Evolution in biological and non-biological systems: the origins of life.

Isaac Salazar-Ciudad

Grup de Genòmica, bioinformàtica i evolució. Departament de Genètica i Microbiologia, Universitat

Autònoma de Barcelona.

Isaac.salazar@uab.cat

Tel: +345812730

Fax: +345812387

Developmental Biology Program, Institute of Biotechnology, University of Helsinki, PO Box 56, FIN-00014,

Helsinki, Finland.

Keywords: Origins of life, generative systems, compartment-first, evolution, development

A replicator is simply something that makes copies of itself. There are hypothetical replicators (for example

self-catalyzing chemical cycle) that are suspected to be unable to exhibit heritable variation. Variation in any

of their constituent molecules would not lead them to produce offspring with those new variant molecules.

Copying, such as in DNA replication or in xerox machines, allows to re-make any sequence and then

sequence variations are inherited. This distinction has been used against non-RNA-world hypotheses:

without RNA replication systems have no capacity to exhibit heritable variation. However, is copying the

only way to have heritable variation? This article suggests that evolution can happen without anything being

copied and that, in fact, RNA copying is too complex to arise spontaneously as the first pre-biotic systems.

There would then be a historical gradation of systems from non-alive to alive in which the capacity to have

heritable changes would progressively increase and in which copying, as in template-based RNA-replication

would be a crucial but relatively late event.

In addition, this article proposes a way by which RNA replication could have arisen, after the arising of

systems able to evolve, as a crucial innovation and how that innovation spread over existing evolutionary

systems and became a major driver of subsequent evolution.

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Main text:

There are a large diversity and disparity of hypotheses about the origins of life. There is no consensus

definition of life or about how life should be recognized. There is, however, a number of features that are

found in all known living beings and are used to describe the last universal common ancestor of those

(LUCA). These include, for example, a nucleic acid based inheritance, nucleic acid copying based on nucleic

acid complementarity, nucleic acid coding for proteins, ribosomes made of RNA and protein, protein

enzymes, NTPs as energy currency, a lipid membrane, and the existence of a nearly universal genetic code to

name a few.

Hypotheses about the OOL often disagree on the order of appearance of those LUCA features and on

the nature of the intermediate stages in the historical path to life-as-we-know-it (LAWKI). In LAWKI, and

presumably in LUCA, all these features are highly imbricate and can not exist in isolation. An obvious

challenge in the study of OOL is to conceive how some of those basic features could exist without the others.

Each hypothesis can be described as a set of intermediate stages and a set of explanations for the transition

between those stages. Those can be integrated, or most often associated, within groups of hypotheses that are

similar or more or less coherent with each other. According to Luisi (Luisi, 2007) the most widespread set of

hypotheses, or at least the ones having the largest share of OOL scientists, can be classified under the

compartment first (Oparin 1938; Segré et al., 2001; Hunding et al., 2006), the RNA-first (Crick, 1968; Orgel,

1968; Woese, 1968) and the more heterogeneous metabolism first (Muller, 1985; Deamer 1997; Huber and

Wächterhäuser, 1998; Smith and Morowitz, 2004) hypotheses. Metabolism and compartment-first

hypotheses propose that self-organizing principles pre-existed hereditary variation by mutations, i.e. template

replication. The RNA-first hypotheses, on the contrary, propose that copying arose before any compartment

or metabolism. This article proposes some outline of how early compartments and metabolisms may have

been able to have some heritable variation, and thus evolve, to reach a level of complexity and variability

from which RNA-replication could arise.

A common terminology that could refer to the multiple, if not most, pre-life stages proposed from the

different sets of hypotheses, could be desirable to cross-compare their coherence and consistence. One of the

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aims of this article is to introduce such a framework. This framework would also be used to review some

analogies with LAWKI implicit in these hypotheses. By doing so this article suggests how some terms

commonly used in biology need to be generalized to consider situations that do not normally occur in

LAWKI but that occurred, according to some hypotheses, in the OOL.

This theoretical framework was originally designed to study evolution in non-biological and biological

systems (such as natural chemical, cultural and artificial systems; Salazar-Ciudad, 2008) and thus this article

could be described as an application of that framework to the OOL. Since evolution before or around the

OOL probably occurred in systems that were not as those in LAWKI, it may be possible to learn something

about them by comparing existing systems that evolve but are not alive (or at least benefit from a common

set of concepts to deal with those diverse systems).

This article starts by critically reviewing some aspects of the three main sets of hypotheses about the

OOL (sections 1 to 3), this review is not extensive, it only focus in those aspects of those hypotheses that

may be based on strong analogies with LAWKI. Then a new set of concepts are introduced in some detail

(sections 4 and 5) and compared with existing ones (sections 6) and examples (section 7).The rest of the

article applies these concepts to the OOL.

