Origins, Worlds, and Life: A Decadal Strategy for Planetary Science and Astrobiology 2023-2032 (2022)

Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

14 Question 11: Search for Life Elsewhere Is there evidence of past or present life in the solar system beyond Earth and how do we detect it? Building upon insights from terrestrial life and our understanding of the diversity of habitable environments elsewhere, as well as significant advancement in biosignature detection technologies, we are poised to conduct a rigorous, systematic search for life beyond Earth in the solar system. 1 Four hundred years after Galileo revolutionized our understanding of our place in the universe, ours could realistically be the generation that triggers another scientific revolution, this time in biology. The search for evidence of life is part of the systematic progression in our understanding of planetary environments, which also encompasses detailed environmental characterization (Figure 14.1). Hence, a well-conceived arc of life detection activities serves to expand our knowledge of planetary environments, whether evidence of life is found or not. In the past decade, past and presently habitable environments beyond Earth were identified, providing a rich spectrum of worlds to explore in the context of astrobiology (Figure 14.2). Some of these environments introduce the potential to understand a biochemistry and/or emergence distinct from that of life on Earth, and thus we might begin to develop a universal theory of living systems. In the coming decade, biosignature searches will require evolution in how planetary systems are studied by new missions, technologies, and approaches to data analysis, shaped by our expanding knowledge of the range of habitats and biochemistries that can produce detectable biosignatures. A comprehensive framework is required to interpret potential biosignatures, abiosignatures, false positives, and false negatives, and promote confidence and consensus in interpretations. This framework needs to leverage past experiences with previous searches for signs of life in Earth and planetary environments and materials (e.g., Viking, ALH84001) to improve our approach of data interpretation and how results are communicated and evaluated, both within the planetary science community and to the general public. Our understanding of Earth life provides a starting point for investigation (Question 9), contrasting the signatures of life on Earth with the prebiotic/abiotic chemistry of primitive bodies. Key areas of research also include the search for and identification of potential biosignatures that are agnostic to life’s molecular makeup or metabolism, and studies of signatures of abiotic processes and phenomena that may define the non-biological baseline and/or mimic biosignatures, affecting biosignature reliability (NASEM 2019). Expanding the search to include the possibility of life ‘not as we know it’ requires further technical and conceptual maturation, including advances in statistical methods, scaling laws, information theory, and probabilistic approaches. Understanding the relationship between the geochemical environment and the prebiotic pathways that can give rise to life requires cooperation among diverse disciplines that extends beyond the traditional platform to include geochemists, atmospheric chemists, geologists, geophysicists, astronomers, mission scientists and engineers, and astrobiologists, among others (Lyons et al. 2020; NASEM 2019). 1 A glossary of acronyms and technical terms can be found in Appendix F. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-1 

FIGURE 14.1 Question 11. The search for life builds upon previous discoveries that span body interiors and surfaces to atmospheres and dynamic habitability (y-axis). Investigations at multiple habitable worlds (x-axis) allows for comparative planetology, leading to comparative biology if life is discovered elsewhere. For further information on where these and other worlds lie on the habitability spectrum, see Chapter 13. FIGURE 14.2 Question 11. Target environments and possible architectures of missions to search for life on other planetary bodies of the solar system. Left: Mission architectures to rocky planets and bodies with an atmosphere include (but are not limited to) atmospheric probes, drones, landers, and rovers. Right: Mission architectures to ocean worlds include (but are not limited to) flyby missions, orbiters, landers, and submersibles. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-2 

Q11.1 PATH TO BIOGENESIS: WHAT IS THE EXTENT AND HISTORY OF ORGANIC CHEMICAL EVOLUTION, POTENTIALLY LEADING TOWARDS LIFE, IN HABITABLE ENVIRONMENTS THROUGHOUT THE SOLAR SYSTEM? HOW DOES THIS INFORM THE LIKELIHOOD OF FALSE POSITIVE LIFE DETECTIONS? Presumably, life originates from a series of abiotic reactions in an organic chemical system, in which prebiotic synthesis of building blocks increases in complexity to ultimately reach biotic synthesis in a living organism. This process of organic chemical evolution develops gradually from simple to complex in terms of both the types of chemical reactions that occur and the diversity of compounds produced. Organic chemical evolution is highly dependent on the geochemical environment: factors such as pH, salinity, temperature, pressure, and the abundance of chemical precursors influence which reactions are favored, the rates at which they proceed, and the lifetimes of reactive intermediates and products available for subsequent chemistry. By understanding the extent and history of organic chemical evolution in the various habitable environments throughout the solar system, we can identify where other worlds might lie along the path to biogenesis (Q10.4), with a spectrum of possible end-members ranging from habitable (but uninhabited) environments to fully inhabited worlds like Earth (Q9.1d; Sutherland 2017). A thorough understanding of abiotic organic chemical evolution across a diverse set of geochemical conditions informs the likelihood of an independent emergence of life, and also helps reduce the likelihood of a false positive result in the search for life beyond Earth. Research in the emergence of life on Earth and knowledge of extant biochemistry can guide system- level analyses to differentiate between extraterrestrial biotic and abiotic chemistry (Q9.1c). While abiotic chemistry can utilize multiple pathways to generate a particular compound, biotic chemistry is characterized by evolved chemical systems that capitalize on a distinct subset of precursors among the abiotic possibilities. In the last decade, new approaches have emerged to understand complex networks of interacting molecules in chemical and biological systems. These approaches, which form the basis of systems chemistry and systems biology, need to be incorporated into astrobiology-focused missions by including analyses of potential precursors associated with any molecular target. It is not enough to simply search for a biosignature; an understanding of environmental processes that might result in false positives, such as high concentrations of abiotic organic compounds, is needed to interpret potential biosignatures as a robust biological signal. To assist in these analyses, significant support for research in prebiotic and general organic chemistry will be needed to enhance our knowledge of abiotic synthetic routes to the building blocks of life and to uncover possible pathways that other forms of life might have adopted. In concert with such efforts, contextual environmental and/or geological information needs to be gathered in order to understand what other features in the environment might provide added confidence to the assessment of the biogenicity of a potential biosignature. A complete picture of the organic chemical evolution in habitable environments requires a thorough understanding of the organics present in and on planetary bodies as they formed (Questions 2 and 3), and processes that might have modified them over time (Questions 4-6 and 10). The organic inventories of meteorites, interplanetary dust particles (IDPs), asteroids and comets are also important, in that these exogenous sources of organic matter can act as “chemical seeds” for the organic evolution of habitable environments, and also represent important abiotic chemical backgrounds against which signs of life need to be identified. Laboratory experiments designed to simulate the conditions and processes that occur in extraterrestrial material environments, such as cosmic ice irradiation and low-temperature photochemistry and geochemistry, are an important complement to the characterization of astromaterials (Hoehler et al. 2020). These experiments are advantageous in that they can help create a system-level understanding of the dynamic processes that give rise to organic inventories in astromaterials (Ditzler et al. 2020). PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-3 

Q11.1a What Is the Organic Molecule Inventory of Habitable Environments Throughout the Solar System, Including Complex Organic Molecules That Can Serve as Prebiotic Building Blocks of Life? The first step to assessing the extent of organic chemical evolution in a habitable environment is understanding what organic molecules exist there now, and in what geochemical context. Such work is underway (Table 14.1) but incomplete (Q10.4). Of great value would be a comprehensive inventory of organic molecules that includes both volatile and condensed phases, as well as soluble and insoluble (e.g., kerogen) fractions and abundant and trace species. Knowledge of the chemical structures (i.e., functional groups, binding motifs) of these organic compounds is important to ascertain synthesis mechanisms and reactivity. The distribution of organics within the habitable environment, particularly with respect to key habitability parameters such as energy sources, pressure or temperature, can provide important clues regarding their origin. The co-location of certain organic molecules with others, or depletion of one molecule with respect to another, may be diagnostic of the chemical pathways that dominate in that region of the environment, though such interpretations are necessarily convolved with careful assessment of the physicochemical properties of that region (see Q11.1c), which can affect the sorption and volatility/fugacity/solubility (and hence detectability) of organic molecules. This also applies to inorganic species, which may catalyze or otherwise facilitate certain organic chemical reactions. Q11.1b What Is the Extent of Molecular Complexity (e.g., Size, Heteroatom Diversity, Structure, Pathway Assembly Index) and Degree of Organization (e.g., Isomeric Preference, Polymerization) That Can Be Generated Abiotically Under Habitable Conditions? How Does This Compare to Prebiotic Experiments to Date? The degree of molecular complexity reflects the extent of organic chemical evolution in an environment and can possibly serve to discriminate biotic from abiotic systems. Key indicators of molecular complexity include, but are not limited to, the size and composition (arrangement, type and ratio of atoms, including diversity of heteroatoms) of molecules, as well as their chemical structure (molecular geometry, presence of functional groups, intramolecular interactions such as folding conformations, and binding motifs). The pathway assembly index—a probabilistic measure of the number of steps needed to construct a molecule (Marshall et al. 2017)—can also be useful in distinguishing biotic from abiotic molecules, though more work is needed to determine if all complex abiotic molecules will pass this test. Abiotic generation of organic molecules in unhabitable conditions also needs to be considered, as such materials could be inherited by habitable environments at a later stage. Biology is the dominant source of organic matter on Earth, where it has typically masked the signal from prebiotic / abiotic chemistry in all surface and near-surface environments (Q9.1). Therefore, the extent of molecular complexity that can be achieved through purely abiotic means, under widespread habitable conditions, is a challenge to assess through the study of natural terrestrial samples (Barge et al. 2020), although in the subsurface of both marine and continental systems, environments exist where low rates of biotic processes are such that abiotic signatures (particularly for abiotic H2, CH4, ethane, acetate, formate and other simple organic compounds) have been identified (Sherwood Lollar et al. 2014; McDermott et al. 2015; Lang et al. 2018). Work in the laboratory—where the influence of biology can be controlled, reduced or eliminated—can serve to inform this question, notwithstanding the challenge of mimicking a broad diversity of geochemical conditions in a laboratory setting. Other habitable environments in the solar system, in particular those with no detectable evidence of biology, can serve as prebiotic laboratories on a planetary scale to understand the extent of molecular complexity and degree of organization that can be achieved without the influence of life (Q10.4). Analyses by the Mars Perseverance rover in situ, and of samples returned in the Mars Sample Return campaign, may prove key in this regard. Methods to evaluate the degree of organization of abiotic systems include, but are not limited to, isomeric preference—whether PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-4 

