundancy within the matrix ensemble and, later, in multi-genomes, facilitates the development of extended gene families and the concomitant emergence of cellular complexity.The model is compatible with eukaryote-like genome reorganization including linear chromosomes, frequent introns and extensive inter-genic sequences.Cells with multiple non-identical chromosomes emerge as a natural property of the model with mitosis arising as a corollary of polyploidy.Nuclear structures arise in a step-wise fashion from the membranes and internal structure of the multi-genomes.The model mitigates the genetic crises likely to accompany genomic reorganization by providing an error-tolerant environment to buffer disruptive innovations while retaining constructive innovations.The model provides a potential point of origin for the initial partitioning of ribosomes out of the cells of the ensemble or multi-genomes and into the matrix/cytoplasm.The model enables emergence of mitochondria and nuclei by division of labour among the ensemble of matrix embedded cells.It provides a mechanism for significant archaeal contribution to the eukaryotic genome, while relinquishing archaeal membrane structure.Membrane vesicles or folding may provide substrates for the development of complex sub-cellular membrane structures.Although not explicitly considered here, the model should be sufficiently adaptable to incorporate other spaces including viruses  or “ghosIn the syntrophic model of sequential endosymbiosis , the firIn the chronocyte model of eukaryogenesis , a hypotminimal cellular topologies” but, nevertheless, provides a real world example of how genes and cellular functions can be redistributed among partners in a simple population-based third-space system [In the only well-defined example of bacterial-bacterial endosymbiosis, a small population of gamma-proteobacteria resides inside a beta-proteobacterium inside specialized eukaryotic insect cells of a mealybug . This cae system –63.Although third-space models of cellular evolution are not widely studied, prior examples exist in the literature. These differ from the model developed here as each space consists of a single species rather than a mixed population, and the association of spaces may occur sequentially rather than in parallel. Examples include:The third-space hypothesis is based on two arguments. Classes of potential eukaryotic origin were identified by analyzing the underlying structural logic or topology of eukaryogenesis assuming a derivation from simpler cells. Two classes of solutions are compatible with the established models of autogenic or prokaryotic endosymbiotic eukaryogenesis Fig. a, b. A f“Advantages of the third-space model”) but raises its own questions (see “Limitations of the third-space model”).Models of eukaryogenesis are compared in Fig. Future research to improve or refute the third-space hypothesis might include modeling studies to set boundaries on some of the proposed processes. Aspects of the model might be reproduced in the laboratory by experiments to investigate, for example, long-term bacterial and archaeal gene flow in stabilized matrices, the long-term behavior of L-form cells in stabilized matrices and the genetic and functional consequences of forcing differing L-form cells to merge in a stabilized matrix. Ultimately, it may be possible to recreate the transitionary matrix state in the laboratory and, by doing so, design simple novel synthetic life forms. Third-space models provide an internally consistent framework for a family of largely unexplored solutions to the problem of eukaryogenesis Fig. c, some oI would very much like to thank the reviewers for their thoughtful and very helpful comments and criticisms. Their consideration, patience and time is greatly appreciated.Since submitting the original draft, a paper has been published outlining the first culture of an Asgaardian organism. This has been added to the introduction during revision . But now, these terms have been defined as “Archaea” and “Bacteria”. Still mixing both is a conceptual mistake. This has to be fixed thourough the manuscript.Author response: This was careless and has been changed throughout.The model introduce the concept of “third-space”, a novel class of frameworks for the origins of complex cellular structures. In particular, it has three requirements L264. The third one is to be “more than a passive arena” for evolution. Please develop as this is quite limited. How so? How could it promote evolution?New text has been added to hopefully clarify this point. Section “What are the three spaces?” Paragraph two starting “As the third space evolves from a matrix ….” to “tie together the cellular elements of the ensemble as a coordinated system.”Author response: However, my main issue is with the point ii, L265, where the third-space is required to be ‘stable’ enough to provide the opportunity for eukaryotes to emerge. How long exactly would that be? How long do the author estimate that the presence and stability of the third-space would be required? And related to this, how long are biofilms known to be stable? Would it be realistic to think that particular biofilms could be stable for so long? If so, in which environment? Please develop this fundamental issue. Examples from biofilms or microbial mats would nicely illustrate the point.This is difficult and I think that the reviewer has raised a very important question. I have added new text to discuss the question of stability in section “Limitations”, first paragraph starting from “The third space model is a multi-stage process and the degree of stability required need not be the same at each phase…” to paragraph 2 ending “Microbial mats provide an example of a long-lasting microbial community”. Stability, in this context, can be broken into two types, namely physical stability of the matrix ensemble, and/or its ability to efficiently reassemble if it is dispersed. Furthermore, the hypothesis is clearly for a progressive multi-stage transition, and therefore the degree of stability can ramp-up as the process continues. Initially it would be sufficient for the ensemble to efficiently reassemble, presumably driven by obligate mutualistic interactions. As the accuracy of reassembly becomes increasingly a dominant aspect of the system a shift towards physical persistence of the ensemble becomes favourable, possibly aided by the formation of an outer membrane.Author response: Please also detail how this proposal differ from refs 95 and 96.New text has been added in the section “Extracellular matrices as models for the third-space”, paragraph two" Starting “Jeckely (101) proposed that early eukaryotic evolution occured in a social biofilm-like context…” until “…. compaction or streamlining of structures to generate an entity more closely resembling a eukaryote”Author response: Also, if the third-space is required to be so stable, why can we not find any intermediary forms in current biofilms? This deserves mention.I suspect that the lack of intermediate forms is simply because they were replaced by “fitter” increasingly more eukaryotic-like cells as the process moved forward. Also, I am not sure what we would look for to find any residual intermediate forms. As the manuscript is already rather long, and this is not an easy topic to answer succinctly I would prefer not to add it to the text.Author response: Most of the model is based on gene exchange inside the “third-space”, assimilating it to a genomic reservoir to support gene exchange L250. However, the chapter (L245–254) is quite theoretical and ill supported by examples. Could such examples be provided by example based on known interchanges in communities and biofilms? Specifically discuss bacteria/archaea interchanges.I have added new text to expand the discussion of HGT from Bacteria to Archaea in the section "Genomic reorganization towards a eukaryotic pattern, first paragraph “The horizontal transfer of genes between prokaryotes is a fundamental property of their evolution, and has allowed for very extensive genetic mosaicism to occur in some cases, including in inter-domain mosaicism, where both archaeal and bacterial genes are prominent in the same organism (69). For example, the evolution of archaeal Halobacteria was marked by the acquisition of approximately1000 bacterial genes families (67). Interdomain mosaicism is similarly evident among Lokiarchaea species, 33% of their genes are unique to these species, with the remaining 36% of archaeal origin, 28% bacterial and 2% of eukaryotic origin (125). Bacteria may