1. The RNA world

Perhaps the most popular hypothesis about the OOL, at least in genetics textbooks, is the so called RNA

world hypothesis. In its original form (Woese, 1968) it simply proposed that the ancestors of LUCA used

RNA instead of DNA as the material bases of inheritance (as it occurs in some extant viruses). A stronger

version of this hypothesis (Gilbert, 1986) proposes that life started as naked RNA molecules with enzymatic

capacity leading, minimally, to RNA replication. More recent versions also consider some kind of

compartimentalization, although the main focus remains in RNA (Robertson and Joyce, 2012) . This

hypothesis is popular in spite of popular and well grounded criticisms. Just to cite a few, there is the extreme

unlikelihood (effectively 0) by which a ribozyme long and complex enough to be capable to produce RNA

replication could form from random molecular events (Luisi, 2007). In fact, most arguments for the RNA-

world stress that replication allows to have heritable variation and thus it is clear that to have RNA

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replication is advantageous. That some thing is advantageous does not imply that it will arise in evolution

and, in fact, the weakness of this hypothesis is precisely that RNA-replication seems a molecularly too

complex process to arise spontaneously. As it will be explained this dichotomy is representative of a general

binary discrepancy among those evolutionary biologists favouring explanations based on what is adaptive,

the neo-Darwinian school, and those that also consider what is likely to arise (by genetic changes and

development in biology and by random molecular events in the OOL). Two other related critiques, relevant

in the context of this article, are based on a strong, and in some views misleading, analogy with LAWKI or,

more precisely, with how evolution is understood in the classical neo-Darwinian paradigm.

First, self-replication is an unnecessarily precise process for early evolution. Evolution can occur,

although less efficiently, even in systems that produce offspring that does not resemble them closely. RNA-

world hypotheses do not presume that RNA replication was very efficient early on but they do presume that

offspring was produced by copying. Thus, even if the replication process itself may not have been very

efficient there is a simple relationship between genotype and phenotype (an identity relationship) and thus by

copying it is ensured that offspring and parents resemble each other closely (except for error). In LAWKI

parents and offspring have a much lower level of similarity than in the RNA-world but evolution still occurs.

The process of reproduction, in asexual organisms, is sometimes conceptualized as copying, specially in

gene-view approaches, in which the offspring is seen as a copy of the parent (plus environmental effects).

Indeed part of the offspring, what we can call the genotype, is a copy of the parent genotype but the rest of

the phenotype is not produced by copying, it is produced from a cell (a zygote or equivalent vegetative

structure) with a complex spatial structure through a complex process of development. In that sense it is

possible to say that the genotype is copied while the phenotype is re-made in each generation from the

information present in the egg cell and genotype (or other reproductive structures like gemma).

In here it is important to introduce a distinction that is crucial for the rest of this article: copying and

re-making. In re-making, a system, for example a machine or a metabolic cycle, works in such a way that it

can make new systems that resemble it: its offspring. However, those systems are not designed, in the case of

machines, to make copies of themselves, they are simply designed to make new systems with a specific set

of characteristics. Thus, for example, if the parent system (phenotype A) makes a mistake in the production

of the offspring system so that it becomes different (phenotype B), then one would expect two possible

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outcomes: either phenotype B is malfunctioning and can not make any offspring system or phenotype B is

functional but it produces offspring with phenotype A since the system works in such a way that only A

phenotyes are possible. That B leads to B is not, in principle, something to be expected in re-making but in

copying. In copying, such as in DNA replication or in xerox machines, there is an interaction between what

is copied (commonly called the template) and the offspring production process in such a way that, if the

parent changes then the offspring changes in the same way. Notice that both in DNA replication and in xerox

machines only some changes in the parent are transmitted to offspring. In DNA, for example, base changes

from adenine to iosine (a base present in tRNA) can not be copied, in xerox machines paper chemical

composition or 3D structure can not be copied. A similar distinction has been introduced before (Varela and

Maturana, 1974; Zachar and Szathmary, 2010).

In LAWKI reproduction involves both copying and re-making. Offspring's genotype is produced by

copying parents' genotypes (and some mixing if sex is involved) while offspring's phenotypes are produced

by re-making: first the egg cell (or equivalent vegetative structures) by the parent's germinal cells and second

the adult phenotype from that cell through a process of development. In the simplest cases re-making simply

involves the syntheses of normal cell components and cell splitting. This process is regulated by gene

products but does not involve the copying of any cell component per se, the cell (other than the DNA) is

simply made again and again in each cell division.

Even in LAWKI, in which reproduction involves the copying of part of the parent's phenotype (its

genotype), evolution is possible even when there is few resemblance between parents and offspring. Even

with perfect DNA copying the resemblance between parent and offspring in LAWKI tends to be much

smaller than the one expected if both the phenotype and the genotype would be RNA molecules. In sexual

metazoan like humans, for example, parent and offspring tend to resemble each other in much less than 50%.

Each offspring receives 50% of their nuclear DNA from its father and 50% from its mother. In addition to

environmental effects that reduce heritability there is dominance between alleles in a locus and different

epistatic relationship that can arise between different combinations of mother and father alleles. In fact,

heritabilities of traits known to evolve over generations can be as low as 11% (Houle, 1992). A more gradual

and likely hypotheses about the OOL should be based on mechanisms of inheritance that could be less

accurate than in the RNA-world, but require less molecular complexity, and thus are more likely to arise by

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chance, than self-replicating ribozymes.

A second relevant criticism to the RNA world is also related to strong analogies with LAWKI as

understood from the neo-Darwnian paradigm. In the hypothetical RNA-world, RNA molecules would

compete for available resources, in the simplest and most ideal case, for freely available NTPs. If those RNA

molecules are not isolated in some way from each other (for example by micelles, liposomes, geographicallly

or by some environmental boundaries) the most likely outcome would always be that one single sequence

replaces the rest, a “loneliness of the fittest” scenario. This is the sequence that most efficiently gets

replicated. That happens to be a very simple sequence (Fontana and Buss, 1994). Thus, a simple selection

criterion on a simple phenotype inevitably leads to a simple evolutionary dead-end were complexity can not

but decrease. This is not an interesting scenario if one is concerned with the increase in complexity assumed

to be required for the OOL.