molecules will react with or bind to another molecule of only a certain stereoisomeric conformation—as well as the type and number of monomeric units in any macromolecules (i.e., polymerization) and any intermolecular interactions (i.e., quaternary structure). TABLE 14.1. Organic Inventory of Worlds Throughout the Solar System to Date Body Organics* detected or predicted to date Complexity Reference(s) Mercury Possibly cold-trapped volatiles (C, H, O, and N-bearing Low Zhang and Paige 2009; species), aldehydes, amines, alcohols, cyanates, ketones and Delitsky et al. 2017; Hamill organic acids, refractory (tholin-like) organic materials et al. 2020 Venus Possibly HCN, methane, ethane, ethene and benzene Low Johnson and de Oliveira 2019; Mogul et al. 2021 The Moon Possibly methane and other cold-trapped volatiles (C, H, O, and Low Zhang and Paige 2009; N-bearing species) Colaprete et al. 2010 Mars Methane, chlorobenzene, dichloroalkanes; thiophenic, aromatic Low/ Freissinet et al. 2015; and aliphatic compounds Moderate Eigenbrode et al. 2018 Ceres Aliphatic organics, possibly amines Low De Sanctis et al. 2018; Raponi et al. 2021 Europa C≡N, C-H functional groups, refractory (tholin-like) organic Low McCord et al. 1998; Chyba materials and Phillips 2002 Ganymede C≡N, C-H functional groups, possibly refractory (tholin-like) Low McCord et al. 1997, 1998 and Callisto organic materials Enceladus Methane, other hydrocarbons, aromatics, amino acids, small Moderate Postberg et al. 2018; Waite and large (macromolecular) O-, N-bearing organics with et al. 2006; Steel et al. 2017 ethoxy, hydroxyl, and carbonyl functional groups Titan Hydrocarbons, nitriles, aromatics, heterocyclic species, High Brown et al. 2010; Lunine et acetylene, ethylene, cyanoacetylene, other N- and O-bearing al. 2020 species, nucleobases, amino acids, heteropolymeric species up to 10,000 Da, refractory (tholin-like) organic materials Uranian Possibly refractory (tholin-like) organic materials (could also Low Cartwright et al. 2018 satellites be amorphous pyroxene) Triton Ethane, hydrocarbons, acetylene, nitriles, heteropolymers, Low Thompson et al. 1989; refractory (tholin-like) organic materials Quirico et al. 1999 Pluto Methane, acetylene, ethylene, HCN, cyanoacetylene, amino Low/ Cruikshank et al. 2019 acids, nucleobases, refractory (tholin-like) organic materials Moderate Comets Acetylene, methane, methanol, formate, methylamine, Low/ Wickramasinghe and Allen ethylamine, PAHs, aromatic nitriles, amino acids, refractory Moderate 1986; Clemett et al. 2010; (tholin-like) organic materials Altwegg et al. 2016 Asteroids Hydrocarbons, polyaromatic carbon, refractory (tholin-like) Low Cruikshank et al. 1987; Chan organic materials et al. 2021; Fink et al. 1992 * An organic molecule is one that contains at least one carbon atom bonded to hydrogen. NOTE: Black text represents organics detected via remote sensing or in situ measurements; blue text represents compounds predicted by laboratory experiments/modeling. Complexity is compared to Earth as baseline (see Chapter 12). PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-5 

If end-member cases of abiotic organic complexity and degree of organization can be established, they would serve as a metric against which habitable environments could be compared in the chemical evolution spectrum spanning fully abiotic to fully biotic. Any unambiguous life detection discovery will require that all plausible abiotic and prebiotic formation mechanisms for the potential biosignature(s) be ruled out (Hoehler et al. 2020); the end-member case of greatest abiotic molecular complexity would serve as an important test for such a claim. Q11.1c What Are the Relevant Chemical Pathways That Can Lead From Prebiotic Chemistry to Biochemistry, and How Does That Transition Depend on the Geochemical State of the Environment? Chemical pathways (links of chemical reactions) in chemical networks are condition-specific; that is, certain pathways will only proceed under certain conditions, e.g., in the presence of a certain reactant or catalyst/substrate, within, for example, a specific pH, temperature or pressure range. The physicochemical conditions of a habitable environment are intrinsically linked to the geochemical state of that environment, and as such the progression from prebiotic chemistry to biotic chemistry through evolution of specific chemical pathways is likely to be mediated by geochemical factors (Q9.2). Mapping which chemical pathways are most likely to lead to biochemistry requires a deep understanding of chemical reaction networks in a variety of geochemical regimes spanning past and present conditions of habitable environments in the solar system. Strategic Research for Q11.1 ● Determine the organic molecule inventory, including extent of molecular complexity and degree of organization, of currently or previously habitable environments throughout the solar system (e.g., Enceladus, Europa, Titan, Ceres, Mars—see Question 10 for full list) with spacecraft in situ and/or remote sensing observations, telescopic observations or sample return. ● Identify relevant chemical pathways that can lead from prebiotic chemistry to biochemistry in currently or previously habitable environments throughout the solar system as a function of geochemical state by characterizing each environments’ geochemistry, physicochemical conditions and associated prebiotic (or potentially biotic) reactants, intermediates and products using spacecraft in situ and/or remote sensing observations, telescopic observations, and via laboratory/experimental and modeling/theoretical research. ● Characterize the extent of molecular complexity and degree of organization achievable for an organic molecule inventory in the absence of life in currently or previously habitable environments throughout the solar system using laboratory/experimental and modeling/theoretical research as well as field work and astromaterial analysis to explore the parameter space (e.g., temperature, pressure, pH, reactants, and catalysts/substrates). ● Ascertain how levels of organic inventory, molecular complexity and/or degree of organization that can be achieved in the absence of life might inform the search for life elsewhere using laboratory/experimental and modeling/theoretical research as well as field work and astromaterial analysis. ● Evaluate possible sources of false positives for organic chemical biosignatures with spacecraft in situ and/or remote sensing observations, telescopic observations and sample return from worlds unlikely to host life (e.g., the Moon, comets, asteroids) and uninhabited regions of previously or currently habitable environments (e.g., the surface of Mars) in an effort to assess the production and/or delivery rate of abiotic organic molecules that might mimic biological organic molecules and establish a baseline against which measurements in habitable environments can be compared. This also includes analysis of astromaterials, as well as theoretical and laboratory approaches. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-6 

