Origins of Life and Evolution of the Biosphere 28: 583596, 1998.
Kluwer Academic Publishers. Printed in the Netherlands.
584 J. CHELA-FLORES

Table I
Evolution of chromosome complexity from macromolecules to prokaryotes

prokaryotes Phormidium frigidum and Lyngbya martensiana (cyanobacteria), but also eukaryotic diatoms (algal protists) are present in these ecosystems. Biological paleoindicators in sediments accummulated in these lakes include cyanobacterial filament sheaths (prokaryotic), as well as algal cells and protozoan cysts (eukaryotic). Hence, it would be important to discuss within the context of a robotic mission how to distinguish amongst the possible constituents of a putative Europan biota. The main objective of the present work is to make a preliminary suggestion for a feasible experiment.

The accelerating evolutionary tempo observed in the Late Proterozoic and Early Phanerozoic Eons may have had a counterpart in corresponding changes in the genomes of microorganisms. Additional arguments in favour of a candidate for such a counterpart are provided, namely, chromosome plasticity. Together with evolution in cellular morphology, there is corresponding evolution in structure, organization, and genetic regulation of the DNA in the nucleoid of prokaryotes. The simplest chromosomes correspond to those of archaea and eubacteria (cf., Table 1). However, maximum complexity is only reached with the first appearance of the eukaryotic chromosome (EC in the Early Proterozoic Eon (Knoll, 1994), some 2.51.6 billion years before the present (Gyr BP). Some clarification should be added regarding the concept of 'complexity' referred to in Table 1: After singlecelled microorganisms have gone beyond prokaryoticity the genome increases in complexity, as exemplified by the requirement of gene dosage in eukaryotes, a property which is illustrated by the gene silencing of female mammals, or in arthropods (the mealy bug, cf., Brown, 1966). Such gene control is almost totally absent in prokaryotes. The word complexity is used only in this sense. The consideration of the evolution from the prokaryotic chromosome (PC) to the EC is forced upon us when we look closer at properties of the contemporary genome of the living cell. The PC is a doublestranded DNA structure usually lacking abundant packaging proteins (histones), an enveloping membrane, and various specialized regions.

SEARCH FOR EXTRATERRESTRIAL EUKARYOTES 585

Table II
Histonelike proteins, which are typical features of the eukaryotic chromosomes, also occur in
some bacterial nucleoids

Amongst characteristic regions missing in the prokaryotes we should recall: • Firstly, nuclear organelles, which are associated with the site of ribosomal RNAcoding genes (nucleoli). Although it should be observed that nucleoli are also missing in some eukaryotes, such as Giardia. )
• Secondly, the prokaryotic chromosome ends are not formed by highly repeated sequences (telomeres), which are characteristic of the eukaryotic cell.
• A third typically eukaryotic feature missing in the prokaryotes are regions, or segments, of the chromosomes in which shutdown inhibition of gene expression (gene silencing) is absent. On the other hand, PCs may consist of a beaded structure, not unlike that of the EC (Griffith, 1976). Even histonelike proteins are known in some bacteria, as shown in Table 11 (archaebacteria will be considered separately in Table V).
The difference between PCs and ECs from the point of view of chromosome size does not help much in distinguishing between these microorganisms either, as PCs are normally smaller than ECs, but some primitive eukaryotes are known to have very small chromosomes too. One example is provided by the rhodophyte Cyanidioschyzon. This seaweed has a genome of only 8 million (M) base pairs (bp) (Suzuki et al, 1992). This tiny genome is only about twice as long as the genome of E, coli. In Table III we compare the genomes of singlecell organisms, metazoans and metaphytes. The problem of eukaryogenesis is rendered still more difficult to define, as the EC has some characteristics which are not common to all eukaryotes. Some possible exceptions are particularly remarkable in lower eukaryotes, such as algal protists: in these cases we may be faced with chromosomes, which seem to lack histories. This is illustrated in Table IV: On the other hand, there may be some further confusion between lower eukaryotes and archaea (besides the examples in Table 11), as there are Some cases of Archaea that are known to have histories, in striking similarity with Eukarya, as we proceed to illustrate on Table V. The case of M. jannaschii is particularly important, as it suggests that Archaea and Eukarya share a deep common evolutionary trajectory, independent of the lineage of Bacteria. Molecular analysis of ribosomal RNA in a variety of organisms,