A different scenario precluding this loneliness of the fittest would be that RNA replication is equally

maximally efficient for a larger number of sequences. In that case a large number of different RNA

sequences could be maintained. For evolution by natural selection to be possible it is then required that those

RNAs are separated in sets and that then some of those sets manage to gather the right metabolites more

efficiently than the rest in a composition specific manner (otherwise there would be no fitness differences

between the sets and no evolution by natural selection). This again implies the pre-existence of

compartments, at least to separate the sets of RNA molecules to allow natural selection to act between them.

Consistently with that argument, recent RNA-worldists also consider compartimentalization (Robertson and

Joyce, 2012).

The selective dead end that is expectable from a pure RNA-world is not usually considered in the

discussion about the RNA-world. This may be related to the specific way in which the process of evolution is

conceptualized in the neo-Darwinian paradigm. Evolution is sometimes described, reduced, or defined as

changes in gene frequencies in populations over generations (Dobzhansky, 1937). Over time the direction of

change is due to mutation, natural selection, drift and other minor forces. At least in current organisms,

however, natural selection acts on the phenotype and not directly on the genotype. At the level of the DNA

sequence the kind of changes that are possible are relatively well known and it is assumed that, overall, all

sequences are roughly equally likely to change. Since heritable phenotypic variation is mostly due to genetic

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variation it is implicitly assumed that all aspects of the phenotype are equally variable and variable in all

directions (just like in DNA) (Salazar-Ciudad, 2006) or, in other words, that there is additive genetic

variation for most traits.

Classically it is assumed that, since phenotypic variation is, in principle, possible in all directions it is

natural selection that is determining the phenotypic direction of evolutionary change. There is plenty of work

explaining why this classical view is inadequate for the study phenotypic evolution (Alberch, 1982;

Goodwin, 1994; Salazar-Ciudad, 2006). It may also be inadequate for the OOL because in its sequel, the

RNA world, it is also expected that random change at the level of sequences is somehow possible in all

directions. This kind of evolution in a mostly RNA world, however, would simply stagnate into a single and

simple highly replicative sequence (as shown in simulations by Fontana and Buss 1994). This is because

selection is a destructive force, it only seems creative by pruning, in some specific direction, existing

phenotypic variation in each generation. The critical factor is then not only selection but also the exact

phenotypic nature of variation: the "mutational" changes. Variation is not possible for all trait combinations

but it has some intrinsic direction of change, or structure, that depends, among other things, on the

mechanisms of development that produce specific phenotypic variation from genetic variation, in the case of

LAWKI (Salazar-Ciudad and Jernvall, 2005), and on pre-life biochemistry and biophysics, in the case of the

OOL.

In the case of the RNA-world the same changes that are seen in LAWKI (at least according to the neo-

Darwinian paradigm) are implicitly assumed to occur. In this case, however, there is nothing yet on which

the RNA could act to make a phenotype do something (other than the RNA molecule itself). In other words

there is barely a functional domain (Moreno and Ruiz-Mirazo, 2009). So either one can produce a really

large range of metabolisms only on the bases of RNA molecules (far beyond the range observed

experimentally so far (Monnard, 2007; Chen et al., 2007)) or one is forced to consider that the earliest

molecular players were not, or not only, RNAs. This does not mean that RNA replication was not a crucial

step in the history of life, it simply was not the first.

2. Compartments-first:

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The compartment first hypotheses have a long tradition (Oparin 1938; Fox, 1972; Segré et al., 2001;

Hunding et al., 2006). Compartment-first hypotheses are the composome approach or lipid-world (Segré et

al., 2001; Hunding et al., 2006) or the related protein-first hypotheses based on the formation of “vesicles”

made of protein-like molecules (Fox, 1972). In here, for simplicity, we will talk about vesicles to loosely

include micelles, liposomes or microspheres, although there are important differences between them (Luisi,

2007). This set of hypotheses propose, in line with previous ones (Oparin, 1938), that early life arose in the

form of amphiphilic micelles or liposomes that were able to differentially incorporate specific molecules

from the environment according to the vesicles' composition. During this process vesicles grow and, after

attaining some critical mass, split (probabilistically and depending on the environment) giving rise to two

offspring vesicles (thus reproduction would be by re-making). There is a large body of experimental work

(Deamer, 1997; Weber, 2005, Luisi 2007) suggesting that the formation and splitting of vesicles is a

relatively likely process in the presence of water and amphiphilic molecules.

Some authors (Hunding et al., 2006) argue that some of those vesicles, what they call composomes

would have membrane domains that would be able to incorporate, in a manner that would be specific of the

set of molecules in each domain, other molecules from the environment and lead to composome growth and

division after a threshold size. This self-organizing capacity would ensure that after composome division

some of the offspring would be able to roughly reconstitute the parent's molecular composition by

differential molecular incorporation from the environment. Other molecules may be able to promote their

own incorporation. If those are sometimes present in the environment they can be fortuitously incorporated

in the composome (Hunding et al., 2006). This changes would then be maintained and lead to inheritance.