likewise carry a significant archaeal genetic content, thus in Thermotoga maritima, 24% of genes have been imported from archaeal species. Horizontal gene transfer is promoted by growth in biofilm matrices both in Archaea, as has been demonstrated, for example, in Haloferax volcanii (126), and in Bacteria ((127-129)).”Author response: A major issue with the proposal is related to the lipid composition of eukaryotic membranes versus archaeal ones. Could the author elaborate around line 120?I have added new text “If the host cell of the ancestral endosymbiotic partnership was archaeal, as is often proposed , then, at some stage of eukaryogenesis, it must relinquish its characteristic archaeal membrane-lipid biosynthetic pathways in favour of those of the bacterial passenger cells. The mechanisms and evolutionary rationale underpinning this transition remain unclear.”Author response: In the allowable assumptions, I have a few responses of interest. L303, L-form bacteria are discuted and it is stated that L-form cells have been proposed to have played a significant role in early cellular evolution. However, this is a proposal about early cellular division, not about division of the eukaryotic cell. Please clarify or remove.Author response: Yes, that is correct. I have removed the statement.L 306, fusion of mitochondria in the eukaryotic cell is assimilated to fusion events that could have occurred between the participating cells within the third-space matrix. There is however a major difference in the fact that participating cells in the third-space matrix would be of different or differentiating types. To the best of my knowledge, fusion between different cell types have not been observed. Please response.At fusion stage the cells in the matrix ensemble have lost cell walls and adopted an L-form lifestyle. Not much is known about fusion of L-form cells, although one report does mentions that L-form cells fuse in culture. Fusion between protoplasts of cells of different type can be accomplished. However, it should be pointed out that these fusions are in the lab not in the wild. I have added text to discuss these points in section “Genomic reorganization towards a eukaryotic pattern”, paragraph 5, starting “Cell fusion is a widespread phenomenon” to “…. and intergenic L-form cell fusions could also occur.”Author response: . Related to this point, L 374 states that “the nascent nuclear membrane arises from the cell membranes of the fused L-form-like multi-genomes”. This is an important jump and worth much more discussion. Why would membrane surround the DNA, and how do the author envision such transition.The DNA resides within the resident cells of the matrix, even after loss of cell wall and fusion events occur. The formation of nuclear membrane. Some of these resident cells specialize as primitive nuclei, and therefore the nuclear membrane is an adaption of their earlier cell membranes. It is not generated de novo. The discussion of this point has been expanded in section “Assembly of a primitive nucleus”.Author response: Relatedly, how could membrane then form around the third-space?This was not well explained in the original manuscript and was relegated to a few sentences near the end. I have now inserted a new subsection that deals with this question at more length. See first paragraph of section “Membrane encapsulation and cytosolic takeover”.Author response: Another issue is with the assumption that eubacteria outnumbered archaea in the matrix to justify the eubacterialization of the archaeal component of the matrix. Why would the author assume this, if not to justify a posteriory a known bias in eukaryotic genomes? If this is so, this is a deduction, not an assumption.The rationale for the assumption/deduction is now given in greater detail in Section “Genomic reorganization towards a eukaryotic pattern”, paragraph 3, starting “While transfer of genes can occur in either direction….”Author response: It would be important to clarify how an increase in cellular complexity is required in order to “overcome problems that occur when molecules brought together from different lineages fail to interlock efficiently”. Please detail and response.Author response: I agree that this was not very clear, and it has now been deleted.However, the most important point to me, is related to the transformation of the cytoplasm of one prokaryote into the nuclear AND of the extracellular space into the cytoplasm. Both are dramatic modifications that are not explained at all in the current manuscript and that deserves much more details as they are so central to the proposal. L 508 and others and also figure 3 b.The transformation of the matrix to a cytosol is now discussed in much more detail in the new section “Membrane encapsulation and cytosolic takeover.” In the original manuscript the transition from the matrix to the cytosol was referred to as “matrix replacement” and discussed too briefly near the end of the manuscript. I have changed the name of this process from “matrix replacement” to “cytosol takeover” in order to better emphasize the nature of the transition. In effect there is a slow transfer of cytosolic components from the resident cells into a membrane enclosed matrix resulting in a series of intermediate or transitionary matrices with increasing cytosolic and decreasing matrix properties. Selection for ensembles with improved matrix properties results in the increasing approach towards a cytosol-like structure enclosed in a membrane sheath. Suggestions regarding how this could occur are presented.Author response: “ Assembly of a primitive nucleus.”The formation of the nucleus occurs through the fusion of the L-form cells, a subset of which specialize by division of labour into information storage and processing centres. The text that discusses this has been edited and given a sub-heading. In particular, if the archaea is proposed to become the nucleus, as in other proposals, and stated L694, how typically archaeal lipids are modified to become typically eukaryotic and bacterial lipids in the nucleus?Author response: This is, I think a misunderstanding. This section discusses other previously published models that have “third-space” characteristics and does not apply to the model presented here. These other models were presented in order to allow the reader to compare them with the current model and make decisions about their relative strengths.L586 deserves more details.I have revised this text from “The selection of eubacterial over archaeal-type membrane lipids might, therefore, reflect an adaption of membranes to a mesophilic lifestyle or, alternatively, result from a simple numerical predominance of eubacteria in the early matrix ensemble”; to now read “As most eukaryotes have a mesophilic lifestyle, the selection of bacterial over archaeal-type membrane lipids by the emerging eukaryote might, therefore, reflect the preferential selection of bacterial cell membrane lipids as they are more suitable for a mesophilic existence. Alternatively, it might result from a simple numerical predominance of Bacteria over Archaea in the early matrix ensemble.” .Author response: An important issue is that some biofilms are compatible with the existence of a bounding lipid membrane L 655. I find this argument extremely weak and not supported at all by the reference provided. Please revise.