586 J. CHELA-FLORES

Table III
Genome size is a poor indicator of an earlyevolving eukaryote

Table IV
Some eukaryotes may lack histones

Table V
Some archaea that are known to have histones

SEARCH FOR EXTRATERRESTRIAL EUKARYOTES 587

together with the molecular clock hypothesis (GogartenBoekels et al., 1994), suggest that life emerged early in Earth's history, even before the end of the heavy bombardment period. Arguments based on the formation of bioorganics in interstellar dust also point towards a precocious start of life on Earth (Greenberg and Krueger, 1996). In those remote times life may have already colonized extreme habitats, allowing at least two prokaryotic species to survive the large impacts that were capable of nearly boiling over an ocean. It seems reasonable to conjecture that in a brief timespan life may evolve given the right conditions, such as the environment postulated for Europa (cf., Sec. 1).

The most likely cause for the evolution of complex chromosome structure seems to be regulation of gene expression, a process which has reached its maximum expression in eukaryotes (Maynard-Smith, 1993). In this context, for a considerable time now, it has been evident that the integration of proteins complexed with DNA ('chromatin') has played a fundamental role in the regulation of gene expression (Littau et al., 1965). Some chromatin replicates its DNA late in the S phase of the cell cycle (Lima-De-Faria and Jaworska, 1968); it is also darkstaining, due to a very specific physical property, its high degree of DNA packaging. In order to differentiate such a special state of chromatin from its, less dense counterpart (euchromatin'), we refer to chromatin in the highly packed state as 'heterochromatin'. This is a well established concept in genetics, but for completeness we review some standard definitions that are needed in the present work. It is convenient to introduce the concept of a dense form of chromatin, which could be due to its specific DNA sequence. One such instance of chromatin contains highly repetitive DNA, which is associated with heterochromatization. A closely related state of chromatin, which is the result of regulation rather than structure, is sometimes found in a higher state of DNA packaging. Such chromatin is referred to as 'facultative heterochromatin'. We reserve the term 'constitutive heterochromatin' to chromatin that remains condensed through interphase. These detinitions will contribute to a better appreciation of the proposal in Sec. 6.

4. 1, POSSIBLE DEGREE OF EVOLUTION OF LIFE IN THE SOLAR SYSTEM

Having defined the problem of eukaryogenesis an important question to address below is how can we subject to experiment the existence of eukaryotes outside the bounds of the evolutionary pathway that has taken place on Earth. Prior to discussing eukayogenesis, in the first place it should be discussed how the different positions are possible regarding the question of extraterrestrial life. It is against this background that the validity of the main conjecture of this paper should be tested, namely, sufficient aspects of the question of eukaryogenesis have been understood in order to approach the search for life in Solar System exploration with the more advanced approach of enquiring, not only whether life is present in other environments such as the Mars permafrost or in the possibly molten ices of the Jovian satellite Europa, but more than that, the correct approach is to ask which is the degree of evolution of the putative extraterrestrial life that various space missions may encounter. This question is not idle: a unique moment in the development of the sciences of the origin of life is approaching with the new vigorous missions that now are in the course of planning by the main space agencies. Time and funding are crucial in this endeavour, so if the question of the degree of evolution can be faced from the start, every effort to do so should be made.

4.2. APPROACHES TO THE QUESTION OF EXTRATERRESTRIAL LIFE

Different positions are possible regarding the question of extraterrestrial life. They can only be distinguished from each other by experimentation either in situ, or by samplereturnmissions. We list a few of them:

4.2.1.

The more widely accepted belief on the nature of the origin of life is that life evolved according to the principles of deterministic chaos. Evolutionary developments of this type never run again through the same path of events. Kauffmann is the leading proponent of this possibility in his various recent contributions (Kauffmann, 1993).

4.2.2.