Q11.2 BIOSIGNATURE POTENTIAL: WHAT IS THE BIOSIGNATURE POTENTIAL (I.E., THE RELIABILITY, DETECTABILITY, AND SURVIVABILITY OF BIOSIGNATURES) IN HABITABLE ENVIRONMENTS BEYOND EARTH? WHAT ARE THE POSSIBLE SOURCES OF FALSE POSITIVES AND FALSE NEGATIVES? Here, the committee defines “biosignature potential” as relating to the properties of a given habitable environment that act to enhance the probability of biosignature preservation and detection, including whether the properties of the habitable environment lend themselves in some way to the discrimination of biosignatures from a background of non-biological processes that might otherwise render them improbable or non-unique. Several important traits to be considered when assessing the functional merit of a potential biosignature include its reliability, detectability and survivability (Meadows 2017; Meadows et al. 2018; NASEM 2019). The reliability of a biosignature weighs the probability of life having produced it (its biological prevalence), against the improbability of non-biological processes producing it (its abiotic prevalence). Currently, the basis set for evaluating the biological prevalence of a potential biosignature is limited to our experience with the terrestrial biosphere, which is potentially limiting when considering potential biosignatures of life on non-Earth-like worlds (see Q11.3c). On the other hand, the abiotic prevalence of a potential biosignature (or abiosignature) relies on our understanding of abiotic processes operating over a broad range of geochemical conditions (see Q11.1). The detectability of a biosignature depends on the quality and magnitude of its signal, which are functions of the biological potential of the environment (see Question 10), and the level to which that biosignature can be quantified with current technology. For example, the reported detection of phosphine (PH3), a potential biosignature in the atmosphere of Venus (Greaves et al. 2020) has come under scrutiny as the absorption feature in the reprocessed data does not meet standard criteria for being statistically significant (Snellen et al. 2020). The survivability of a biosignature depends on whether it can be preserved long enough for analysis, or whether its decomposition products can be traced back to the identity of the original biosignature. Biosignature detectability and survivability are environment-specific; therefore, a thorough understanding of the environmental context is critical for biosignature evaluation. Q11.2a What Characteristics Indicative of Life Can Serve as Definitive Biosignatures in Environments Beyond Earth? As described in Question 9, biosignatures can be defined broadly as objects, substances, and/or patterns whose origin specifically requires a biological agent (Des Marais et al. 2003), and can manifest in a broad diversity of observables including, but not limited to, cell-like morphologies; sedimentary fabrics; organic molecular structures, abundance distributions of organic molecules; chirality in classes of molecules; mineral properties; stable isotope patterns in minerals and organic compounds; relative abundance of atmospheric gases; or temporal changes (e.g., daily, seasonal) in global planetary properties (Des Marais et al. 2008). Experience from the study of life on Earth suggests that these potential biosignatures can be grouped into three generic types: (1) chemical, (2) morphological, and (3) physiologic/metabolic. Chemical biosignatures result from life’s selectivity towards specific compounds (organic and inorganic) and from life’s capability to ‘invent’ complex organic molecules that fill important biological roles (e.g., biopolymers). Morphological (structural) biosignatures may emerge from life’s fundamental cellular architecture (though may not be required; see Q11.4a), and/or from the complex web of competitive and cooperative interactions between organisms (e.g., sedimentary fabrics), and their interactions with the environment. Physiologic/metabolic biosignatures are direct manifestations of the activity of living organisms (e.g., mobility, growth) including when they have the capacity to interact with and alter the environment at rates that are significantly faster or different than equivalent abiotic processes (e.g., chemical catalysis). For a given element, the relative alteration of different isotopic forms for that element (most commonly 13C, 12C) have frequently been used as biosignatures, despite an increasing recognition of the range of abiotic processes that may produce similar depletions in the 13C signatures and hence “mimic” PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-7 

life (NASEM 2019). Studies of biosignatures produced by life on Earth provide a foundation for the search for similar biosignatures in other habitable environments of the solar system (Question 9). The building blocks of life can have various abiotic origins and are likely to permeate the solar system and other planetary systems in our galaxy. This is to be expected, as abiotic chemistry provided the feedstock for prebiotic chemical evolution that ultimately gave rise to life at some point in the early history of Earth. A complex abiotic organic chemical background represents one possible form of abiosignature, i.e., chemicals, features, and/or processes that define the non-biological background against which signs of life needs to be resolved. This is made even more challenging by the fact that, in some cases, such abiosignatures may mimic biosignatures. Thorough characterization of the environment, including the extent of molecular complexity and degree of organization achievable in abiotic conditions for that environment (Q11.1b), can support assessment of the biogenicity of a potential biosignature even in a complex abiotic chemical background. Q11.2b What Are the Relationships Between the Physical and Chemical Properties and Processes Operating in a Habitable Environment, and the Potential Amount of Biomass That Might Be Present? How Might This Drive the Detectability of Any Biosignatures Present? The chemical inventory and fluxes of metabolically useful redox couples, the environmental conditions (pH, Eh, temperature, presence/absence of sunlight), and availability of substrates that can facilitate catalysis are among the important factors that will determine whether a habitable world supports the production of a large (in terms of total biomass) and productive biosphere, or a biosphere that is limited in terms of its productivity, total biomass and areal / geographic extent. Predictions regarding the potential size and extent of the biosphere on a habitable world would provide useful guidance for mission planning, sampling, and instrument design, and inform assessments of the susceptibility of a biosignature search to false negative outcomes. Studies of Earth’s biosphere provide a framework for thinking about biological productivity, and ultimately the rate of biomass production, that can be supported by an environment. A useful metric of biological productivity is “Net Primary Productivity (NPP)”: the quantity of CO2 fixed into organic matter per unit time, minus the quantity of organic carbon oxidized to CO2 by autotrophic respiration. Focusing specifically on Earth’s oceans, modern marine photoautotrophs can access virtually unlimited sources of water, sunlight, and inorganic carbon to support an NPP estimated at ~1016 grams of carbon/year (Field et al. 1998; Bender et al. 1994), such that marine NPP is limited by the availability of other nutrients including dissolved phosphorus, fixed nitrogen, and sometimes iron. Models of the early marine biosphere suggest that NPP would have been limited early on by the availability of inorganic geochemical electron donors, such as Fe2+ and H2, limiting NPP to ~1013-1015 grams carbon/year (Ward et al. 2019; Kharecha et al. 2005; Canfield et al. 2006). The geological record of carbon isotopes in carbonate and organic carbon indicate that the fraction of Earth’s surface carbon budget buried as organic matter in sediments has increased through time (Krissansen-Totton et al. 2015), consistent with the notion of an increasingly productive biosphere. Similar assay and bioenergetics-based approaches to modern subsurface environments provide additional useful guidance for estimates of non-photosynthetic subsurface biological productivity and biomass (e.g., Spear et al. 2005; Lollar et al. 2019), and these concepts have been extended to estimate the potential biomass of, e.g., the modern martian subsurface (Weiss et al. 2000; Sholes et al. 2019), and other habitable environments beyond Earth (e.g., Higgins and Cockell 2020; Cable et al. 2020; Europa Lander SDT Report 2016). While such modeling efforts require assumptions about the metabolic strategies employed by organisms living in the system, and the fluxes of nutrients available to them, they have the potential to yield estimates of biological productivity and perhaps even predictions regarding the composition of relevant stable isotopic reservoirs on other worlds. Such predictions can be useful in developing measurement requirements (biosignature type and expected concentration) for astrobiology- focused science objectives. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-8 

Q11.2c How Do the Physical and Chemical Properties and Processes in a Habitable World Affect the Survivability of Biosignatures? When considering the likelihood that a biosignature can be detected in a habitable environment, it is also important to consider how the physical and chemical conditions that make an environment habitable also act to modify, enhance, or erase evidence of inhabitation. NASEM (2019) speaks to this issue in the context of the survivability of biosignatures, or “taphonomy”, which is the field of study concerned with those processes that result in the formation, preservation, alteration, and destruction of biosignatures. This issue is also discussed in detail in Section 9.5. Our understanding of the processes that affect the biosignature potential of a habitable environment necessarily have “baked in” assumptions about the nature of the biological system one suspects is or was present. The search for the remnants of an extinct, non- photosynthetic, lithotrophic microbial biosphere on Mars, for example, would require an exploration of the unique chemical and physical conditions favoring biosignature production and preservation in that environment, distinct from those that might favor the successful search for an atmospheric biosignature on Venus or an exoplanet. Accordingly, the trade space for understanding how the chemical and physical processes in a habitable world affect its biosignature potential is vast, and needs to be narrowed on the basis of assumptions about the biosphere that produced it, and the environment(s) in which the signature(s) of that biology have resided in order to avoid false negatives that result from an under-appreciation of the specific environmental factors that might act to modify or erase a potential biosignature. Q11.2d How Can We Best Devise a Formal Framework for the Interpretation of a Set of Biosignature Measurements in a Given Environment? To account for uncertainties in biosignature potential and the possibly unique nature of life elsewhere (Q11.3c), a search for evidence of life is more likely to succeed if it is based on multiple, mutually supporting lines of evidence as well as on their environmental context (Figure 14.3). Building on well- established tenets such as multiple working hypotheses (where every rational explanation of the phenomenon is included and impartially evaluated; Chamberlin 1890, 1897), frameworks that allow correct interpretation of the significance of each potential biosignature, according to their own possible sources of false positive and false negative signals, provide a more rigorous standard of proof. In addition, such frameworks can help identify sets of measurements that together discriminate between a biotic or abiotic origin for potential biosignatures with high statistical significance (NASEM 2019). Some preliminary concepts for interpretive frameworks have been posited recently for possible life detection mission scenarios, considering uncertainties arising from environmental or instrumental factors and where valuable but limited opportunities exist to collect additional samples or conduct follow-up analyses. At the most basic level a framework may be represented as a decision tree, wherein questions about acquired data may lead to increased or decreased probabilities or “scores” associated with life detection (e.g., Vago et al. 2017). Thorough, yet flexible, decision trees may prove especially useful during a mission, where selection of samples or measurement parameters can be guided by “real-time” analysis of collected data. Where data increase in volume and complexity and become increasingly cumbersome to manage, such as on remote missions with restricted communication, science autonomy will increasingly be required for life detection (Theiling et al. 2021). Machine learning and other tools may provide an expanded practical framework for interpreting biosignature measurement data in such conditions. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-9 