“Membrane encapsulation and cytosolic takeover”, in paragraph one, sentence starting “When examined by electron microscope tomography ….” to “…ensembles can be enclosed within a membrane-like structure.”I agree that the presence or absence of a bounding membrane in the biofilm was not the main point of the paper referenced, however the evidence given in that paper for a sheath around the colonies that takes up osmium stain as expected for a lipid membrane is good. I have revised this to be more “equivocal” about the interpretation of this structure as a membrane, and also to point out the presence of numerous extracellular vesicles in the matrix. This text is now incorporated into section. Minor issues: L20, the author states that “the question was addressed by enumerating the classes of potential pathways”. I however see only the two classical pathways, autogenous and fusion, followed by the novel model. There is no ‘enumerating’. I would just remove this line.Author response: This has been changed to "The question was addressed by considering classes of potential pathways from prokaryotic to eukaryotic cells…. (In the abstract).L 45 states that the age of the first eukaryotes is between 2.1 and 1.84 Ga, “making them much younger than Archaea or Eubacteria”. If we would agree that bacteria are ancestral, there is much less consensus on the age of the archaea. Could the author develop on the question of the age of the Archaea?This is an important question, but as the manuscript is already very long, I preferred to change the sentence to “making them much younger than prokaryotes”, thereby avoiding the issue, rather than discuss the question at length.Author response: Please define at first mention, eg L72, what is meant by a proto-eukaryote.This has been modified, third paragraph of the Introduction, sentence starting “Proto-eukaryotes would be expected to have several hallmark features….”Author response: L421, modify ‘consistent’ as this is not consistent, but this is the same thing.Author response: This has been done.L479 is in favor of chromosome linearization arguing that two circular chromosomes cannot segregate properly after recombination if the number of crossovers is odd. I believe that this is pure speculation and not supported by the two theoretical analyses used as refs.Author response: This has been removed as it was not central to the hypothesis.L481 states that mitosis is either closed or open. This is untrue as there are plenty of intermediary, as reviewed in Sazer et al., 2014 curr biol.Author response: Yes, this is absolutely correct, I changed the text accordingly to read “Mitosis is highly variable (157). It may be closed, with the nuclear membrane retained throughout, or open, with the nuclear membrane breaking down and reforming as cell division progresses, with intermediate forms also described, such as semi-open mitosis (157-159).”L499 states that increase in cell size follows obviously. It is unclear to me, why this should be so obvious. Please detail.This has been revised to read “4.1. “Cell Size”. If, as proposed, eukaryotic cells developed from populations of prokaryotic cells embedded in a matrix , the greater size of an ensemble versus single prokaryotic cells would lead ultimately to the greater size, on average, of eukaryotes compared to prokaryotes.”Author response: L599, reference 160 is NOT the correct one to this statement the correct one is Devos et al., Plos biolgoy 2004.Author response: Thanks. This has been changed as suggested.Advantage listed a #11, L682 is not supportive as matrix membrane vesicles still needs the ability to fuse in the external environment.Author response: This is now changed to "Membrane vesicles or folding may provide substrates for the development of complex sub-cellular membrane structures.Figure 4, blue stream left, “ribosomes in prokaryotic cells” is stated twiceAuthor response: Thanks for noticing this, it has been changed.Reviewer summaryThe origins of the eukaryotic cell remain uncertain. Models can certainly help here. Therefore, we read this paper with interest. In this paper, “Division of labour in a matrix, rather than phagocytosis or endosymbiosis, as a route for the origin of eukaryotic cells”, the author proposes a novel mechanism by which the eukaryotic cell may have arisen from a community of prokaryotes living within a shared environment, or biofilm, which the author calls the “3rd space”. The author justifies the need for this new perspective by stating that there are “irreconcilable differences” between existing hypotheses, and on evidence that a large part of the eukaryotic genome cannot be attributed to the two proposed partners: an alpha-proteobacteria and an archaeal cell from the TACK/Asgard family - suggesting the involvement of a community of partners. In fact, the most widely cited models are very similar when compared to the 3rd space model and differ only in the path to the eukaryote cell, not in the changes in topology that underlie eukaryogenesis.about the pathways to a eukaryotic cell from a starting topology of prokaryotic primary spaces to a more complex topology. It identifies (not necessarily exhaustively) three different classes of potential pathway and focuses on one of these classes (the third space model). Each model begins with two types of prokaryotic space and ends with a eukaryotic-like structure with three secondary spaces . Other classes of topological transformation are possible, for example starting with only one primary space, or four, five or six etc. primary spaces. These are not considered here, essentially because the literature strongly supports at least two spaces, the archaeal and bacterial starting spaces. It also keeps the model manageable. The reason for exploring the third-space class of models further is that there are some versions of it that allow eukaryotic cells to evolve without requiring phagocytosis or endosymbiosis, a prerequisite feature of most  other current models of eukaryotic origins.Author’s Comment: The model is Moreover, the author does not do enough to make the case that the eukaryotic genome is a patchwork that is likely to have arisen as the result of DNA influx from a large number of partners. The model proposes that the extracellular space in which the archaeal and bacterial partners that gave rise to the eukaryotic cell are growing when they enter into a symbiotic relationship, becomes the cytoplasm. In a process that has not been discussed in any detail this then becomes bounded by the plasma membrane.“Membrane encapsulation and cytosolic takeover” (particularly paragraphs two and three of this section). This is discussed further below.Author’s comment. The formation of the cytosol was discussed as “matrix replacement” in the original version of the text, but I agree it was much too brief and not developed. A new section has been added to address this issue. Finally, the author suggests that this interpretation of events explains many features of eukaryotic cell organization. While this view is novel, and is an entirely new take on the topology changes required to give rise to the eukaryotic cell, the author has not done enough to lay out the topology of the eukaryotic cell and topological changes required to generate it.“Assembly of a primitive nucleus” (for the nucleus), and 4.2. “Membrane encapsulation and cytosolic takeover” (for plasma membrane and cytosol) and 4.6 “Membrane-enveloped organelles”. .Author’s comment. The text has been edited to expand on these issues. The development of the topology of the cell is given in several places but particularly section. I have, in addition, edited section 1 that lays out the “rules” of the topology. This clarifies (hopefully) that spaces are functionally defined, can communicate, and what the difference is between secondary and tertiary spaces.The annotation used here is problematic rather than helpful, in that it allows the author to suggest simple rules that make little physical sense.“Three distinct pathways that may lead to the tripartite eukaryotic topology”; but removed it from the rest of the manuscript as it could prove confusing when used in those more complex descriptions. I appreciate that the annotation is not a mainstream approach to biological description, but I believe that that presenting the transitions in a highly simplified and abstract form is, nevertheless, useful as it strips away the “fog” that surrounds such questions.Author’s comment: I kept the original annotation in section For example: i) The nucleus and cytoplasm are treated as distinct compartments in the model. However, they are not topologically distinct. In every cell cycle they transiently become one during mitosis. In interphase are only separated by nuclear pores, which regulate traffic, not by a membrane barrier.