Thus, globally there is the chance for effective statistical reproduction and inheritance of some variation (in

what has been called compositional inheritance; Shenhav et al., 2005) and populational growth in some

lineages. Reproduction could also be accomplished through simple specific chemical synthesis of the

amphiphilic molecules from environmentally available precursors leading to vesicle growth and splitting

(Fellerman and Solé 2007; Luisi 2007; Fernando and Rowe, 2007; Zhu and Szostak, 2009)

This hypothesis has the advantage that those vesicles could in principle easily arise from early earth

geochemistry (Deamer, 1997; Luisi 2007) and that it proposes a simple mechanism of inheritance with low

heritabilities. It proposes a mechanism of reproduction that is much simpler than replication and thus much

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more likely to arise from random molecular events than RNA replication. In either case the range of

reactions, and the range of natural catalysts, that can lead to those amphiphilic molecules (for example lipids

and amphiphilic polypeptides) is much larger than the range of reactions and catalysts that can lead to

nucleic acids and, specially, to their replication.

3. Metabolism first hypotheses:

The previous sets of hypotheses are based on analogies (or alleged homology) with extant genetics (the RNA

world) or extant cell biology (the compartment hypotheses). There is a third set of hypotheses in itself quite

diverse, that directly focuses on metabolisms: on analogies with extant metabolic diversity. In broad terms

those hypotheses focus on the question of the origins of the energy in early life (Deamer, 1997; Lane et al.,

2010), on the questions of the origins of the chemical constituents of life (Smith and Morowitz, 2004) and on

their chemical order (Kauffman, 1993; Huber and Wächterhäuser, 1998). In general this set of hypotheses is

complementary to the other two sets in the sense that they tend to focus on bioenergetics and chemical

composition rather than on the mechanisms of variation and inheritance. The RNA-world hypothesis is also a

metabolic hypothesis but based only on the reactive capacity of RNA. The compartment-first hypothesis is

also concerned with metabolism but in the context of the compartment itself.

4. Generative systems:

Evolution is defined, in here, as changes in a lineage over generations. A lineage is a temporal sequential

chain of systems in which one system generates the next. In here the first system is the parent system and the

next is its offspring system. Thus, "changes in a lineage over time" means that the structure, or phenotype, of

those systems changes across generations. "Generates" means in this context that this system, called in here a

generative system, is physically independent from the system it generates and is, at least, causally

responsible for the characteristics of the offspring system that the latter requires to be a generative system.

Thus, this definition encapsulates the recursivity implicit in lineages and thus in evolution: a generative

system is a system that can generate another generative system. This definition is, however, delimited by the

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requirement of physical independence and intergenerational causality.

Physical independence means in this article that once the offspring system is formed, changes in the

parent system will not necessarily lead to changes in the offspring system. Physical independence is simply

required to distinguish between evolution and development and what is part of a generative system and what

is not. Thus, which parts belong to a generative system is established using the criterion of physical

independence: changes in parts that are physically dependent are defined as development and changes in

parts that are physically independent are defined as evolution (if those parts belong to the same lineage).

Causally responsible simply means in the context of this article (not in general since there are many

different definitions of causality referring to different things) that some of the characteristics of the offspring

generative system are due to some characteristics in the parent generative system. This implies, in here, that

variation in those characteristics in the parent should lead to a range of specific changes in the offspring.

These characteristics should include those that allow the offspring system to generate its offspring (those

characteristics will be called, in here, the kernel). These characteristics, however, do not need to be the same

in every generation process. There is nothing in the definition of generative system specifying that: parent's

and offspring's kernel do not need to resemble each other, it is simply required that one is causally

responsible for the other. The generation process may involve the transmission of information between

generations as in the DNA in LAWKI (or RNA in some viruses) or re-making (as in the case of epigenetic

inheritance in LAWKI; Jablonka and Lamb, 2005, 2006) or as in composomes (Hunding et al., 2006) or

others. This does not imply that each generative system can only have a single parent and, in fact, lineages

can branch backwards giving rise to a reticulate phylogeny. Notice that to understand this concept properly

one needs to consider that information in here is understood as organization or structure and it does not

imply the existence of a code, as in the genetic code. Thus, those uncomfortable with that use of the word

information could replaced it by the word organization (Salazar-Ciudad, 2008), structure or pattern (simply a

spatio-temporal distribution of items, like cells or molecules, to which one can arbitrarily assign types, like

liver cells or actin molecule) in the rest of this article.

The concept generative system applies even to completely hypothetical systems that exemplify, as

extreme cases, what is a generative system (precisely because of being totally imaginary and unconstrained

by LAWKI). An intelligent robot able to build other intelligent robots would be a generative system if it

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builds them in such a way that these can also build robots able to build robots, and so forth. This does not

imply that there is any information transmitted between generations: each robot could learn by itself from

experiments or other sources how to build the new generation. In this exotic example there would be change

in the phenotypes (or structure) of the robots over generations. Each individual phenotype will be caused by

that of its parents, although the mechanisms by which those are generated, and the phenotypes generated,

may change from generation to generation. Obviously there is nothing granting that there would always be

an actual production of offspring (the generation process may fail or undesired by some robots). In fact, thus,

the categorization of a system as a generative system should always be regarded as a hypothesis for a given

environment. This is just like in LAWKI: nothing warranties the survival and reproduction of any individual

living being.

The definition of evolution provided in here is in plain contrast with many definitions used in

biology since it does not imply or require nucleic acids, copying or even the conservation or similarity

between generations. There is a pervasive perception that in evolution something has to be conserved, or

passed through generations, and that at the same time something has to change in that thing that is being

passed from generation to generation. Thus, there is a long-held contradiction in which evolution is defined

as both change and conservation. Then which is the minimal degree of conservation required to consider that

there is evolution? In here it is suggested that what needs to be conserved is the fact of having a lineage (and

thus of being a generative system). Thus, the apparent contradiction of other definitions of evolution can be

solved if one defines evolution solely on the bases of change and uses the concept of lineage introduced in

here to define in what is this change considered.