FIGURE 14.3 Question 11. A formal framework for interpretation of biosignature measurements would build on prior knowledge of biological and abiotic signatures and utilize data, environmental context and biological potential to construct a probabilistic model robust to false positives and false negatives. Strategic Research for Q11.2 ● Determine which attributes of terrestrial life can serve as definitive biosignatures in the search for life beyond Earth by theoretical, field, and laboratory research activities that inform on the range of morphological and chemical (organic, isotopic, mineralogical, atmospheric) signatures produced by living systems, with a complementary emphasis on what contextual evidence is required to confidently recognize a potential biosignature. ● Constrain the biomass and/or bioenergetic potential of habitable environments throughout the solar system through measurements of environmental factors (e.g., pH, temperature, salinity, redox potential) and fluxes (e.g., energy, nutrients) with spacecraft in situ and/or remote sensing observations, or returned sample analysis; and theoretical, field, and laboratory research activities to understand how modern and ancient environmental properties and processes on Earth relate to biomass production to inform the development of life detection strategies and technology (see also Question 9). ● Characterize the range of processes that affect the production and preservation of detectable biosignatures in habitable environments by theoretical, field, and laboratory research activities that inform us about the pathways and rates of biosignature production, preservation, and destruction, be they morphological or chemical (organic, isotopic, mineralogical, atmospheric) in nature. Tie those studies to in situ, remote, and/or telescopic measurements of specific environmental properties of habitable worlds in the solar system (e.g., Enceladus, Europa, Titan, Ceres, Mars—see Question 10 for full list) that might act to enhance, preserve, or destroy biosignatures. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-10 

● Establish a comprehensive, standardized framework for evaluation of biosignatures, including the potential for abiosignatures, false positives, and false negatives, through community-level dialog and consensus, supported by laboratory/experimental and modeling/theoretical research as well as field work. Q11.3 LIFE DETECTION: IS OR WAS THERE LIFE ELSEWHERE IN THE SOLAR SYSTEM? Recent decades of planetary exploration have witnessed a small revolution in our understanding of habitability conditions elsewhere in the solar system (Figure 14.4). It is now widely accepted that long- lived habitable environments existed on the surface of Mars early in the planet’s history, and that habitable conditions may have persisted until the present in the subsurface. There is compelling evidence that a habitable ocean exists beneath the surface of Enceladus, and possibly also beneath the surfaces of Europa and Titan. Motivated by these discoveries, we are poised to address the question of whether there is, or ever was, life elsewhere in the solar system, with scientific rigor. FIGURE 14.4 Question 11. The search for life has been revolutionized in the past decades by pivotal discoveries such as the identification of what is now recognized as a third branch in the tree of life (Archaea) and confirmation of subsurface liquid water oceans within several moons of Jupiter and Saturn. The search for evidence of life beyond Earth builds upon and extends the traditional objectives of planetary sciences, complementing comparative planetology and extending to a new potential field: comparative biology (Q11.4a). The magnitude of the question requires the coming together of scientists from a wide range of disciplines, and the combination of a diversity of perspectives and approaches. The search also needs to satisfy the scientific criteria of experimentation, address testable hypotheses, and allow modification of those hypotheses based on observation and measurement (NASEM 2019). This applies equally to cases where life may have gone extinct, detectable through its imprint preserved over time, or cases where life is currently viable, and may even comprise a population of metabolizing organisms, albeit not necessarily in a way immediately familiar to us (Q11.3c). To minimize the likelihood of a false negative or a false positive result, optimal life-detection strategies would target multiple, orthogonal, mutually supporting lines of evidence, and carefully select sets of PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-11 

candidate biosignatures based on several key criteria (Q11.2). Assessments of the presence or absence of potential biosignatures in a habitable environment (present or past) need to be congruent with the physicochemical setting and ought to be informed by the biological potential of that environment (Figure 14.5 and Question 10). In addition, life-detection mission designs need to include comprehensive contamination control approaches informed by acceptable levels and types of contamination derived from top-level science and mission requirements. This effort would start as part of mission concept development, and contamination awareness and control would be addressed in detail throughout all mission phases. Finally, the novelty of the measurements needed for life detection and the rigorous standards of evidence mandate tailored approaches in the conception, maturation, and deployment of instruments and instrument suites (Hoehler et al. 2020). FIGURE 14.5 Question 11. To provide evidence of life above the threshold established by abiotic processes in a given environment, multiple independent biosignatures (B) are needed, as well as a thorough understanding of the physicochemical setting (C). Q11.3a Are There Chemical, Morphological and/or Physiologic/Metabolic or Other Biosignatures in Currently Habitable Environments in the Solar System? The continued exploration of planetary bodies of the solar system is revealing a broader range of potentially habitable solar system environments than previously anticipated (Question 10). Data gathered by the Cassini spacecraft suggests that the subsurface ocean of Enceladus currently meets the requirements to sustain life (Cable et al. 2020). The Europa Clipper and Dragonfly missions will help constrain the biological potential of Europa’s and Titan’s subsurface oceans, respectively. The exploration of Venus (VERITAS; DAVINCI) and Mars (Curiosity; Perseverance) will help establish whether localized habitable regions currently exist within these seemingly uninhabitable worlds. Once habitable environments are identified, the search for evidence of life represents the logical next step, and also the greatest challenge. The search needs to be conducted thoughtfully and with an open mind concerning potential outcomes, balancing the stringency and inclusivity of the observational strategy applied to a given environment. Stringency sets criteria for the quality and robustness of a biosignature detection, amidst potentially confounding conditions or background signals from the planetary environment, and thus seeks to minimize potential false positive results such as a “life-like” abiotic pattern or response. Inclusivity emphasizes consideration of a wide range of possible alien biosignatures (chemical, morphological and/or physiologic/metabolic), not relying solely on Earth life as a guide, as well as their prevalence and detectability in the given environment. As such, inclusivity seeks to minimize potential false negative PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-12 

results, where life could be “missed” for lack of the ability to detect or recognize it. These concepts apply equally to cases where life may have gone extinct, detectable through its imprint preserved over time (Q11.3b). The search for evidence of life requires tailored technology solutions (see the Technology chapter). The novelty of the measurements needed for life detection and the rigorous standards of evidence we will demand of them mandate new approaches in the conception, maturation, and deployment of instruments and instrument suites. Specifically, because the preferred route to arriving at defensible conclusions is to employ multiple orthogonal lines of life detection, future missions will need to be designed holistically, potentially having instrument providers engage with each other very early in the process (i.e., pre-Phase A) to propose integrated instrument suites. The most sensitive life-detection experiments require sample acquisition, sample handling and sample analysis (Figure 14.6); therefore, in situ detection of life is best advanced by integrated suites of instruments or single instruments that permit multiple analytical techniques, including nondestructive approaches, to be applied to the same collected materials (NASEM 2019) (see the R&A chapter). Each step forward in this effort will also require a continuing vigilance against forward contamination from Earth (McKay et al. 2020). FIGURE 14.6 Question 11. Examples of instruments that can be used to search for chemical/molecular biosignatures, organized by their range of detection limits and sample processing requirements. Note that sample preconcentration can improve detection limits for some instruments. Figure updated from Mars 2020 SDT Report. (FTIR, Fourier transform infrared; GC, gas chromatography; LC, liquid chromatography; MEMS, microelectromechanical system; MS, mass spectrometry; OW, ocean worlds; TOF, time-of-flight; UV, ultraviolet). *Detection limits depend on sample matrix and instrument architecture. **Some instrument designs require wet chemical processing. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-13 