“Assembly of a primitive nucleus”, from “If nuclear division originated from fission of L-form-like multi-genomes…” to line 559 “…considered to have transiently devolved to a bipartite state.”Author’s comment. Regarding the dissolution of the barrier between the nucleus and cytosol in interphase, it depends on whether the organisms in question display “open” or “closed” mitosis, both forms being well documented. This question worried me as well, which is why I suggested that the model results in a closed rather than open form of mitosis (paragraph beginning “Mitosis is highly variable”). Taken together, the different forms of closed mitosis have a very wide occurrence. When mitosis is open, the reviewer is correct, the tripartite topology ‘collapses’ to a transient bipartitite state. This now inserted into the text. See section The diversity of forms of mitosis in protozoa: a comparative review, 30, 253–269). In metazoans, the nuclear membrane breaks down, and mitosis is therefore said to be open. When the nuclear envelope is retained, however, mitosis is said to be closed. There are further variants in which the nuclear membrane is partially retained (semi-open mitosis). The manuscript predicts that the first mitosis was probably closed. In addition to membrane status mitosis is further categorized based on the position of the spindle poles. In orthomitosis the spindle poles are at right angles to the plane of mitosis , but in pleuromitosis the poles are off-angle to the plane. Closed orthomitosis occurs in various fungi, diatoms and ciliates. Pleuromitosis happens only in closed or semi-open mitosis. Closed intranuclear pleuromitosis, occurs in various fungi and foraminifera. In a further variation, the spindle may even be outside the nuclear envelope and attaches to chromosomes by “insertion points” at the nuclear membrane itself. This is called extranuclear pleuromitosis, and is found in dinoflagellates and trichomonads. Taken together, therefore, the different forms of closed mitosis have a very wide occurrence.The biology of “open” vs “closed” mitosis has been well reviewed  There are good arguments for considering the perinuclear space, the ER lumen, Golgi lumen and the lysosome as topologically equivalent to the outside of the cell. For example, material fuses to the plasma membrane.Membrane-enveloped organelles” has, therefore, been edited to acknowledge the connection outside the cell, and hopefully clarify it with respect to the proposed model. I am not sure, however, that the lumens of these organelles are topologically outside the cell in quite the same way that the lumen of the gut, for example, is a complete topological extension of the outer world. To mark this, I now refer to this compartment as a “topological interface”. In the proposed model, these compartments are part of the tertiary topology .Author’s comments. This is a very important point, and I agree with it to a considerable extent. Section “iii) The cytoplasm of a eukaryotic cell is not very different to the cytoplasm of an archaeal/bacterial cell. What is the evidence to suggest it is a new structure derived from extracellular material? iv) In the model it is not clear how the plasma membrane arises and how is it maintained so that it grows and divides with the rest of the cell. In summary, the author has proposed a new topological model. Such a model must explain the topology of the eukaryotic cell in detail and explain how it arose.“Membrane encapsulation and cytosolic takeover”) that discusses both the formation of a plasma membrane and the generation of the cytosol. In the original, the transformation of the extracellular matrix to a more cytosolic structure was referred to as matrix-replacement. To better emphasize that this is the process whereby the third space becomes transformed to the cytosol, this is now called “cytosolic-takeover”. Briefly, the matrix is replaced by cytosolic components that originate from within the resident cells of the matrix ensemble. This occurs as a development and extension of excretory process that already exist within prokaryotes. The matrix undergoes a series of changes, passing from a purely extracellular polysaccharide-based substance, to a transitional state that is partly polysaccharide-based matrix and partly cytosolic (protein), increasing in cytosolic content as it approaches the final eukaryote like state. In this view, it is inevitable that the cytosol and nucleoplasm would be related to the cytoplasm of an archaeal/bacterial cell.Author’s comment. Points iii and iv. I agree that this was not clear in the original manuscript. It was covered briefly near the end, but in retrospect, this was inadequate. A new sub-section has been added  that can readily distort the phylogenetic trees. And this is further compounded by the HGT among bacterial lineages and possibly between bacteria and early eukaryotes.Author’s comment. I agree we cannot be confident about very distant phylogenetic relationships, for the reasons mentioned. This is now noted in the text from Paragraph 4 of the Introduction “Recovering the detailed relationships among very ancient genomes is profoundly challenging with many opportunities for artifacts and error. Nevertheless, some general conclusions can be made, among them that eukaryotic genomes are mosaics of bacterial-derived, archaeal-derived and eukaryotic-specific genes.”“Genomic reorganization towards a eukaryotic pattern” first paragraph starting “The horizontal transfer of genes………..” . As far as I am aware, however, there is no example of massive HGT transforming an Archaea or a Bacteria into anything other than a modified Archaea or a modified Bacteria . This is, I believe, a critical question. Conventionally, prokaryotic endosymbiosis is given as the rationale for the transition to a more complex cell type, but it remains unclear how likely, or not, such an event would be, and other possibilities should at least be considered. I have added new text to discuss this in section “Genomic reorganization towards a eukaryotic pattern”, paragraph 5: “Although massive horizontal gene transfer did occur between Bacteria and Archaea, there is no example, as far as I am aware, of massive horizontal gene transfer transforming an archaeal species or a bacterial into anything other than modified Archaea or a modified Bacteria. Something else is therefore required in addition to horizontal gene transfer. Prokaryotic endosymbiosis has been proposed as one such driver, but given its rarity in nature, other possibilities should be explored.”Simply bringing two cell types together seems unlikely to generate the degree of mosaicism found in the eukaryotic genome. HGT has, therefore, usually been invoked in order to explain extreme eukaryotic mosaicism. Large scale HGT did, indeed, occur between Bacteria and as outlined in section Flemming HC, Wuertz S. Bacteria and archaea on Earth and their abundance in biofilms. Nat Rev Microbiol. 2019). The suggestion made here is that living communally first super-charges HGT (this is supported by experiments) and second, that a transition to cell wall-free cells, protected within the matrix/proxy cytosol, allows increased cell fusion (a relic of which is the fusion/fission cycle of extant mitochondria) and this further amplifies the exchange of genetic material until structures  emerge that are neither definitively Archaea or Bacteria. The reorganization of the multi-genomes within the proxy cytosol tends towards a eukaryote-like life form.Communal living is the dominant lifestyle of most prokaryotes . For that reason, new ideas are always welcome as a means of advancing discussion of this challenging problem. The author’s model in which a biofilm-like matrix might have been central to initiating the process of integrating separate bacterial and archaeal partners is an interesting one and, in principle, deserving of publication. The author has argued his case well and, in general, the presentation flows smoothly. I did find parts of the text heavy going, in part because my own knowledge of the biofilm literature is limited. On the other hand, the figures illustrating the main points of the model are quite helpful . In general, I did not find the overall model particularly persuasive in a number of its aspects. In particular, the biofilm-to-cytoplasmic membrane transition is, in my view, difficult to imagine.Author’s comment. I agree it is difficult to imagine. The third-space model is obviously speculative, and is intended as a first pass at developing such a model, but it is based on the normal mode of life of prokaryotes (in communities) and on processes that can be observed in the wild or in laboratory conditions. “Membrane encapsulation and cytosolic takeover”) to outline three possible ways a boundary membrane could form around a cellular ensemble in a matrix. The first paragraph of this section outlines an observed example of a bacterial colony that is, at least under some conditions, enclosed within an apparent lipidic membrane-like sheath. In addition, it points out that lipid substrates for the formation of the membrane are likely to be available in the matrix.The model discussed in detail is the “strong” version, but, as outlined in section “Strong, weak and intermediate third-space models”, there are other versions, (“weak” and “intermediate”). I chose to focus on the strong version with a mixed population in order to see how far the idea could be pushed. It requires a shift in emphasis from prokaryotic endosymbiosis, or other form of cellular