The biological validity of this seemingly heterodox definition can be easily understood by tracing

forward the evolutionary process. Currently there are a set of characteristics that are shared by all living

beings and there is always a large number of things that are conserved in each instance of generation in any

living being lineage. In principle, however, biological evolution could lead, in a distant future, to the change

of those characteristics in such a way that there would not be a single characteristic shared by all the

descendants of current living organisms. This is analogous to the common evolutionary situation in which

some species in a taxonomic group lose the characteristics that were originally used to define that taxonomic

group (Ghiselin 2005: 91); like annelids that are not segmented or chordates that have no notochord. So there

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is no foreseeable reason by which future evolution in current living beings should always lead to something

being conserved between generations (eventually even between two generations). Thus, it is change in a

lineage that is , or should be, used to define evolution, not change and conservation. In fact, from the

definition of generative systems, the last characteristic that could be lost in the descendants of all living

beings would be, precisely, the characteristic of being a generative system (after that there would not be,

accordingly, evolution).

5. Evolutionary systems:

An evolutionary system is a generative system in a lineage that can evolve. This would depend on the

environment in which this generative system is found (e.g. even if a living being, like a bacteria, is likely to

be a generative system on earth it is unlikely to be so if placed in the core of a star) and thus hypotheses

about evolutionary systems imply a generative system plus an environment. Notice that evolutionary systems

can lead to started-ended evolutionary histories and there is nothing (and there should not be) in the

definition of evolution precluding that. Thus, tumor cells in their original body can be considered as

independent evolutionary systems within their environment (the host body) that simply lead to relatively

short started-ended evolutionary histories (Salazar-Ciudad, 2008; Godfrey-Smith, 2009) that end with the

death of the host and the tumoral cells. This allows to scientifically study all instances of evolution without

being initially constrained by their relative temporal length or their outcome being us or other familiar

species.

The environment can affect the evolution of a generative system in a number of ways. For the purposes

of this article only natural selection and mutations will be considered. Mutations are defined in the present

framework as any change in the structure of a generative system that is due to the environment. Notice that

this definition is different from the standard one in genetics; it includes it but also considers changes in

systems without copying and changes that will not be inherited (like in phenotypic plasticity). These can

either lead to changes in the phenotype of the offspring, or lead to no phenotypic changes.

A closed mutation is a change that causes that all the successive offspring is inevitably changed (in

respect to how the phenotypes in that lineage would have been without that mutation). Mutations in which

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that does not happen are called, in here, open mutations. An open mutation could be, for example, an

environmental change that transforms a vesicle of some type (for example of type A) into one of another type

(type B) but in which the type B vesicle can generate only vesicles of the A type. In closed mutations B

would produce B. Intuitively unless a generative system has a rather peculiar functioning the default

expectation is that most mutations would be open and only systems such as DNA copying would allow a

large number of closed mutations. Even in DNA, in fact, most mutations are open mutations: unless they

change one DNA base into another. To explain the concept of closed mutations a biological example of

closed mutation would be used:

In most animal egg cells there is a spatial asymmetry in the distribution of specific long-lived

mRNAs or proteins. Those are strictly required for the early spatial partition of the embryo body into parts

(for example to differentiate the anterior from the posterior part of the body). An environmental change that

would eliminate one of those mRNAs would likely lead to (as in many experiments) to an altered adult

phenotype. That mutation would be considered as closed if it leads to a viable and divergent adult and if that

adult produces some egg cells that specifically lack that same mRNA or protein in the same location. For

multicellular organisms there is a long chain of causal molecular interactions between an egg cell and the egg

cells in the next generation so that kind of change seem extremely unlikely. For unicellular organisms there

are examples of those kinds of mutations (see section 7).

6. Generative systems versus replicators and reproducers:

A very popular concept in the study of the origins of life, especially from the RNA-world school of thought

and other gene-centred approaches to evolutionary biology, is that of replicators (Dawkins, 1976; Hull, 1988;

Sterelny et al. 1996). A replicator is simply something that leads to something resembling it. The concept of

generative system is different from the concept of replicators in three fundamental ways:

First, the concept of generative system does not require that parent and offspring resemble each other

(thus it is more general).

Second, the concept of generative systems only considers systems that can actually evolve. This is

not the case for replicators since non-informational replicators are classified as not being able to evolve

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(Zachar and Szathmary, 2010) because they can not pass to offspring any kind of acquired changes (in their

terms there is no heredity). This leads to these authors to discard, as a principle, that evolution, and the OOL,

could happen without copyng. The requirement of causality in the definition of generative system implies

that every generative system (in a given environment) needs to have variation in the form of closed

mutations. This is because causality is defined on the bases of specific variation in the parents leading to

specific variation in the offspring kernel. In that sense those systems are not directly relevant to evolution.

However, the concept of generative systems and closed mutations conceptually considers the possibility that

some cross-catalytic metabolic cycles or networks (or other systems discussed in here) could have some

closed mutations and thus, without being actual informational replicators (there is no copying), could evolve

(and eventually lead to an increase in complexity or variation that would allow the arising of copying).

In the view presented in here, the problem with the replicators concept comes from a strong analogy

with LAWKI that leads to define evolution on the bases of copying and natural selection, an not on the bases

of change in a lineage on which selection may or may not act. That perception is reinforced by the fact that

copying is the most efficient known system of inheritance at the molecular level (often it is also the only

considered in spite of others being known; Jablonka and Lamb, 2005).