Q11.3b Are There Chemical, Morphological, or Other Biosignatures in Previously Habitable Environments in the Solar System? Mars witnessed a protracted period of habitable conditions early in its history, with relatively mild surface temperatures and widespread bodies of liquid water with physical and chemical conditions compatible with biology. A detailed geologic record of this early habitable period has been preserved on the surface, as shown by data gathered by the Curiosity rover at Gale crater. A search for evidence of past life on Mars is now underway at Jezero crater with the Perseverance rover, and is a top priority objective for the analysis of samples cached by the rover, which will eventually be brought to Earth as part of the Mars Sample Return campaign. While physiologic/metabolic biosignatures (which would indicate organisms that are alive today) may no longer be present, evidence of past life in the returned samples can be ascertained from chemical and morphological fingerprints left behind. To support that objective, data gathered by Perseverance may help constrain biosignature survivability (Q11.2) and offer a deeper understanding of the geologic evolution of that environment, particularly in terms of physical (temperature, pressure, transport) and chemical (e.g., oxidation, photodissociation, radiolysis) processing that biosignatures may have undergone over geologic timescales. Studies of biosignatures produced by ancient life on Earth (Question 9), such as in Archaean sediments, can inform the search for similar biosignatures in martian sedimentary deposits. Q11.3c How Might We Develop the Scientific Understanding to Recognize Life ‘Not As We Know It’? The search for evidence of life beyond Earth is necessarily informed by life “as we know it” (the particular case of terrestrial life, Question 9), which increases the potential likelihood that we would not detect life “not as we know it” in an otherwise inhabited world. Acknowledging that terrestrial life might be but one example of all possible forms of life in the universe, we need to recognize the possibility of life with biology that differs in a significant way from the terrestrial model in form and/or function. There is a recognized need to improve our understanding of which biosignatures are likely to be prevalent across all forms of life, in order to reduce the risk that our missions and technologies will be too narrowly focused on the features of life “as we know it” from Earth. The goal of such research is to expand the ability to search for any life through exploration of a broader definition of life based on activity, with less dependence on assumptions about structure and specific biogeochemistry—specifically, more universal or agnostic biosignatures (NASEM 2019). From least to most radical (and therefore most to least likely), potential biochemical differences include (NRC 2007): ● Different lipid molecules for membranes. Major categories of living organisms on Earth are distinguished in part by their use of different lipids, suggesting that alternative patterns compatible with other metabolic pathways are likely. ● Different sets of amino acids or nucleobases than the ~20 and 5 used by terrestrial life, respectively, to encode information (Young and Schultz 2010; Hoshika et al. 2019). ● The opposite enantiomer of amino acid and sugar chirality (i.e., mirror life), and/or a genetic polymer other than a polyanionic backbone”. ● Different genetic molecules. Life on Earth has switched genetic molecules at least once (from RNA to DNA); alternatives may be numerous (Cleaves et al. 2019; Zhou et al. 2021). ● Alternative essential elements with similar chemical properties (e.g., elements within the same column of the periodic table or transition metals with similar oxidation states, reactivities and/or binding affinities). ● A solvent that is liquid at temperatures and pressures other than those of liquid water. In the solar system, two environments with liquids not dominated by water are the hydrocarbon (methane- ethane) lakes fould in Titan’s polar regions and sulfuric acid clouds in the atmosphere of Venus. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-14 

Both have been hypothesized to be able to harbor life as we don’t know it (Lunine et al. 2020; Limaye et al. 2018), although limitations of organic compounds in these environments (such as low solubility in Titan’s lakes and instability in the Venus’s atmosphere) may pose insurmountable challenges even to novel/alien biochemistries.. Other potential solvents include formamide, ammonia and carbon dioxide, but no planetary environments are yet known to contain such liquids as the dominant component. Accordingly, life “not as we know it” can fall along a sliding scale from less to more radical departures from the basic terrestrial model. Strategies to search for evidence of life need to strike the right balance between inclusiveness of alternative biologies (e.g., by considering novel and/or agnostic biosignatures) while adhering to known biological principles (e.g., a chemical system that can undergo Darwinian evolution). Understanding the spectrum of possible departures from the terrestrial model, and how those might impact our selection of potentially habitable environments and the interpretation of potential biosignatures therein, is an important aspect of astrobiology. Q11.3d If We Don’t Find Evidence of Life in a Habitable Environment, What Would It Take to Convince Ourselves That There Truly Is or Was No Life Present There, Rather Than Possibly Not Having Detected It (A False Negative)? When searching for evidence of life, the probability of a false negative result is highest in environments where potential biosignatures occur at very low abundance (e.g., due to low productivity or to degradation/destruction processes), operating at a very low (or even dormant) metabolic state, or where life is not distributed homogeneously (i.e., biological oases amidst an abiotic landscape). False negatives due to low biological signals can be constrained based on contextual information. But false negatives due to heterogeneous distributions of biological signals are more difficult to recognize, because spatial heterogeneity can occur at any scale, up to planet-wide. Research in various environments on Earth suggests that heterogeneous distributions of life are nevertheless not arbitrary. Biological oases typically occur in areas where resources (water, nutrients, energy) are locally more abundant, or where lethal environmental conditions (e.g., radiation, excessive temperatures) are somehow mitigated. Life signatures can be relatively diverse and abundant in those oases, but quickly vanish with distance or time. Often, biological oases are associated with specific substrates or physical environments (rocks, sediments, subsurface layers, fracture surfaces) whose chemical or physical properties provide a survival advantage to organisms. As such, correct interpretations of a negative result require adequate understanding of the spatial variability in resources and environmental conditions. Research in terrestrial environments can inform how spatial variations in resources and environmental conditions can shape the distribution of life in the landscape (Question 9). From these studies, models can be developed that predict potential ‘hotspots’ or blooms of life as a function of resources and environmental conditions. Such models can then inform the most likely locations to find evidence of life on a planetary body, and how the likelihood of finding evidence of life changes spatially. Similarly, models taking into account putative metabolisms in a certain environment could inform protocols for instigating a ‘bloom’ in a collected sample, if nutrient-starved organisms were lying dormant. Strategic Research for Q11.3 ● Develop and validate effective life detection payloads that support the search for evidence of life beyond Earth by maturing end-to-end technologies for sample acquisition, sample handling/ preparation and sample analysis, and by prioritizing the early integration and validation of these technologies and instrument suites. ● Search for evidence of present life in environments beyond Earth that currently have a high biological potential by looking for multiple, independent biosignatures with spacecraft in situ PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-15 

observations or in samples returned to Earth, informed by laboratory/experimental and modeling/theoretical research as well as field work of currently habitable environments on Earth. ● Search for evidence of past life in environments beyond Earth that had a high biological potential in the past by looking for multiple, independent biosignatures with spacecraft in situ observations or in samples returned to Earth, informed by laboratory/experimental and modeling/theoretical research as well as field work of ancient life on Earth. ● Search for evidence of life “not as we know it” in environments beyond Earth by looking for multiple, independent biosignatures with spacecraft in situ observations or in samples returned to Earth, informed by laboratory/experimental and modeling/theoretical research of putative organisms that might utilize unique biochemistry or alternative essential elements or solvents. ● Determine the optimal sampling strategy to minimize the likelihood of a false negative life detection measurement (or set of measurements) due to heterogeneous distributions of biological signals or other factors by employing laboratory studies and modeling/theoretical research in conjunction with field studies of biological oases and environments with severe nutrient/energy limitations on Earth. ● Develop and implement analysis techniques to evaluate the likelihood of forward contamination in life-detection missions through models of contamination transfer from spacecraft surfaces and by improving hardware protection/cleaning methods, in order to meet the stringent contamination requirements imposed by life-detection science. Q11.4 LIFE CHARACTERIZATION: WHAT IS THE NATURE OF LIFE ELSEWHERE, IF IT EXISTS? With terrestrial life as the sole example, biology is unlikely to evolve into a universal science in the same way as physics, chemistry, and geology have evolved with the respective advent of telescopic astronomy (17th century), spectroscopy (19th century), and planetary exploration by spacecraft (1960s). For these fields, we now have a predictive understanding. But the discovery of life on another world would allow us, for the first time, to conduct comparative biology beyond the boundaries of Earth, and to address some of the most fundamental questions in biology: Is there a general theory of living systems? Is life a common cosmic phenomenon? What is life, how does it emerge, and what are its universal means of operation? All life on Earth is genetically related and likely represents only one data point among the possible instances of biology. Characterizing life elsewhere, if it exists, would allow us to begin distinguishing attributes specific to life on Earth from those that are shared across instances of biology (Figure 14.7). An important challenge in this process will be to recognize life beyond Earth as a truly independent biology, with no ancestral links to life on Earth due for example to biological exchange by impacts. Our ability to characterize life beyond Earth if/once it is discovered will be key to addressing this challenge. Three scenarios could be possible. First, life could be discovered that is so similar to Earth life that it would be challenging to rule out terrestrial contamination without extensive phylogenetic analysis. Second, identification of extraterrestrial life whose biochemistry overlaps significantly (but not completely) with terrestrial life might point to a shared origin and later dispersion through planetary exchange. Third, we may find extraterrestrial life that has sufficiently different biochemical attributes from any known Earth organism. This case may point to a separate origin of life and imply that living systems can readily emerge under appropriate physical and chemical conditions; alternatively, the two systems could share a common origin but have diverged so greatly over time that any trace of common evolutionary heritage has been obscured. Each scenario would have significantly different implications about the prevalence of life in the universe or the likelihood of it being transported across planetary havens. Coupled with a detailed understanding of its environment, inspection of biological properties and processes beyond Earth would provide insight into how a biosphere arises and co-evolves with its host world. Such insights into universal PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-16 

properties, emergence, prevalence, and workings at scales from microscopic to global could bring within reach a definition of life and a universal theory of biology. Since the onset of robotic exploration of the solar system, the knowledge gained via such exploration and concomitant advances in the understanding of what life requires, what constitutes evidence of life, and how to build instruments to search for it has poised humanity on the verge of finding and characterizing either life beyond Earth, or habitable worlds with no evidence of life. Either way, these discoveries, for which the groundwork can be undertaken in this coming decade, are bound to bring answers to some of humanity’s most profound questions. FIGURE 14.7 Question 11. Development of the scientific understanding to recognize and characterize life “not as we know it” would help us understand the nature of life via comparative biology. Q11.4a How Might Comparative Biology Be Implemented on a Small Sample Size (N=2) If Evidence of Life Is Discovered Elsewhere in the Solar System? Characterizations of life beyond Earth would take place at multiple levels (e.g., biochemically, structurally, metabolically), each one likely to produce profound insights on the nature of biological systems. For example, the cell is the basic structural, functional, and biological unit of all modern terrestrial organisms. However, life elsewhere could exist in an acellular evolutionary form (e.g., with only a protein coat as in viruses, see Q9.2), or it could be based on a different cellular architecture. It is unknown whether all living systems require a cellular organization altogether. Similarly, for all their metabolic diversity, terrestrial organisms extract energy from a limited subset of physical and chemical sources (see Question 9). However, life elsewhere might be able to exploit other sources of energy (e.g., different wavelengths and intensities of radiation) reflecting adaptation to their local environment. Finally, all life on Earth shares the same biochemical framework based on a universal set of molecular building blocks (e.g., amino acids, nucleobases, sugars, lipids) decorated with essential elements such as N, P, and S that confer a broad range of molecular structures and functionalities. This particular biochemical framework operates optimally within a range of physical and chemical conditions (e.g., temperature, salinity, pH) (Q9.2). The biochemistry of life elsewhere could be identical to Earth’s, and be bound by a similar range of environmental conditions, or could be based on a different subset of building blocks and essential elements, and operate optimally under conditions that are prohibitive to terrestrial life. In order to understand this potential variability, the discovery of evidence of life beyond Earth ought to be followed by dedicated investigations into the nature of those life forms. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-17 

