In this work, we address the difficulty of reliably identifying traces of life on Mars. Several independent lines of evidence are required to build a compelling body of proof. In particular, we underline the importance of correctly interpreting the geological and mineralogical context of the sites to be explored for the presence of biosignatures. We use as examples to illustrate this, ALH84001 (where knowledge of the geological context was very limited) and other terrestrial deposits, for which this could be properly established. We also discuss promising locations and formations to be explored by ongoing and future rover missions, including Oxia Planum, which, dated at 4.0 Ga, is the most ancient Mars location targeted for investigation yet.

Mars exploration motivates the search for extraterrestrial life, the development of space technologies, and the design of human missions and habitations. Here, we seek new insights and pose unresolved questions relating to the natural history of Mars, habitability, robotic and human exploration, planetary protection, and the impacts on human society. Key observations and findings include:

1. – high escape rates of early Mars' atmosphere, including loss of water, impact present-day habitability;

2. – putative fossils on Mars will likely be ambiguous biomarkers for life;

3. – microbial contamination resulting from human habitation is unavoidable; and

4. – based on Mars' current planetary protection category, robotic payload(s) should characterize the local martian environment for any life-forms prior to human habitation.

Some of the outstanding questions are:

1. – which interpretation of the hemispheric dichotomy of the planet is correct;

2. – to what degree did deep-penetrating faults transport subsurface liquids to Mars' surface;

3. – in what abundance are carbonates formed by atmospheric processes;

4. – what properties of martian meteorites could be used to constrain their source locations;

5. – the origin(s) of organic macromolecules;

6. – was/is Mars inhabited;

7. – how can missions designed to uncover microbial activity in the subsurface eliminate potential false positives caused by microbial contaminants from Earth;

8. – how can we ensure that humans and microbes form a stable and benign biosphere; and

9. – should humans relate to putative extraterrestrial life from a biocentric viewpoint (preservation of all biology), or anthropocentric viewpoint of expanding habitation of space?

Studies of Mars' evolution can shed light on the habitability of extrasolar planets. In addition, Mars exploration can drive future policy developments and confirm (or put into question) the feasibility and/or extent of human habitability of space.

Since their discovery, submarine hydrothermal vent systems have been pointed out as important places where chemical evolution on Earth could have occurred; and their role in the process has been highlighted. Similarly, some hypotheses have considered these systems in origin of life scenarios. In this way, many experiments have been developed, and the knowledge about these systems has increased. Due to their complexity, many experimental simulations have only included a few of the geochemical variables present in these environments, pressure and temperature. Other main variables have hardly been included, such as mineralogy, thermal and pH gradients, dissolved ions and/or redox reactions. As it has been understood, the dynamism and heterogeneity of these environments are huge, and it comprises different scales, from single vents to full hydrothermal fields. However, the vast majority of experiments focus on a specific part of these systems and do not include salinity, mineralogy and pH gradients. For this reason, in this paper, we pointed out some considerations about how this dynamism can be interpreted, and included in some models, as well their importance in prebiotic chemistry experiments and their extrapolations regarding the hypothesis about the origins of life.

Bacillus pumilus SAFR-032, an endospore-forming bacterial strain, was investigated to determine its methylation pattern (methylome) change, compared to ground control, after direct exposure to space conditions onboard the International Space Station (ISS) for 1.5 years. The resulting ISS-flown and non-flown strains were sequenced using the Nanopore MinION and an in-house method and pipeline to identify methylated positions in the genome. Our analysis indicated genomic variants and m6A methylation increased in the ISS-flown SAFR-032. To complement the broader omics investigation and explore phenotypic changes, ISS-flown and non-flown strains were compared in a series of laboratory-based chamber experiments using an X-ray irradiation source (doses applied at 250, 500, 750, 1000 and 1250 Gy); results show a potentially higher survival fraction of ISS-flown DS2 at the two highest exposures. Taken together, results from this study document lasting changes to the genome by methylation, potentially triggered by conditions in spaceflight, with functional consequences for the resistance of bacteria to stressors expected on long-duration missions beyond low Earth orbit.