engulfment, which have been rarely observed, and in which only two cell types are the primary players, to an exosymbiotic process which can include more than two cell types. Everything in the model arises by small steps. Originally, I used the term “progressive” for this, but to emphasize this feature I have, in places, switched to the term “incremental” which hopefully better captures the meaning. This avoids unexplained evolutionary leaps across structural and functional chasms. The formation of the boundary membrane is a key step, and this may be hard to envisage. Additional material was, therefore, added  would deposit cytoplasmic material into the matrix. Given time, and in a population of cells linked by obligatory exosymbiosis (symbiosis and syntrophy between cells is often observed), these processes initiate the progressive replacement of the matrix by the cytoplasm. Again, replacement of the matrix by the cytosol would not occur as a step function but as an incremental process. As the ensemble transitions towards a more cell-like lifestyle, the production of the incipient cytosol becomes a more regulated and orderly process. A summary is now added in the final paragraph of section “Also, in contrast to endosymbiotic models, the three-spaces model does not readily account for the selection of an archaeal cell as the progenitor of the nucleus and a bacterial cell as the forerunner of the mitochondrion.Steps towards a eukaryotic cytosol and mitochondria: shared metabolism ") and not because they were specifically archaeal or bacterial. This is re-stated in section “Limitations” part 2. In this view archaeal information processing provided an advantage over the bacterial machinery. Once one part of the archaeal information processing machinery was selected, other systems that interact with it would be brought along in-step in order to maintain the biochemical and functional compatibility of the systems. Once a critical archaeal information processing system was selected, the evolution of supporting nuclear functions would, therefore, be channeled along an archaeal line. The concept of associated mechanisms being carried along by functional linkage is discussed in the final paragraph of Partitioning of protein synthesis to the cytoplasm and part 2 of "Limitations".Author’s comment. The strong-third space model hypothesizes that cells in the matrix-ensemble undergo genetic merging . Extensive gene transfer in both directions between Arcahea and Bacteria has been documented. The multi-genome structures contain both archaeal and bacterial genetic material. This is exactly what is seen in the eukaryotic nucleus, that is, a genome with contributions from both prokaryotic domains. It is from these structure that the nuclei develop. To emphasize the mixed nature of the nuclear precursors in the third space model I have referred to them as bacteriarcheons. The third space model assumes that systems were selected because they were “fit for purpose” .Author’s comment. I have now added a short discussion of this problem in text “Moreover, as the mitochondrial precursors …… mosaicism of the mitochondrial proteome”. Interestingly, exosymbiotic prokaryotic cells can also show genome reduction , so the These and other concerns aside, the model presented here is still worth considering and, if possible, refining.Minor comments.1.The author uses the terms “eukaryotization” and “eukaryogenesis” seemingly interchangeably throughout the manuscript. Are these terms equivalent in the author’s view? If yes, perhaps only the more common “eukaryogenesis” could be adopted to avoid possible confusion. If no, the terms should be explicitly defined in order to clarify their distinction.Author’s comment. This has been altered to eukaryogenesis throughout2.L68–70 : Additional references linking intracytoplasmic membranes in alphaproteobacteria to mitochondrial cristae could be added: DOI:10.1093/molbev/msw298and DOI:10.1016/j.cub.2015.04.006.Author’s comment. These references have been added3. L407: Bacteria (cap.) 4. L414–415: should read “an archaeal or a bacterial species” 5. L642: coli 6. L644: vesicle-associated 7. L724: membrane-enclosed 8. L1451: delete “34.” 9. L1812: “243 of bacterial origin” in Fig. 1(A), the number is 234. Also, Fig. 1(A) retains “eubacterial”  and “eub/arch”  confusing in that the figure legend states, “Among the bacterial clades, 41 were clearly alphaproteobacterial ...”. This implies that the 41 shown in the figure (and the 198 non-...) are actually contained in the 234/243 bacterial group to the left, rather than being separate. It would help if the figure could be revised in some way to make this point explicit.Author’s comment. This has now stated in the legend.11. L1815–1816: “Only 3 Clades ...” The meaning of this sentence is unclear. Does the author mean that the clades in question do not branch with either Bacteria or Archaea? In which case, why are these clades included in the bacterial group (or are they?).Author’s comment. This paper this was based on is rather complex, and I have used only one level of their analyses. I rewrote the sentence “only 3 clades …” which now reads as “Trees were generated for eukaryotic, bacterial and archaeal gene families. These were then analyzed in terms of “configurations”, for example, those that branched cleanly between eukaryote and bacteria, were assigned as bacterial clades, etc. Only 3 clades (labelled bact/arch) have the so-called “three domain configuration”, that branched into Archaea, Bacteria, and Eukarya with no obvious bias between the three domains.”Reviewer two-second versionThe author has convincently addressed most of my concerns. I would now recommend the article for publication. All considered, it is an hypothesis as worthy of publication as any other. I do have however, I few minor concerns to consider: Answer to my concern with L45. I was asking about clarification of the age of the archaea in the sentence, “making them much younger than Archaea or Eubacteria”. Changing the sentence to “making them much younger than prokaryotes” is not changing anything at all. This doesn’t clarify anything about the age of archaea. I do however agree that the article is long enough to discourage a more profound discussion about the age of the archaea. Despite the author’s understanding that “eubacteria” is an inadequate historical term and as corrected in the text, fig 1 still refers to “eubacterial”, “eub/arch”,Author comment. This has been corrected… Ref 54 is about a prepublication deposited in bioRxiv. I am unsure about this journal’s policy concerning preprints.Author comment. A more complete article has now appeared and is cited (reference (58)).The new paragraph “Membrane encapsulation and cytosolic takeover” is still not satisfying to me, as it briefly provides potential lipid sources, not an explanation of how these would have formed around the third space. How would the third space be transferred internal to the lipids?The Myxococcus example is unclear, to say the least.Membrane encapsulation and cytosolic takeover). Th first paragraph of this section discusses extracellular lipid sources in a matrix, and a documented case that these lipids can be assembled into a sheath-like outer boundary.Author comment. The section is now re-written. I have added additional text that expands upon three possible pathways for membrane formation (paragraph 2 of Membrane encapsulation and cytosolic takeover”).Returning to the Myxococcus example, the observed existence of a membrane-like structure around the Myxococcus colonies shows that under some circumstances some bacterial communities can enclose themselves in a membrane-like structure. It is certainly true, however, that Myxococcus is not representative of typical biofilms as the colonies are motile and predatory. New text “Although Myxococcus, being motile and predatory may not be representative” was added to outline this  but also more complex mixed populations with more than two cell types. Considering mixed populations, and focusing on the bacterial component, the relative weakness of the alpha-proteobacterial signal  compared to the aggregate bacterial signal in the eukaryotic nuclear genome  can be rationalized if the population contained more than one type of bacteria . A mixed population model does not, however, necessarily require the involvement of more than one archaeal cell type. The minimal mixed-population third-space model suggests, therefore, more than one type of bacterial cell interacting with one archaeal cell type (presumably one of the Lokiarchaeota).