Q11.4b How Do We Discriminate Between an Independent Origin of Life Elsewhere in the Solar System and a Shared Ancestry with Earth Life? The above alternative means by which biological structure and function may be achieved could be used to distinguish life that shares ancestry with Earth life (up to the point of a last universal common ancestor— LUCA; Q9.1f) from life with a more distant relationship or independent emergence. Finding a new metabolic strategy but the same biomolecules as on Earth would suggest adaptation of terrestrial life transported to a new world. The identification of a cell membrane with new lipids would suggest a new category of organism, but one not necessarily more distinct than the three categories of organisms on Earth are from one another. In contrast, alternative biomolecules or elements would point to a more distantly related or unique instance of life. More generally, among the different types of biosignatures (chemical, structural, metabolic/physiological; Q11.2a), only certain chemical (organic) biosignatures, or informational evidence derived from them, might truly reveal an independent emergence. The latter include biopolymers whose sequence changes randomly through time (nucleic acids, peptides, membrane lipids) and their specific building blocks (including specific molecular properties such as chirality). Even if alternatives as described in Q11.3c are found, there will be a degree of uncertainty in our assessment of an independent origin of life due to our limited understanding of the pre-LUCA biochemical nature of life on Earth. Another clue can be taken from the likelihood of material transfer (and of biological viability in this material during the transfer) between Earth and another inhabited world. The presence of rocks from Mars and other bodies in the meteorite collection show that this likelihood is nonzero; in the case of Mars specifically, the time when Mars was most habitable (late Noachian) would have coincided with a period of large impacts occurring on Earth, meaning that life could have spread from one potentially habitable world to another. Transfer between Earth and any inhabited environments in outer solar system ocean worlds and dwarf planets is less likely, however, due to low impact probabilities and high impact velocities of ejected material, as well as the barrier presented by ice shells that encase subsurface oceans (Question 5). Q11.4c What Does the Presence or Absence of Life on Other Worlds Tell Us About the Emergence of Life in General? Proposed environments for the emergence of life on Earth, likely during the early Archaean, include surface aquatic environments (lakes, shallow marine settings), ephemeral playas with wet-dry cycling, hydrothermal volcanic fields at the surface, hydrothermal vents on the ocean floor, and subsurface fracture networks (Q9.1). All have attributes that would be favorable or challenging to prebiotic chemical evolution (e.g., stability, longevity, energy supply, dilution), and the scarcity of early Archaean geologic and fossil records limits our capacity to constrain the actual environmental setting where life could have begun on our planet. The search for evidence of life on other worlds is a means to test some of these origin of life hypotheses. For example, the absence of life in an otherwise habitable ocean world would rekindle ideas for an origin of life in environments with low water/rock ratios (Deamer and Damer 2017). But the discovery of life in an ocean world would lend credibility to theories that life on Earth could have originated in deep hydrothermal vents on the ocean floor. Therefore, characterizing life beyond Earth could shed light on the unknown circumstances of life’s emergence on our own planet, at least in terms of likelihood and environmental setting. The likelihood of life emerging and persisting on a habitable world is unknown, but our presence on Earth shows that it is not zero. An instance of biology on another world would help us understand whether life is an unavoidable outcome in the evolution of worlds that harbor habitable environments for sufficient time (one billion years or less; van Kranendonk et al. 2018), whether it requires an improbable combination of circumstances (Ward and Brownlee 2000), and/or how frequently life transported from another world takes hold. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-18 

Q11.4d How Have Life and Its Host World Co-Evolved? Determining the biochemical and metabolic characteristics of any extraterrestrial life would help us understand how it may have adapted to its environment, and perhaps the impact it has had on that environment from microscopic to global scales. Life on Earth has been shaped by the environment, including multiple mass extinction events that profoundly changed the phylogeny and ecology of the surviving groups. But terrestrial life has also had a profound impact on its environment, causing significant changes in the composition of the atmosphere, ocean sediments, and the upper layers of the crust (Q9.4). Is this co-evolution of life and the environment an intrinsic characteristic of inhabited worlds? If life ever existed on Mars, whose geologic history is in some sense comparable to Earth’s, was it able to significantly modify the environment? Did it retreat to cryptic niches as the planet became increasingly colder and dryer? Or did it succumb to one or more mass extinction events? If life exists in an ocean world, has it modified the chemistry of the ocean? Has it been shaped by events outside its sheltered confines? Or does it represent an end-member instance of static biology in a largely unchangeable environment? The extent of a biosphere and of its effects likely depends on its overall energy and nutrient supply (see Question 10 and Q11.2). Evidence to date suggests that any life elsewhere in the solar system has not, and cannot have, had as much of an effect on its host world as—even microbial—life on Earth, which has shaped our oxygen-rich atmosphere (Q6.2). However, the global influence of biology is the prime line of evidence being pursued to search for life on extrasolar planets. The next (ultimate?) level of study would examine the biochemistry and physiology of any discovered organisms. Given the experimental rather than observational or analytical nature of such investigations, current and foreseeable technology likely limits them to approaches involving humans, either with returned samples or, for Mars, in situ human exploration, rather than solely via in situ robotic exploration. Strategic Research for Q11.4 ● Investigate the chemical environments of Europa and Enceladus that are relevant to potential biochemistry through measurements of possible metabolic reactants and products (organic and inorganic compounds), reaction conditions (e.g., temperature, pressure, Eh, pH), rates and catalysis (e.g., mineral surfaces, trace elements), as well as the structure of biomolecules at the surface and in waters sourced from the subsurface. ● Determine whether any life present in martian materials might share ancestry with Earth through measurement of any biomolecules as part of the organic chemical inventory to assess their function (including, for ancient life, their pre-degradation form) and what they reveal about the co- evolution of Mars’s life and climate. ● Prepare for characterizing life in the subsurface of ocean worlds by determining the heterogeneity of thicknesses of ice shells via planetary mission data as well as validating and deploying emerging technologies for life characterization, and maturing technology for accessing the subsurface for exploration, by work in the field and in the laboratory. ● Develop viable solutions to technical challenges for curation of samples to be returned from Mars and ocean worlds that will preserve their scientific integrity, including biosafe curation coupled (for ocean worlds) with cold conditions. ● Characterize any form of life discovered beyond Earth, and investigate its relatedness, or lack thereof to life on Earth through measurements of its biochemical, morphological, and/or physiological/metabolic traits with spacecraft in situ observations or in samples returned to Earth. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-19 