Third, in the case of cyclic metabolic networks with closed mutations and in general for “A leads to

B, B leads to A” lineages the concept of replicators is a bit difficult to materially specify. If A leads to B and

B to A, is it each pair of A and B molecules that is “replicating” itself? But then each A and B molecule is

indistinguishable so those pairs are not always replicating themselves as such (or it is unclear which is

replicating which). This problem is not present in the concept of generative system (Salazar-Ciudad, 2008) or

in accounts by Godfrey-Smith (Godfrey-Smith, 2009): A is a generative system and so is B and each leads to

a specific offspring system (B in the first case and A in the second). This is possible precisely because

parent's and offspring do not need to be similar (thus again the concept of generative systems being more

general and independent from the analogies borrow from LAWKI).

The concept of generative systems is vaguely reminiscent of that of reproducers (Griesemer, 2000)

and to Godfrey-Smith discussion on which entities can evolve (Godfrey-Smith, 2009), although reproducers

are not defined as precisely as generative systems and include things that are not included in generative

systems (and do not include things included in generative systems). A reproducer is a unit of multiplication,

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hereditary variation and development in which a parent generates a offspring that is able to develop to

generate its own offspring (similarly to a generative system as in here). Generation in reproducers is not

defined on the bases of physical independence, kernel information and causality. Godfrey-Smith does not

consider the former two things but does consider causality between generations similarly than in here.

Material overlap is not required for generation in generative systems but it is required in reproducers

(although not in Godfrey-Smith work). This is probably due to the concept of reproducers being designed to

deal with LAWKI (where there is always a material overlap between parents and offspring) while generative

systems being designed to deal also with non-biological systems.

7. Examples of closed mutations:

There is extensive empirical evidence of epigenetic inheritance in LAWKI (Jablonka and Lamb, 2005, 2006)

and in some cases these may allow that some of their variation is also inherited. This possibility has not been

systematically studied and again it is quite likely that only some changes are closed mutations (this may

depend in complex ways in the chemical nature of the systems and changes involved).

The most compelling empirical examples of non-genetic closed mutations are found in the

morphology of ciliates (Sonneborn, 1964; Frankel, 2008). Experimental micro-surgical manipulations, no

genetic change involved, leading to major morphological changes (such as: increases in the number of ciliary

rows, changes in the polarity of ciliary rows or mouth duplications) have been shown to be inherited to

offspring. This is, for example, that a two-mouth cell will lead to a two-mouth cell offspring. Those changes

can even occur naturally and lead to viable long-lasting lineages with, for example, two mouths. Thus, there

are closed mutations and, potentially, a path to evolution without genetic changes. Those examples occur,

however, in highly sophisticated cells. Simpler examples can be found in prions.

Prions are infectious agents consisting of a single misfolded protein that leads to the misfolding of

other existing copies of the protein. Each prion protein nicely fits the definition of generative system. The

correctly folded protein is normally expressed in sane individuals. Either spontaneously (Parchi et al., 2011)

or through ingestion of misfolded proteins, correctly folded proteins get in contact with misfolded proteins.

This leads to the transformation of the correctly folded forms into the misfolded forms. Strictly speaking

15

only the misfolded forms are generative systems since they are the ones that lead to other misfolded proteins

(properly folded proteins do nothing in that sense). This is enough according to the definition because there

are several misfolded forms that can spontaneously change into each other by rare environmental changes

(closed mutations), and those specifically generate themselves by getting in contact with correctly folded

proteins. Notice that there is no genetic change involved and that prions causally generate other prions and

can exhibit closed mutations. In this way, prions have been empirically shown to evolve (Li et al., 2010).

Although these are clear proofs of concept they are unlikely to be directly relevant for the origins of life: life

did not arise from prions.

There are some possible examples of closed mutations in the systems proposed by the compartment-

first hypotheses. The incorporation, from the environment, of molecules that happen to be able to self-

enhance their incorporation (or synthesis) in a composome would be, de facto, closed mutations that would

be retained by that composome's lineage. There are mathematical models (Shenhav et al, 2005; Vasas et

al.,2010) that try to explore the possible evolutionary dynamics and the actual capacity to produce reliable

inheritance of composomes relative to copying systems, but those mathematical models pre-specify, ad hoc,

the range of possible reactions and thus are uninformative about the actual range of possible chemical

reactions and about the likelihood of those kinds of inheritances. Other mathematical models (Fernando and

Rowe, 2007), however, suggest that in similar systems where there is no differential incorporation but an

intra-vesicular metabolic network, vesicles can evolve to become more complex through a process in which

there is inheritance without copying through “avalanches”.

In even more abstract terms similar examples of closed mutations have been proposed by King

(King, 1980) and Wächterhäuser (Wächterhäuser, 1994). According to King metabolic cycles could fuse

leading to substraction, addition or simple juxtaposition of metabolic networks. Wächterhäuser proposes that

catalytic cycles can grow in complexity in a heritable manner if new molecules, arising either from the

environment or by mutations in existing ones, are able to change the cycle in such a way that its production

is catalyzed (as in avalanches). In these cases there is no single mechanisms allowing a whole class of closed

mutations (as in RNA replication) but each of those would occur rarely and by its own mechanism that could

be highly specific of each metabolic network.

16

8. Three kinds of generative systems in LAWKI:

In the cells of LAWKI there are at least three kinds of generative systems that are currently highly

interdependent. These three kinds of generative systems would be similar to the kind of OOL systems

proposed by the three main sets of hypotheses described above. The suggestion that those three sets of

systems may have been slightly more independent originally or that their relationships were different in the

past has been put forward before in different forms (Dyson, 1985; Bedau and Cleland, 2010; Luisi, 2007).