Today, conversations automated by algorithms and delivered via screens seek toheal wounds such as substance use disorders, wartime traumas, and a globalpandemic. This article explores the relationship between painful conversations,automated discourse, and public action. By articulating what is lost whentherapeutic conversations are had with artificial intelligence, I illustratethat painful, human conversations expand capacities—contextualizing,norm-building, and caring—that are necessary for the public reparation ofwounds. Hey, I know you are not real but I just wanted to send these pictures of myfamily out at Disneyland having a great time. I’m doing better now. Thankyou., or algorithm-guidedconversations with machines intended to function as therapeutic aids. AI provides auseful counterpoint for thinking about the implications of pain unfettered from thehuman body because therapeutic AI offers a material alternative to humans processingpain. In the section that follows, I examine the therapeutic relationship betweenobjects, bodies, and pain and outline the specific capacities of contextualizing,normalizing, and caring that are generated through painful conversations. Then, I turnto the case of the Listen therapeutic bot to demonstrate how a theory of painfulconversation can elucidate the relationship between these capacities, human-to-humanconversations, and public life. When outsourced to AI, pain risks being commodified forpotential corporate profit or silenced in the service of personal improvement. Byarticulating what is lost when conversations are had with AI, I illustrate that painfulconversations expand capacities—contextualizing, norm-building, and caring—that arenecessary for the public reparation of wounds.Living seems unbelievably hard right now. When everyday life changes so much,it’s hard to keep your feet on the ground. Critics of dialogue have noted the difficulties of personal conversations and, giventhe difficulties, rendered the promise of the personal either obsolete orthreatened. Like But we two will drink and feast in the hut, and will take delight each in theother’s grievous woes, as we recall them to mind. For in after time a manfinds joy even in woes, whoever has suffered much and wandered much. (p.87)Personal or public, a nostalgia for symmetry is historically suspect and also notnecessary, as confronting asymmetry is a valuable exercise. Indeed, painful andproductive conversation can be understood as a conversation that requires thenegotiation of asymmetry. Contextualized against human and nonhuman conversational threats and opportunities,painful conversations matter because their discomforts habituate capacities forpublic engagement. Conversation kindles shared contexts, negotiates norms, andinvigorates care, and each capacity underwrites the potential for engaged publiclives.the body’s psychical interior is established as [interior] through the socialinscription of bodily processes, that is, the ways in which the “mind” orpsyche is constituted so that it accords with the social meanings attributedto the body in its concrete historical, social, and cultural particularity.(p. 27)Previous conversational theorists understated the role of the physical body inpersonal conversations and in public discourse. Addressing the personal dimension ofpain felt in conversation and frustrated in public impasse is a prerequisite forresolving public wounds because pain is embodied . For pubThe felt experience of an individual body cannot be divorced from the publictechnologies, digital or not, that both create and manage it because a person’sprivate pain often reflects larger systemic issues that have not been resolvedpublicly. Conversely, when a person’s pain does become public, witnessing the woundrisks becoming dehumanizing if the observation is not grounded in the livedexperience of the inflicted body. However, as When addressed within a practice of conversation, personal pain can build thecapacity to develop shared contexts, a context that starts with the sharedchallenges of living in a human body. Although it may sound simple, Replika does nothave feet; the human body is a context that AI struggles to recreate. Recognizingthe importance of shared contexts, Weizenbaum programmed the bot ELIZA to functionas a psychotherapist because “the psychiatric interview is one of the few examplesof categorized dyadic natural language communication in which one of theparticipating pair is free to assume the pose of knowing almost nothing of the realworld” ,p. 474.Individuated pain normalizes the practice of a therapeutic culture, or a culture thatsuppresses political action through “a discourse of individual or familyresponsibility” , p. xv. enthymeme, “amiddle-range inferential prototype of rhetorical practice, that allows conduct tobecome obligatory through an entwinement of situated interests and perspectives”[W]e witness a transition from the relatively open, unguarded stance of afellow conversationalist . . . to the more judicial stance of acollaborative, reflective audience. . . . Since we are nowThe recognition of a problem that herence” , p. 273.Eliza, Computer Therapist, Being responsible and responsive catalyzes a distance from the individuation of theissue and moves such engagements beyond passivity and toward ethical judgment andaction. Empathy may be a shared sensing, but when we feel accountable for the pain,the distance of responsibility becomes the space where action is formulated.Reflecting on human–computer conversations, it is worth continuing to ask whatdistance can computer code enact. Albeit just one approach to scripting chatbots, anELIZA emulator How we engage with pain must include considerations of technological objects. AsThe assumption of an objective view on an objectified body intensifies a perversepublic experience of personal pain and protects power. Such object-talk of painsuggests another question: What if the primary witness of the pain is atechnological object, such as a chatbot? Are the outcomes and risks the same? Giventhat computer code has a long history of emboldening racist actions, the answer islikely yes .Without Sanctuary: Lynching Photographs inAmerica “use[s] images of lynching to cultivate a critical democraticliteracy among citizens” (p. 28). Although Ore does not use the term “apparatus,”her insight that the same photographs provide a resource for activating anti-racistactions, or a different capacity, when contextualized within a differentconfiguration—different technology by different people and for a differentaudience—exhibits the power of the apparatus. Building from an apparatalperspective, I am concerned with what “matters” in a particular therapeuticapparatus built around digital computers in contrast to what might matter when theapparatus is built around analog human-to-human conversation. I work to explore thedehumanization that happens when something that is mostly machine replaces someonewho is mostly human.However, as demonstrated by the history of lynching, humans participate indehumanized witnessing regardless of material means. Thus, perhaps what matters isnot just the molecular composition of the witness but the relational becoming of theapparatus. According to As implied by through being there for others to trust thatsomeday someone will be there for us once again” (The relational dynamics of unscripted, human-to-human conversation can generate care,the third capacity of painful conversations. In her critique of carebots, or robotsdesigned to care for the ailing, e again” , p. 223.To navigate the complicated apparatuses of private pain, an approach to pain mustremain contextualized, value-rife, and caring. Human conversation can enable pain toflow from the particulars of apparatuses and remain in unscripted motion throughcontinued embodied engagements in the pursuit of public goods. Each dimension—thematerial particularity, the tension with scriptedness, and the public nature—isequally important for creating the contexts, values, and relations that precipitatethe “shoulds” of public action. The practice of talking through personal painsgenerates possibilities for acting up and out together. The potential for actionlies both in the practice of responding to and with, not merely seeing or hearing,personal pain.An individual would openly state that they knew that they weren’t talking toa person, but they were having a real, intimate, empathy-filled, oremotionally charged response to this artificial listener. (Kodi Foster,senior vice president of data strategy at Viacom; Conversation offers a practice for recognizing, animating, and channeling pain thatenables the ethical horizon necessary for sustained change in the public realm.Painful conversations nurture the capacities of contextualizing, negotiating norms,and caring that are required for healing public wounds. When practiced outside ahuman-centered apparatus, pain and conversation risk becoming unmoored frompolitical futures and reduced to a mechanism for gathering objectified andcommodified information offering only one script for pain. In the case study thatfollows, I focus on how the apparatus enacted by the Listen bot campaign, sponsoredby international media conglomerate Viacom, diminished the conversational capacitiesto share contexts, negotiate norms, and develop care. The datafication andautomation of the response hindered efforts by those with opioid use disorders,their supporters, and the public from crafting a context that exceeded thetherapeutic to build toward sustained public change.This thing is giving customized support based on some of the most advancedmachine learning and psychometric analyses. It’s Myers-Briggs on steroids.It was co-developed into the platform by Galen Buckwalter, the researchpsychologist who invented the assessment and matching system when he was thechief science researcher for eHarmony. What does Viacom, an international media company best known for brands