A possibility for similar evolutionary pathways on different planets of the Solar System has been defended recently (Davies, 1996). Indeed, even if some authors may consider this to be a remote possibility, Davies bases his work on the increasing acceptance that catastrophic impacts have played an important role in shaping the history of terrestrial life. Thus there may be some common evolutionary pathways between the microorganisms on Earth and those that may have developed on Mars during its 'clement period' (roughly equivalent to the early Archean in Earth stratigraphy). The means of transport may have been the displacement of substantial quantities of planetary surface due to large asteroid impacts on Mars.

4.2.3.

Even in spite of the possibility raised above, in 4.2.2, many researchers still see no reason to assume that the development of extraterrestrial life forms followed the same evolutionary pathway to eukaryotic cells, as it is known to have occurred on Earth. Moreover, a widespread point of view is that it would seem reasonable to assume that our ignorance concerning the origin of terrestrial life does not justify the assumption that any extraterrestrial life form has to be based on just the same genetic principles that are known to us. In sharp contrast to this position of common genetic principles for deciding how different would be the outcome of the origin of life elsewhere, De Duve has pointed out lessons that we should learn from the very principles of genetics that lead to the traditional expectation (cf., Section 4.2.4 (De Duve, 1995)). Amongst the various positions regarding the possible implications of our current understanding of life's origins, De Duve's approach is original and may be said to add a fourth point of view to the discussion of the various possible ways to approach the question of expectations of the nature of extraterrestrial life.

4.2.4.

Everyone is in agreement that the final outcome of life evolving in a different environment would not be the same as the Earth biota. But De Duve has broken new ground when he raised the question used as a title for the present Section 4: How different would such alternative extraterrestrial evolutionary pathways be? This has led to a clear distinction that there is no reason for the details of the phylogenetic tree to be reproduced elsewhere (excluding the possibility raised in paragraph 4.2.2). The tree of life constituted by the Earth biota may be unique to planet Earth. On the other hand, there is plenty of room for the development of differently shaped evolutionary trees in an extraterrestrial environment, where life may have taken hold. But "certain directions may carry such decisive selective advantages as to have a high probability of occurring elsewhere as well" (De Duve, 1995).

The evidence gathered together up to this point in the present paper, from an understanding of the appearance of life on Earth, leads to believe that the appearance of prokaryotes is a process that occurs within a short time span in an Earthlike planet or satellite (cf., the hints of the appearance of life, even during the heavy bombardment period, in Sec. 2). Therefore, certain extraterrestrial environments may have encouraged eukaryogenesis as well. Data available from a large number of earth and life science disciplines lead to the conjecture that: Whenever planets or satellites of a given solar system have the appropriate volatiles (particularly water and oxygen), not only prokaryotic life will occur, but eukaryogenesis will also take place. This conjecture has the merit that it is subject to experimental verification in the foreseeable future, as we shall argue in the present section. Primitive eukaryotic organisms, namely those lower eukaryotes which share some common features with prokaryotes, have been studied in considerable detail. Before we can demonstrate the above conjecture, many difficulties must be overcome in the eventual design of an assay to identify eukaryoticity in an extraterrestrial environment. (The questions raised by SETE begin to be important in view of multiple forthcoming space missions.) For this purpose we discuss some properties of organisms for which it is difficult to assign radically different physical parameters such as size of the genome, inner mambrane enveloping its DNA, or cellular size (as we have illustrated in Table III): • One taxon of eukaryotes with morphological features that are not radically different from prokaryotes can be found amongst the members of the family Cyanidiophyceae. These organisms are rhodophytes, commonly known as red algae (Seckbach, 1995). In particular, Cyanidium caldarium has a primitive cellular structure, in spite of the fact that phylogenetic analysis based on ribosomal RNA points towards microsporidians and particularly Giardia lamblia as the most likely candidates for primitive eukaryotes. The morphology of C. caldarium nevertheless makes this organism a clear example of the difficulties that would be faced by a robotic mission (cf., Section 5) to distinguish in clear assays between prokaryotes and those eukaryotes whose parameters have not radically evolved from the prokaryotic cellular plan (cf., Table 111). Other remarkable properties of C. caldarium are that it may live at temperatures of up to 57C and it shows better rates of growth and photosynthesis when cultured in an 'atmosphere' of pure C02 • Silicification experiments of microorganisms have led to the identification of one artefact, which can cause confusion in the identification of the transition organisms (Westall et al., 1995). It has been observed that an artificial nucleus is formed during the process of fossilization. If such a phenomenon is preserved in the natural fossil record, then it can lead to a confusion with the eukaryotes. • A prokaryote in symbiosis with the ciliated protozoan Euplotidium itoi has ultrastructure which is more complicated than the majority of prokaryotes (Rosati et al., 1993). Strictly from the point of view of planning future SETE experiments, the most striking feature of the symbionts of E. itoi is the presence of 'basket tubules', consisting of the protein tubulin, which is normally present only in eukaryotes.