REFERENCES Altwegg, K., H. Balsiger, A. Bar-Nun, J.-J. Berthelier, A. Bieler, P. Bochsler, C. Briois, et al. 2016. Prebiotic chemicals—amino acid and phosphorus—in the coma of comet 67P/Churyumov- Gerasimenko. Science Advances, 2, e1600285. https://doi.org/10.1126/sciadv.1600285 Barge, L.M., L. Rodriguez, J.M. Weber, and B. Theiling. 2020. Beyond “biosignatures”: Importance of applying abiotic / prebiotic chemistry to the search for extraterrestrial life. White paper submitted to the Planetary Science and Astrobiology Decadal Survey. Bulletin of the AAS, 53, 4. https://doi.org/10.3847/25c2cfeb.b66ffca4 Bender, M., T. Sowers and L. Labeyrie. 1994. The Dole effect and its variations during the last 130,000 years as measured in the Vostok ice core. Global Biogeochemical Cycles 8 (3), 363-376. https://doi.org/10.1029/94GB00724 Brown, R. H., J.-P. Lebreton, and J. H. Waite. 2010. Titan from Cassini-Huygens. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-9215-2 Cable, M.L., M. Neveu, H. Hsu, and T. Hoehler. 2020. Enceladus in Planetary Astrobiology, Eds. V. Meadows et al., pp. 217.246, Univ. of Arizona, Tucson. https://doi.org/10.2458/azu_uapress_9780816540068-ch009 Canfield, D.E., M.T. Rosing, C. Bjerrum. 2006. Early anaerobic metabolisms. Philosophical Transactions of the Royal Society of London B Biological Sciences, 361, 1819-1836. doi: 10.1098/rstb.2006.1906. Cartwright, R. J., J. P. Emery, N. Pinilla-Alonso, M. P. Lucas, A. S. Rivkin, and D. E. Trilling. 2018. Red material on the large moons of Uranus: Dust from the irregular satellites? Icarus, 314, 210-231. https://doi.org/10.1016/j.icarus.2018.06.004 Chamberlin, T.C., 1890, The method of multiple working hypotheses: Science (old series) v. 15, p. 92-96; reprinted 1965, v. 148, p. 754-759. Chamberlin, T.C., 1897, The method of multiple working hypotheses: Journal of Geology, v. 5, p. 837- 848 Chan, Q. H. S., A. Stephant, A. Franchi, X. Zhao, R. Brunetto, Y. Kebukawa, T. Noguchi, et al. 2021. Organic matter and water from asteroid Itokawa. Scientific Reports, 11, 5125. https://doi.org/10.1038/s41598-021-84517-x Chyba, C. F., and C. B. Phillips. 2002. Europa as an abode of life. Origins of Life and Evolution of the Biosphere, 32, 47-67. https://doi.org/10.1023/A:1013958519734 Cleaves, H.J., C. Butch, P.B. Burger, J. Goodwin, and M. Meringer. 2019. One among millions: the chemical space of Nucleic Acid-like molecules. Journal of Chemical Information and Modeling, 59, 4266-4277. https://doi.org/10.1021/acs.jcim.9b00632 Clemett, S. J., S. A. Sandford, K. Nakamura-Messenger, F. Horz, D. and McKay. 2010. Complex aromatic hydrocarbons in Stardust samples collected from comet 81P/Wild 2. Meteoritics and Planetary Science, 45, 701-722. https://doi.org/10.1111/j.1945-5100.2010.01062.x Colaprete, A., P. Schultz, J. Heldmann, D. Wooden, M. Shirley, K. Ennico, B. Hermalyn, et al. 2010. Detection of water in the LCROSS ejecta plume. Science, 330, 463-468. https://doi.org/10.1126/science.1186986 Cruikshank, D. P., and R. H. Brown. 1987. Organic matter on asteroid 130 Elektra. Science, 238, 183- 184. https://doi.org/10.1126/science.238.4824.183 Cruikshank, D. P., C. K. Materese, Y. J. Pendleton, P. J. Boston, W. M. Grundy, B. Schmitt, C. M. Lisse, et al. 2019. Prebiotic chemistry of Pluto. Astrobiology, 19, 831-848. http://doi.org/10.1089/ast.2018.1927 Deamer, D., and B. Damer. 2017. Can life begin on Enceladus? A perspective from hydrothermal chemistry. Astrobiology, 17, 834-839. https://doi.org/10.1089/ast.2016.1610 PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-20 

Delitsky, M. L., D. A. Paige, M. A. Siegler, E. R. Harju, D. Schriver, R. E. Johnson, and P. Travnicek. 2017. Ices on Mercury: Chemistry of volatiles in permanently cold areas of Mercury’s north polar region. Icarus, 281, 19-31. https://doi.org/10.1016/j.icarus.2016.08.006 De Sanctis, M. C., V. Vinogradoff, A. Raponi, E. Ammannito, M. Ciarniello, F. G. Carrozzo, S. De Angelis, et al. 2018. Characteristics of organic matter on Ceres from VIR/Dawn high spatial resolution spectra. Monthly Notices of the Royal Astronomical Society, 482, 2407-2421. https://doi.org/10.1093/mnras/sty2772 Des Marais, D.J., L.J. Allamandola, S.A. Benner, A.P. Boss, D. Deamer, P.G. Falkowski, J.D. Farmer, et al. 2003. The NASA Astrobiology Roadmap. Astrobiology, 3, 219-235. https://doi.org/10.1089/153110703769016299 Des Marais, D.J., J.A. Nuth, L.J. Allamandola, A.P. Boss, J.D. Farmer, T.M. Hoehler, B.N. Jakosky, et al. 2008. The NASA Astrobiology Roadmap. Astrobiology, 8, 715-730. https://doi.org/10.1089/ast.2008.0819 Ditzler, M.A., A.C. Rios, M. Nuevo, M. Popovic, R. Mancinelli, D. Summers, J.T. Broddrick, et al. 2020. Beyond targeted searches: The need for system-level approaches to understanding the connection between astrochemistry and the emergence of life. White paper submitted to the Planetary Science and Astrobiology Decadal Survey. Bulletin of the AAS, 53, 4. https://doi.org/10.3847/25c2cfeb.7a06f84d Eigenbrode, J.E., R.E. Summons, A. Steele, C. Freissinet, M. Millan, R. Navarro-Gonzalez, B. Sutter, et al. 2018. Organic matter preserved in 3-billion-year-old mudstones at Gale Crater, Mars. Science, 360, 1096-1101. https://doi.org/10.1126/science.aas9185 Europa Lander Study 2016 Report: Europa Lander Mission. 2016. JPL D-97667. https://solarsystem.nasa.gov/docs/Europa_Lander_SDT_Report_2016.pdf Field, C.B., M.J. Behrenfeld, J.T. Randerson, and P. Falkowski. 1998. Primary production of the biosphere: Integrating terrestrial and oceanic components. Science, 281, 237-240. https://doi.org/10.1126/science.281.5374.237 Fink, U., M. Hoffmann, W. Grundry, M. Hicks, and W. Sears. 1992. The steep red spectrum of 1992 AD: An asteroid covered with organic material? Icarus, 97, 145-149. https://doi.org/10.1016/0019- 1035(92)90064-E Freissinet, C., D. P. Glavin, P. R. Mahaffy, K. E. Miller, J. L. Eigenbrode, R. E. Summons, A. E. Brunner, et al. 2015. Organic molecules in the Sheepbed Mudstone, Gale Crater, Mars. Journal of Geophysical Research: Planets, 120, 495-514. https://doi.org/10.1002/2014JE004737 Greaves, J.S., et al. 2020. Phosphine gas in the cloud decks of Venus. Nature Astronomy 5, pp. 655–664. https://doi.org/10.1038/s41550-020-1174-4 Hamill, C. D., N. L. Chabot, E. Mazarico, M. A. Siegler, M. K. Barker, and J. M. Martinez Camacho. 2020. New illumination and temperature constraints of Mercury’s volatile polar deposits. The Planetary Science Journal, 1, 57. https://doi.org/10.3847/PSJ/abb1c2 Higgins, P.M., and C.S. Cockell. 2020. A bioenergetic model to predict habitability, biomass and biosignatures in astrobiology and extreme conditions. Journal of the Royal Society Interface, 17, 1- 13. https://doi.org/10.1098/rsif.2020.0588 Hoehler, T., W. Brinckerhoff, A. Davila, D. Des Marais, S. Getty, D. Glavin, A. Pohorille, et al. 2020. Groundwork for Life Detection: A perspective contributed by The Center for Life Detection. White paper submitted to the Planetary Science and Astrobiology Decadal Survey. Hoshika, S., N.A. Leal, M.J. Kim, M.S. Kim, N.B. Karalkar, H.J. Kim, A.M. Bates, et al. 2019. Hachimoji DNA and RNA: A genetic system with eight building blocks. Science, 363, 884-887. https://doi.org/10.1126/science.aat0971 Johnson, N. M., and M. R. R. de Oliveira. 2019. Venus atmospheric composition in situ data: A compilation. Earth and Space Science, 6, 1299-1318. https://doi.org/10.1029/2018EA000536 Kharecha, P., J. Kasting, J. Siefert. 2005. A coupled atmosphere-ecosystem model of the early Archean Earth. Geobiology, 3, 53-76. https://doi.org/10.1111/j.1472-4669.2005.00049.x PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-21 