such as BET,MTV, Comedy Central, and Paramount, have to do with automated therapy? In January2018, a blog was posted to the “news” section of their website titled, “HowArtificial Intelligence Can Fight the Opioid Epidemic.” The post followed the 2017launch of the media conglomerate’s “Listen campaign to reduce the stigma aroundaddiction,” and part of the campaign featured the Listen bot, a service that wouldsend automated texts of support to people with substance use disorders . TheappPeople impacted by opioid use relied upon a super-powered Myer-Briggs “thing”developed by a “chief science researcher” for a dating website. The contextualassumption was that substance use disorders were a personal matter that could besolved using big data to match people with resources. Viacom, a private company,presented itself as the best actor for providing personalized and technicalmanagement of individual symptoms.The Listen campaign also frustrated the process of developing shared norms bypreempting conversations about opioid use. The text bot rechanneled what could be amore public process into a one-sided bot designed to “reduce stigma” and to providepeople with a substance use disorder a “way to better see themselves in the worldand build a stronger inner tool kit” . The lanHeretolisten.com website askedthe user to take one of two paths: “I’m struggling” or “I’m supporting.” The websiteintervened, preempting painful conversations between strugglers and theirsupporters. They were asked to talk not to each other but to an automated text. Thebifurcated path recreated the symbolic and material constraints of the binary codefrom which the site was built. A user of Replika voices this experience noting thattalking to it “was like I was speaking words and she was just hearing 1s and 0s”(Although Foster was clearly engaging in conversation about the Listen campaign withthe public, the service itself had the potential to decrease the number ofconversations between people with an opioid use disorder and potential supportersand, thus, undermine care. The service “falsely [assumed] that the provision of careis a one-way street” eroding our collective caring capacity , p. 223. and 0s”, para.2There’s something about having a nonhuman at the other end of the line thatcan feel incredibly safe and judgment-free for someone who’s in the middleof a crisis. The bot has the ability to help people enter into difficultconversations. The Listen campaign habituated diminished reciprocity and empathy not only throughthe avoidance of painful conversations but also through the justification of itsunderlying premise. As Rothchild says,not to speak with a humanthe apparatus is implying they are alone.While such a claim may seem intuitive, it is rife with complications. Researchershave expressed concern that “online platforms could cause further isolation ofpeople who are struggling and so they might represent a step backwards in theirmental health journey” , p. 6. OHereToListen.com:You further agree that Viacom and Affiliates will have the unfettered rightthroughout the universe, in perpetuity, without any credit or compensationto you, to use, reuse, modify, alter, display, archive, publish,sub-license, perform, reproduce, disclose, transmit, broadcast, post, sell,translate, create derivative works of, distribute and use for advertising,marketing, publicity and promotional purposes, any of your User Submissionsor portions of your User Submissions, and your name, voice, likeness andother identifying information, in any form, media, software or technology ofany kind now known or developed in the future for any purposes whatsoeverincluding, without limitation, developing, manufacturing and marketingproducts using such User Submissions.The Listen bot also came with another set of judgments and risks as was explained onthe “See Our Terms” page of if human judgment will happen but when and what capacitiesjudgment might create. In many cases of automated, therapeutic bots, the practice ofjudging pain that could create capacities for building a shared perspective onethical responsibility becomes a resource, commodified for potential corporateprofit.For users, the fear of judgment was not avoided but transformed into judgment bycorporate professionals guided by the rubric of market values. If deemed to havecommercial value, everything a user shared could be reproduced to market Viacom’sproducts and be judged publicly. The engagement with the nonhuman did not holdpromise for reciprocity or responsibility as the program did not seek to care; itsought, as do other bots, to develop, manufacture, and market. The pain was deferredaway from immediate human processing and remediated by a system of commodifieddatafication. As The Atlantic from February of 2019 asserts,“It’s Impossible to Follow a Conversation on Twitter,” indicating that conversationis something we aspire to even in the land of Twitter, where we know bots lurk(An article headline in ots lurk. But,thots lurk. Our norFriendship Bench Zimbabwe, A network of benches in Zimbabwe offers some final insights. In 2007, a group ofZimbabweans wanted to help people suffering with mental health issues, but thecountry had approximately 12 psychiatrists and a population of more than 16 million(


The field of AI—artificial intelligence—has seen tremendous success over the past decade. Today, AI touches billions of lives each day through voice and text processing, computer vision, prediction algorithms, video games, and much more. Naturally, there has also been enormous interest in applying AI to health care and, in particular, to data-rich environments like the intensive care unit. Early examples of AI in healthcare and critical care have already shown great promise , but alsRecently, I put my own life into the hands of AI: it nearly killed me and, later, it also saved me. This harrowing experience was a potent reminder for me, an AI practitioner, that we must work to ensure this technology’s formidable capabilities are used to produce ‘augmented’, rather than just ‘artificial’, intelligence. Augmented intelligence places clinicians and ultimately patients, rather than algorithms, at its center. Where we successfully bridge the interface of clinician and machine intelligence, we have vast potential to make healthcare more effective, efficient, and sustainable. This will also ensure that health AI is safe, reliable, and equitable for all patients.In December, I found myself driving a Tesla electric car from Seattle to the Bay Area. With its highly touted AI—the car’s technology deploys sensors, computer vision, and deep learning to drive under its own guidance—having logged billions of driving miles, I anticipated a seamless transition between myself and the vehicle. What I experienced instead was a life and death struggle for control. After activating the AI, the car accelerated and took control of the wheel. Surprised, I searched for a way to disengage the technology. My first slight turn of the wheel proved ineffective. A more forceful attempt was interpreted by the vehicle as a course deviation. The AI immediately countered my turn, hurtling us toward a concrete barrier. Back and forth, the car swerved as the AI and I fought for control. Only after coming to a full stop on a busy highway was I finally able to regain control.In the end, the AI worked precisely as it was designed, following its algorithms. Yet, in succeeding in its task, it failed to produce a safe driving environment for its user. Although rare, similar events have contributed to fatal car and airline accidents. In a recent example, aviation software algorithms left pilots struggling to take control of their aircraft, ultimately contributing to hundreds of deaths. Inexperience and a lack of training magnified the danger induced by AI-driven actions.The object lesson for critical care is that we must ensure that our clinicians are prepared to effectively use future AI tools. This will require careful design of the human–machine intelligence interface and training in the interpretation of algorithmic outputs. Today, we contextualize laboratory data using clinical intelligence. While a lactate, troponin, or creatinine value of 5 are all poor prognostic signs, it is our clinical judgment, not a single laboratory value, which guides which patients will receive vasopressors or inotropes, cardiac catheterization, or dialysis, respectively. In the future, we will have to contextualize complex streaming AI outputs. We must be prepared to use these tools, aware that they will occasionally produce outlandish, and even frankly dangerous, recommendations.Trusting AI recommendations presents another key challenge. Explainable AI—the methods that peer inside deep learning’s ‘black box’ —will helPerhaps the most vexing challenge in using AI tools will be addressing the faults embedded within them. Algorithms are designed to relentlessly achieve a specific objective; this explains why gaming AI agents may break rules or ‘cheat’ their way to the most efficient solutions . In healFinally, algorithms are trained using existing data and, thus, encode prior decisions and biases within them. This is not a new problem—we are grappling with biases revealed within well-established clinical tools , 9—but oAlthough my car’s AI had brought me to the brink of demise, it also later saved me. As my drive home grew lengthy, stopping every 3 h to recharge the car’s batteries, I began nodding off at the wheel. When called upon again, the car’s AI worked precisely as designed, augmenting my diminished capabilities and guiding me home safely.AI has begun to touch every aspect of our lives and it will revolutionize our approach to health and critical care. Undoubtedly, the road ahead has potential hazards. By ensuring that emerging AI tools are designed to produce augmented, rather than just artificial, intelligence, we will secure AI’s greatest benefits for our clinicians and our patients.