I have described, in an abbreviated form, the necessary concepts from molecular biology and genetics that are needed to formulate the details of a SETE experiment. First of all we recall that what is required from the point of view of exobiology is to identify possible tests for eukaryoticity capable of being carried out in a robotic mission. To understand the rationale for such an experiment requires some definitions, which have already been introduced in Sec. 3. It is clear that regardless of its molecular basis, the packing of selected regions of the genome into heterochromatin is a uniquely eukaryotic form of gene regulation that is not available to bacteria (Alberts et al., 1989). It has already been seen, in the abovementioned section, that blocks of heterochromatin replicate late during the S phase. This is radically different from 'housekeeping genes' (i.e., those genes that are expressed in all cells), which replicate early during the cell cycle. This suggests that a sequence of fluorescence micrographs be taken of a dividing cell that may be encountered in solarsystem exploration, and be relayed to an Earthbound laboratory for inspection. Indeed, it is well known that staining eukaryotic chromosomes with a DNAbinding dye ('Giemsa staining') leads to the identification of a eukaryotic hallmark, namely, late replicating heterochromatic dark bands (cf., first paragraph of Sec. 3). The distinctive banding patterns probably reflect the tight and specific folding of the chromosome in the later stages of cell division (Bradbury, 1992; ChelaFlores, 1994), These condensed chromosome segments can subsequently be studied with fluorescence microphotography (Watson et al., 1987). The opposite case (gene activity) is radically different as it is generally correlated with early replication and lightstaining bands. Simpler tests in other environments of the Solar System may be conceived, for instance to look for the mode of cell division, which in the case of micropaleontology on Earth has given excellent results: A population of organic walled microstructures from the Archean Swaziland System have given convincing indicators of biogenicity by the persuasive preservation of various stages of binary division (Knoll and Barghoorn, 1977). However, by restricting the experiment to such simple tests, the opportunity for distinguishing between prokaryotes and eukaryotes is lost as bacteria normally undergo binary division; but so do the eukaryotes, yeast and the alga C caldarium. The proposal of the present paper is to focus attention on the degree of evolution rather than a simpler less embracing test should be seen in the context of the possible space missions, whose purpose would be to land in the Solar System environment to be probed for new forms of life, say the Jovian satellite Europa. There is a compromise to be solved between the engineering problem of miniaturization of the equipment designed to test for the degree of packaging of the chromosomes (heterochromatin), and the payload that would be possible to transport. In a preliminary discussion (Trowell et al., 1996), the space available is severely limited.