Some authors (Gánti, 2003) relate the origin of life precisely to the coming together of those three kinds of

systems. However, as it will be explained, to consider each of those as different kinds of generative systems

and to include the concept of closed mutations may facilitate the study and understanding of such early

systems.

Although most inheritance comes from cells' DNA, DNA requires, at least the two other systems: the

cell and its metabolism. Thus, even if cells' proteins (coded by the DNA) can reproduce the cell structure

they can do it only from an initial condition that is itself a cell (Varela and Maturana, 1974). Thus, every time

there is reproduction both the DNA and the cell structure are inherited (the DNA being copied and the cell

being re-made) although most closed mutations will always be in the DNA. Allegedly the structure of the

cell in the offspring is affected by the genes in the offspring but that again provided that the parent cell had a

given epigenetic structure to start with. The epigenetic structure is understood in here as the actual molecules

and their spatial distribution and associations. Thus, there is in LAWKI a cyclic interdependence between

genetic and epigenetic information but none of them can be said to be fully explainable from, or reducible to,

the other (Griffiths and Gray, 1997; Oyama, 2000). Notice that information is generally understood in here as

organization not as RNA or DNA sequence.

In this article the epigenetic system is separated into cell structure (roughly the compartments and the

cytoskeleton and its bacterial homologue) and metabolism. Most metabolic reactions in the cell are catalyzed

by specific genetically-encoded enzymes but in absence of precursor metabolites nothing will arise from

those enzymes. In fact, it is not even that any given cell can reconstruct all, or even most, its metabolism on

the basis of a single metabolite and genetically encoded enzymes. As it has been recently shown by

metabolic scope analysis (Ebenhoh et al., 2004) and by genomic analysis (Kun et al., 2008) in all organisms

17

there is always some molecules that can not be synthesized de novo nor can they be incorporated from food.

They have to be present in at least one copy in the parents' cellular material contribution (like in the gametes

or gemma). An intuitive example of that would be ADP. This implies that there is a continuous causal chain

of reactions going back (and branching/converging back) from the metabolism in each current living

organism and some metabolism in early life or before.

9. Individuality and fusion in vesicles:

If life comes from those three partially independent generative systems the questions are: which of

them arose first? How did they arise? How were they related to each other originally? And how did their

relationship evolve into the one found in LAWKI? The question of how each of those generative systems

arose has been described in the original proposal of each of those hypotheses. As described (Hunding et al.,

2006; Luisi, 2007) vesicle-like entities are relatively likely structures in early earth geochemistry that may, in

some cases, directly classify as generative systems. Metabolic generative systems could originate

independently or after, and within, vesicles while nucleic acid copying, as it will be suggested, may have

appeared later within vesicles. In addition, since those early vesicles allegedly arose spontaneously and

relatively easily (Hunding et al., 2006; Luisi, 2007) from purely abiotic matter (mostly water and some

anphiphilic molecules) it can be expected that they arose in large numbers (at least locally, globally their

abundance may have been insignificant). In fact, large numbers of vesicles can arise in multiple conditions

by mixing water and different amphiphilic molecules (Fox, 1972; Luisi, 2006).

It has been proposed that in early life (Norris and Raine, 1998; Hunding et al., 2006; Jalasvuori and

Bamford, 2008) individuals could fuse with relative ease and quite promiscuously. This could have three

consequences for the evolution of vesicles. This would produce a constant recombination of the different

kinds of vesicles that could exist at a given time. If fusion occurs less often than growth and fission, then a

large variability in vesicles is allowed, and that should facilitate the capacity to adapt by natural selection.

Fusion among vesicles may also imply that from early on there were predation/symbiosis relationship

between pre-life generative systems (and globally some kind of ecology). Some vesicles have been suggested

to be able to grow and reproduce due the differential inclusion of specific molecules from the environment

18

and divide by fission after reaching a range of thresholds sizes (Norris and Raine, 1998). The promiscuous

fusion scenario implies that vesicles may also grow by fusion with other vesicles in a process reminiscent of

predation. The composition of those vesicles may differentially affect the range and frequencies of vesicles

with which a given vesicle could fuse, just like in the composome hypothesis but at the level of whole

vesicles, as suggested by Norris and Raine (Norris and Raine, 1998). Natural selection in this context simply

implies that vesicle that are more able to grow (by precursor incorporation or vesicle fusion) and undertake

fission in a given environment become more abundant over time. Notice that at this early stage predation,

simbiosis and sex would be difficult to differentiate.

10. The invention of the genotype:

According to the views just presented evolution started without copying and thus without nucleic acids.

Thus, for that hypothesis to be tenable an explanation for the arising of nucleic acid replication is required. In

addition to being stored in RNA, single NTP molecules are universally used as energy currency in LAWKI.

This suggests that this energetic function may be rather old. To explain why RNA replication may have

arisen in vesicular generative systems I will first assume that NTPs could be acquired in some vesicles that

had some sort of metabolism (it is not even required that NTPs were frequent). Some short (very few bases

long) oligonucleotides arising by spontaneous polymerization could become involved in catalyzing some

reactions. They could also affect the catalytic activity or specificity of some of the catalysts in a vesicle, a

more likely scenario given the relatively modest catalytic capacity of short oligonucleotides compared to

other biomolecules such as oligopeptides (Monnard, 2007; Chen et al., 2007). This association could, for

example, simply thermally stabilize the catalyst in a larger range of temperatures (to slightly different

extents, depending on the sequence of the oligonucleotide) as it has been suggested on the bases of chemical

experiments (Ehrensvärd, 1962). They could do that simply by binding, possibly non-covalently, to the

catalysts existing in a vesicle (as it is often the case in LAWKI).