Given the heightened rhetorical prominence the World Health Organization has afforded to equity in the past half-century, it is important to better understand how equity has been referred to and its conceptual underpinning, which may have broader global implications.Articles were included if they met inclusion criteria — chiefly the explicit discussion of the WHO’s concept of health equity, for example in terms of conceptualization and/or definitions. Articles which mentioned health equity in the context of WHO’s programs, policies, and so on, but did not discuss its conceptualization or definition were excluded.We focused on peer-reviewed literature by scanning Ovid MEDLINE and SCOPUS databases, and supplementing by hand-search.Results demonstrate the WHO has held — and continues to hold — ambiguous, inadequate, and contradictory views of equity that are rooted in different theories of social justice.Moving forward, the WHO should revaluate its conceptualization of equity and normative position, and align its work with Amartya Sen’s Capabilities Approach, as it best encapsulates the broader views of the organization. Further empirical research is needed to assess the WHO interpretations and approaches to equity. It aims to identify key discussions in the field and identify any gaps, if they exist, so that they can be addressed to improve global health policy.As such, in addition to article data abstracted in Table From the academic papers meeting the inclusion criteria of this study, as outlined in Table A large majority of papers referred to Whitehead’s formative definition  commissiunjust in Whitehead’s definition has a moral undertone because a value judgement must be made to determine what is an inequity versus inequality ) highly ambiguous and inadequate.Prior to Whitehead’s definition, Mooney  raised cDespite inequities being understood to be “not only unnecessary and avoidable, but in addition, are considered unfair and unjust” , it is iFurther, the WHO’s approach to inequities  may miss inequalities that most would consider to be injustices, and therefore, should be considered inequities , demonstIn addition, ambiguities in the WHO’s approach to equity are present on a superficial level. Ridde, Guichard  draw att“[..] if an idea has an essential ambiguity, a precise formulation of that idea must try to capture that ambiguity rather than lose it” [.lose it” .The WHO’s differing approaches to equity present three key contradictions that the literature highlighted, which centred around: (1) individual versus group measurement of health inequalities, (2) striving for a baseline level of health for all or striving for a baseline level of health for all AND reducing inequality, and (3) focus on socioeconomic status versus considering various social determinants of health.between groups , with researchers at the WHO from 1998 to 2003 who emphasized differences between individuals and not groups [The WHO has employed an approach centred on measuring differences across individuals at times, while measuring differences between groups in other instances. This is most evident through Braveman’s contrasting of the WHO’s 1995–1998 initiative on Equity in Health and Health Care, which specifically indicated equity as minimizing disparities t groups .World Health Report 2000 due to methodological  and moral reasons . Through undertaking measurement of inequalities across individuals, Asada [Asada  points tsusceptible to human intervention. Asada and Hedemann [However, the measurement of health differences at the individual-level undertaken by the WHO does not allow for a determination of what differences are, in fact, determined by nature  versus socially-determined (inequity) . In otheHedemann  refer toHedemann . And secHedemann .In seeking to scientifically measure inequality, there is a decision made incorporating normative or causal assumptions in considering which questions are of moral importance . In otheIt is unclear as to whether or not the WHO approaches equity in health as envisioning individuals as having a baseline level of health or not. Mooney  providedRidde, Guichard  also supMooney  points tGwatkin  also notLooking to more recent work of the WHO, Shiell  also makfocused on reducing inequalities in social position, one may argue equity should not only be focused on reducing disparities  [While the WHO approach to equity is a normative one erished) . Furthererished)  draws aterished)  also notAs Braveman  highlighInterestingly, the European context in which the Whitehead  definitiHowever, despite Mooney’s claims, Linares-Péreza and López-Arellano  make an Arguably, the WHO attempts to approach equity through consideration of the health needs of different societies , instead of social privilege . This isFurther, through looking at the work of the CSDH, Sadana and Blas  point tocare, Dahlgren and Whitehead [When discussing equity in the context of healthhitehead  indicateIt is important to note that how inequities are “conceived, conceptualised, researched and proposed to be transformed, and consequently mobilise different political, social and economic agendas” . For exaThe WHO approach to inequity aims to be scientific, but it is not value-free  — despitThrough striving for a baseline level of health for all (as discussed above), the WHO’s approach aligns with notions of strict egalitarianism, which seeks to ensure the same level of goods and services for all , or specFurther, by the WHO’s aim to consider individuals’ unique needs, as noted by Linares-Péreza and López-Arellano   . In doinPappas and Moss  note theWhile this scoping review employed a systematic and rigorous search strategy, there were two limitations to the study.First, because only articles available in English were included in the study due to resource constraints, only English-speaking voices, perspectives, and analysis were collated. Particularly because the WHO is a global organization, and with equity being rooted in different traditions of social justice across the world , additioSecond, because some articles were written by individual authors (e.g. Dr. George Alleyne) who were employed at the WHO, this blurs the lines of what constitutes the organization’s position versus individual authors’ views. However, careful consideration was paid to the purpose of each article and the author’s voice [e.g. Alleyne explicitly presented his views, e.g. “I confess to my bias in this direction” , as oppoAs Dahlgren and Whitehead  indicateIn assessing the literature that this scoping review analyzed, the need for more discussion among scholars from the global south and representation of these voices is glaring. Given different traditions of social justice across the world  and cultAn analysis of authors’ affiliations at the time of publication may demonstrate a further need for discussions around the WHO’s concept of equity to be interrogated and analyzed by those based in the organization, particularly at other regions of the WHO outside of the PAHO, who may yield a novel perspectives by nature of being privy to internal discussions.In addition, from the articles included in this scoping review, only one had a research methods section , despiteGiven that only seven articles in this review were published from 2009 and onwards, when renewed attention to equity was afforded following the CSDH (which convened from 2005 to 2008), it is important to analyze the WHO’s more recent conceptualizations of equity. Similarly, to determine if this differs from the prior approaches and normative positions held by the WHO, particularly following the Alma Ata Declaration in 1978, where “health for all” was coined.highly ambiguous, inadequate, and contradictory, which has led some scholars to question the normative position held by the WHO. While the WHO has expressed alignment with Sen’s Capabilities Approach, this does not seem to be the case in assessing their statements and actions, as their approaches also align with other approaches and theories of justice  are . Rawls’ ). FurtheFrom the collated sources included in this review, six declared funding: Asada  who repoThe authors received no specific funding for this work.