Krissansen-Totton, J., R. Buick, and D.C. Catling. 2015. A statistical analysis of the carbon isotope record from the Archean to Phanerozoic and implications for the rise of oxygen, American Journal of Science, 315, 275-316. https://doi.org/10.2475/04.2015.01 Lang, S.Q., G.L. Früh-Green, S.M. Bernasconi, W.J. Brazelton, M.O. Schrenk, and J.M. McGonigle. 2018. Deeply-sourced formate fuels sulfate reducers but not methanogens at Lost City hydrothermal field. Scientific Reports, 8, 755. https://doi.org/10.1038/s41598-017-19002-5 Limaye, S.S., R. Mogul, D.J. Smith, A.F. Ansari, G.P. Slowik, and P. Vaishampayan. 2018. Venus’ spectral signatures and the potential for life in the clouds. Astrobiology, 18, 1181-1198. https://doi.org/10.1089/ast.2017.1783 Lollar, G.S., O. Warr, J. Telling, M.R. Osburn, and B. Sherwood Lollar. 2019. ‘Follow the Water’: Hydrogeochemical Constraints on Microbial Investigations 2.4 km Below Surface at the Kidd Creek Deep Fluid and Deep Life Observatory, Geomicrobiology Journal, 36, 859-872, https://doi.org/10.1080/01490451.2019.1641770 Lunine, J.I., M.L. Cable, S.M. Horst, and M. Rahm. 2020. The Astrobiology of Titan in Planetary Astrobiology, Eds. V. Meadows et al., pp. 247-266, Univ of Arizona, Tucson, DOI: https://doi.org/10.2458/azu_uapress_9780816540068-ch010 Lyons, T.W., K. Rogers, R. Krishnamurthy, L. Williams, S. Marchi, E. Schwieterman, N. Planavsky, and C. Reinhard. 2020. Constraining prebiotic chemistry through a better understanding of Earth’s earliest environments. White paper submitted to the Planetary Science and Astrobiology Decadal Survey. https://arxiv.org/abs/2008.04803 Marshall, S.M., A.R.G. Murray, and L. Cronin. 2017. A probabilistic framework for identifying biosignatures using Pathway Complexity. Royal Society Philosophical Transactions A, 375, 2109. https://doi.org/10.1098/rsta.2016.0342 McCord, T. B., R. W. Carlson, W. D. Smythe, G. B. Hanson, R. N. Clark, C. A. Hibbitts, F. P. Fanale, et al. 1997. Organics and other molecules in the surfaces of Callisto and Ganymede. Science, 278, 271-275. https://doi.org/10.1126/science.278.5336.271 McCord, T. B., G. B. Hansen, R. N. Clark, P. D. Martin, C. A. Hibbitts, F. P. Fanale, J. C. Granahan, et al. 1998. Non-water-ice constituents in the surface material of the icy Galilean satellites from the Galileo near-infrared mapping spectrometer investigation. Journal of Geophysical Research: Planets, 103, 8603-8626. https://doi.org/10.1029/98JE00788 Meadows, V.S. 2017. Reflections on O 2 as a biosignature in exoplanetary atmospheres. Astrobiology, 17, 1022-1052. https://doi.org/10.1089/ast.2016.1578 Meadows, V.S., C.T. Reinhard, G.N. Arney, M.N. Parenteau, E.W. Schwieterman, S.D. Domagal- Goldman, et al. 2018. Exoplanet biosignatures: Understanding oxygen as a biosignature in the context of its environment. Astrobiology, 18, 630-662. https://doi.org/10.1089/ast.2017.1727 McDermott, J.M., J.S. Seewald, C.G. German, and S.P. Sylva. 2015. Pathways for abiotic organic synthesis at submarine hydrothermal fields. Proceedings of the National Academy of Sciences, 112, 7668-7672. https://doi.org/10.1073/pnas.1506295112 McKay, C., A. Davila, J. Eigenbrode, C. Lorentson, R. Gold, J. Canham, A. Dazzo, et al. 2020. Contamination control technology study for achieving the science objectives of life-detection missions. NASA STI Program, NASA/TM-20205008709 Mogul, R., S. S. Limaye, M. J. Way, and J. A. Cordova. 2021. Venus’ mass spectra show signs of disequilibria in the middle clouds. Geophysical Research Letters, 48, 7. https://doi.org/10.1029/2020GL091327 National Academy of Sciences, Engineering, and Medicine (2019) An Astrobiology Strategy for the Search for Life in the Universe. The National Academies Press, Washington, D.C., https://doi.org/10.17226/25252 National Research Council. 2007. The Limits of Organic Life in Planetary Systems. Washington, DC: The National Academies Press. https://doi.org/10.17226/11919 PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-22 

Postberg, F., N. Khawaja, B. Abel, G. Cholet, C. R. Glein, M. S. Gudipati, B. L. Henderson, et al. 2018. Macromolecular organic compounds from the depths of Enceladus. Nature, 558, 564-568. https://doi.org/10.1038/s41586-018-0246-4 Quirico, E., S. Doute, B. Schmitt, C. de Bergh, D. P. Cruikshank, T. C. Owen, T. R. Geballe, and T. L. Roush. 1999. Composition, physical state, and distribution of ices at the surface of Triton. Icarus, 139, 159-178. https://doi.org/10.1006/icar.1999.6111 Sherwood Lollar, B., T.C. Onstott, G. Lacrampe-Couloume, and C.J. Ballentine. 2014. The contribution of the Precambrian continental lithosphere to global H2 production. Nature, 516, 379-382. https://doi.org/10.1038/nature14017 Sholes, S.F., J. Krissansen-Totton, and D.C. Catling. 2019. A Maximum Subsurface Biomass on Mars from Untapped Free Energy: CO and H2 as Potential Antibiosignatures. Astrobiology, 19, 655-668. https://doi.org/10.1089/ast.2018.1835 Snellen, I.A.G., L. Guzman-Ramirez, M.R. Hogerheijde, A.P.S. Hygate, and F.F.S. van der Tak. 2020. Re-analysis of the 267 GHz ALMA observations of Venus. Astronomy and Astrophysics, 644, 1-3. https://doi.org/10.1051/0004-6361/202039717 Spear, J.R., J.J. Walker, T.M. McCollom, and N.R. Pace. 2005. Hydrogen and bioenergetics in the Yellowstone geothermal ecosystem. Proceedings of the National Academy of Sciences, 102, 2555- 2560. https://doi.org/10.1073/pnas.0409574102 Steel, E. L., A. Davila, and C. P. McKay. 2017. Abiotic and biotic formation of amino acids in the Enceladus ocean. Astrobiology, 17, 862-875. http://doi.org/10.1089/ast.2017.1673 Sutherland, J.D. 2017. Opinion: Studies on the origin of life—the end of the beginning. Nature Reviews Chemistry, 1, 0012. doi: https://doi.org/10.1038/s41570-016-0012 Theiling, B., W. Brinckerhoff, J. Castillo-Rogez, L. Chou, V. Da Poian, H. Graham, S.S. Hosseini, et al. 2021. Non-robotic science autonomy development. White paper submitted to the Planetary Science and Astrobiology Decadal Survey. Bulletin of the AAS, 53, 4. Thompson, W.R., S.K. Singh, B.N. Khare, and C. Sagan. 1989. Triton: Stratospheric molecules and organic sediments. Geophysical Research Letters, 16, 981-984. https://doi.org/10.1029/GL016i008p00981 Vago, J.L., F. Westall, Pasteur Instrument Teams, Landing Site Selection Working Group, and other contributors. 2017. Habitability on Early Mars and the Search for Biosignatures with the ExoMars Rover. Astrobiology, 17, 471-510. https://doi.org/10.1089/ast.2016.1533 van Kranendonk, M. J., V. Bennett, and E. Hoffmann. 2018. Earth’s Oldest Rocks. 2nd edition. Elsevier Science, Cambridge, MA. 1078 pp. ISBN: 978-0-444-63901-1 Waite, J.H., M.R. Combi, W.-H. Ip, T.E. Cravens, R.L. McNutt, W. Kasprzak, R. Yelle, et al. 2006. Cassini Ion and Neutral Mass Spectrometer: Enceladus plume composition and structure. Science, 311, 1419-1422. https://doi.org/10.1126/science.1121290 Ward, L.M., B. Rasmussen, and W.W. Fischer. 2019. Primary productivity was limited by electron donors prior to the advent of oxygenic photosynthesis. Journal of Geophysical Research: Biogeosciences, 124, 211–226. https://doi.org/10.1029/2018JG004679 Ward, P.D., and D. Brownlee. 2000. Rare Earth. Copernicus Books, Springer, New York, NY. https://doi.org/10.1007/b97646 Weiss, B.P., Y.L. Yung, and K.H. Nealson. 2000. Atmospheric energy for subsurface life on Mars? Proceedings of the National Academy of Sciences, 97, 1395-1399. https://doi.org/10.1073/pnas.030538097 Wickramasinhe, D. T., and D. A. Allen. 1986. Discovery of organic grains in comet Halley. Nature, 323, 44-46. https://doi.org/10.1038/323044a0 Young, T.S., and P.G. Schultz. 2010. Beyond the canonical 20 amino acids: expanding the genetic lexicon. Journal of Biological Chemistry, 285, 11039-11044. https://doi.org/10.1074/jbc.R109.091306 PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-23 

Zhang, J.A., and D.A. Paige. 2009. Cold-trapped organic compounds at the poles of the Moon and Mercury: Implications for origins. Geophysical Research Letters, 36, 16. https://doi.org/10.1029/2009GL038614 Zhou, Y., X. Xu, Y. Wei, Y. Cheng, Y. Guo, I. Khudyakov, F. Liu, et al. 2021. A widespread pathway for substitution of adenine by diaminopurine in phage genomes. Science, 372, 512-516. https://doi.org/10.1126/science.abe4882 PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION 14-24 

Q12 PLATE: Artist’s depiction of Kepler-186f, an Earth-sized planet orbiting a distant star in the habitable zone, discovered in 2014. Additional exoplanets can be seen closer to the star. SOURCE: NASA Ames/SETI Institute/JPL-Caltech. PREPUBLICATION COPY – SUBJECT TO FURTHER EDITORIAL CORRECTION