Even if those effects may have been, and could have been expected to be, rather modest they

immediately impose a small but important advantage if those oligonucleotides can roughly replicate. Very

short (even 2 or 3 bases) oligonucleotides could replicate by simple spontaneous ligation of NTPs brought

19

together by specific complementary base pairing to the original oligonucleotide. The crucial advantage of

oligonucleotides would be that some of the chemical changes altering any of those oligonucleotides (for

example changes of one of the four bases into another one) are themselves heritable (so the variation is also

inherited: closed mutations). This is because the above mentioned way of replication could be expected to

work roughly as efficiently for any oligonucleotide of a given (small) size. Changes, by mutation, in those

oligonucleotides would directly increase the molecular diversity in a vesicle in a heritable manner. This way

the system could accumulate functional genetic heritable information without requiring a specific mechanism

to replicate long RNA sequences.

Over time those aspects of a metabolism that are affectable by those oligonucleotides could evolve

more easily thus leading to a genetic drive (Salazar-Ciudad, 2008). In other words, in those lineages in which

complexity has increased (for example in the lineages allegedly leading to LAWKI) it can be expected that

this increase in complexity (for example an increase in the number of different metabolites) would occur, in

larger and larger proportions through time, on those parts of the phenotype that can be affected by the

genotype.

Nucleic acid copying may be the simplest (that means requiring less molecular complexity and then

more likely to arise by chance) way to produce a systematic increase in the number of closed mutations from

a re-making system able to evolve slowly by closed mutations. RNA replication may have arisen simply

because it was the simplest copying mechanism from the molecules available in those early systems. It could

also be that there were other, yet unidentified, ways of copying leading to an increase in closed mutations (as

suggested in Egholm et al., 1993). Those were simply not found by early generative systems (then the use of

nucleic acid copying in LAWKI would be a simple frozen accident) or were found and later became extinct.

11. The origins of viruses:

The copying of oligonucleotides and the later arising of mechanisms to copy larger and larger

oligonucleotides leads to the origin of the third kind of generative system (after compartments and

metabolism). This is in fact an evolutionary system enclosed in an environment that is itself an evolutionary

system. To the extent that the mechanisms controlling the fission of a vesicle and the copying of its

20

oligonucleotides may not have been originally coupled, there can be an evolutionary conflict between the

two generative systems. All available NTPs could go to oligonucleotide copying. In those cases, there would

be a short process of evolution, within a vesicle, leading to the overabundance of the oligonucleotide

sequences that are most rapidly copied (and the concomitant end of the vesicle's capacity to re-make).

In other vesicles a way to find a compromise between the vesicles' evolutionary interests and the

oligonucleotides' evolutionary interests could arise. It could be, for example, that the oligonucleotides bound

to the molecules they affect, for example polypeptides, are more likely to replicate or have longer half-lifes

(for example because there is some inespecific degradation of the unbound oligonucleotides as in LAWKI).

Then, since the number of those molecules could be regulated by the vesicle's metabolism oligonucleotide

growth in number would be coupled with vesicle growth and no conflict arises.

The possibility that early vesicles fused rather promiscuously has an important consequence in here.

Even if the arising of oligonucleotides affecting metabolism could be seen as not especially likely, the

possibility of fusion implies that, once they appeared, this advantage could easily spread through large

populations of vesicles and through geographical space. In other words, copying nucleic acids could spread

over earth's populations not just by the classical Darwinian evolutionary replacement of lineages but by a

process that is more analogous to the spreading of a disease. In that context, the oligonucleotides that would

be more able to make copies of themselves in a wide range of vesicle hosts would be more likely to remain

over generations. Other attributes, like promoting fusion, dispersion or being stable in a larger range of hosts

and environmental conditions would also be selected positively (just like in current viruses). In that sense the

co-evolution between viruses and cells could be seen as being as old as life itself (cells being some kind of

symbiosis between some kind of virus and come kind of composome).

There is a recurrent debate in the study of OOL on whether the OOL is a unique and very unlikely event

or, on the contrary, something likely or even inevitable (Raup and Valentine, 1983, Kauffman, 1993). The

hypothesis presented here suggests a mixed possibility in which early steps would be rather likely, for

example vesicle formation, and would occur multiple times independently (with fusion and then a multiple

OOL). Later stages, like the acquisition of RNA molecules and rudimentary copying, would be much less

likely. Once those unlikely stages are reached, however, they would confer a larger capacity for heritable

variation and thus would be more likely to persist over generations. In addition, the “infective” capacity of

21

oligonucleotides (by vesicle fusion) may have promoted that the vesicles with oligonucleotides become the

dominant form of life. The key is, in here, the link between those two kinds of stages. The first ones are

likely and set the conditions for the second ones to arise and the second ones allow the first ones to remain

over generations in evolution (instead of appearing and disappearing over time).

Acknowledgements:

I thank Miquel Marín Riera, Miguel Brun Usan, Kepa Ruiz-Mirazo and Irepan Salvador Martinez for useful

comments. This work was supported by the Ramón y Cajal program (RYC-2007-00149) from the spanish

ministry of science and innovation and by the Finnish Academy.

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