A related unavoidable question for future space missions is to investigate whether the first steps towards multicellularity have also been taken. However, the origin of metazoans cannot be inferred from the fossil record on Earth. Indeed, metazoans are too well developed when they make the first fossil appearance (Lipps et al., 1992). The fossil evidence suggests that eukaryogenesis is about 2 Gyr BP, but it should be recalled that it is generally believed that eukaryotes are much older than 2 billion years. It is further believed that appearance in the fossil record is not a good criterion for early divergences. On Earth eukaryogenesis may have been related to the increment of atmospheric oxygen. This is well established from at least two lines of evidence: • There is some evidence for the growth of oxygen in the atmosphere from some sedimentary rocks older than about 2 Gyr BP (bandediron, which are formations, related to anoxic conditions) • There is some complementary evidence from rocks younger than about about 2 Gyr BP (red beds, or shale, coloured by ferric oxide, Their existence demonstrates the presence of a considerable fraction of the present atmospheric levels of oxygen). Consequently, in any exobiological consideration in which such detailed atmospheric evolution remains missing (as sketched above for the well known case of the Earth), the search for life should not be entirely constrained to assays that envisage microorganisms that are a priori assumed to belong to the domains Bacteria or Archaea. Two main conclusions may be drawn from the analysis of data from molecular evolution, genetics, microbiology, geology, paleontology and solar system exploration. • In the search for unicellular organisms in the assays that might he considered in future space missions, the hallmark of eukaryotes should not be overlooked. This may be implemented by a robotic mission that is capable of monitoring cell division, in an attempt to identify late replicating heterochromatic sectors of the chromosomes. • It cannot be excluded that in planets or satellites, where atmospheric evolution may have differed from the Earth, precocious multicellularity may have occurred in prokaryotes, such as stromatolitic colonies of cyanobacteria. However, the presence of either multicellular eukaryotes, such as primitive metazoans or metaphytes cannot be excluded either. If such cases are possible, assays should not be ruled out that may test whether micrometazoan organisms [similar to modem marine larvae (Davidson et al., 1995)] may be present deep in the crust of terrestrial planets such as Mars, or in the Europa ocean. Evident morphological features, such as cellular size, dimension of the genome, or even mode of cellular division are components of potentially ambiguous tests as, for instance, a primitive alga does not diverge significantly from a prokaryote

Table VI
Some difficulties in the traditional approach of identifying eukaryoticity through morphological aspects of the cell

with respect to these three parameters (cf., Sec. 5). In addition, the presence of endosymbiotic organelles does not fare much better in our search for valid eukaryotic hallmarks, as archeozoan eukaryotes lacking organelles demonstrate so forcefully, There is another evident difficulty: the proposed assay should be able to distinguish between, for instance, a fossil of a microsporidianlike eukaryote (lacking organelles) and prokaryotes, such as N. multiformis, which have their nucleoids covered by membranes, These aspects of the problem are included in Table V1. The work described in Sec. 7, emphasising gene silencing in eukaryotes, suggests how to decide whether the putative living extraterrestrial organism has taken the first steps towards full eukaryogenesis, which is the basic cell structure on which organisms on Earth were raised to the level of intelligent beings. The case of extraterrestrial fossilized microorganisms has been discussed earlier in terms of certain biomarkers (ChelaFlores, 1997). The ultimate aim of research ill exobiology, however, continues to be the search for other intelligences in the cosmos (Drake, 1996). To sum up, the main thesis of the present paper is that we ought to discuss the most important questions for the search of extraterrestrial life that may be asked in unambiguous terms. A simpler test such as cell division does not settle the question of the degree of evolution of life in a habitable extraterrestrial environment as, for instance, the mode of division of the eukaryotic alga Cyanidioschyzon is by means of binary fission (Seckbach, 1994c). The reasons for emphasizing heterochromatin have been presented (ChelaFlores, 1998). It seems reasonable to suggest, therefore, that questions addressing heterochromatin are the least ambiguous indicators of eukaryoticity. The engineering problems regarding miniaturization of the current equipment for identifying heterochromatic bands is a separate question. In other words, a subsequent question to answer is whether the minimum space available for performing the experiment is compatible with the maximum space available in the proposed mission, Finally, more practical tests, not involving Giemsa staining, may be possible; but, further discussion of this possibility lies beyond the scope of the present paper

The author would like to thank Drs. J. Peter Gogarten, Giovanna Rosati and Frances Westall for sending him copies of their work. He would also like to acknowledge support from two sources: the International Centre for Theoretical Physics for financial support to attend 8th ISSOL Meeting (I Ith International Conference on the Origin of Life), and IAU for their support that enabled him to attend the 5th International Bioastronomy Symposium (IAU Colloquium No. 161), "Astronomical and biochemical origins and the search for life in the universe". Finally we would like to thank Ms. Katrina Danforth for a critical reading of the manuscript.

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