Philippa Kaur

This paper describes the complexity of gravitational fractals in terms of global and local dimensions. They are presented in Table 1.Table 1 Global and local dimensions of gravitational fractals and attraction basins.Full size tableThe fractal in hexagonal CPT space, shown in Fig. 1, has a very rich structure, and therefore its characterization by means of fractal dimensions requires two approaches: (1) a global approach treating the fractal as a complex whole and (2) a local approach which allows us to determine the dimension of its fragments which are particularly interesting from a research perspective (see also Table 1). In the subsequent part of the paper, the results obtained are presented and interpreted according to the division in the table.Global dimension of boundaries of gravity attraction basinsTwo types of fractal dimensions have been thus far used in this analysis, i.e., the box and ruler dimensions. Figure 3 shows the distribution of the values of these dimensions determined for the boundaries of attraction as a function of space friction μ.Figure 3Comparison of the variability of the global ruler and box dimensions. Legend: The edge of all attraction basins is a function of the μ coefficient; 1–edges of all basins, 2–entire basins.Full size imageFigure 3 empirically confirms a fact known from chaos theory that whenever a fractal represents full chaos, the ruler dimension may be greater than 2 (Peitgen et al.33, 192–209), whereas the box dimension never exceeds this extreme value. Clearly, for a certain value of μ (in this case μ = 0.19), the numerical values of both types of dimensions are identical.In the bottom part of Fig. 3, line 1 illustrates the variability of the shapes of the attraction basins of individual cities depending on the value of μ, i.e., space resistance. The initially extremely complex shapes of the boundaries are smoothed to take the form of straight lines in the case of a large value of μ (μ = 0.52).In turn, line 2 illustrates not only the boundaries of the attraction basins, but also their internal structure. Clearly, the initially chaotic impacts of individual cities on the agent (μ = 0.005) are gradually smoothed out, so that in the final stage of the process they fully stabilize. This means that each city has a geometrically identical basin of attraction. Hence, if the agent is in the attraction basin of city 1 (purple color), it will always be attracted only by that city. This rule also applies to the other cities. It is obvious that the random process occurring at μ = 0.09 is then replaced by a strictly deterministic one. When chaos becomes complete order (Banaszak et al.15, the numerical values of both types of dimensions appear to stabilize at the level of 1.Global dimension of the boundary of each separate attraction basinFigure 1 also shows the geometric image of the attraction basins of individual cities. They were almost identical, and therefore also the fractal dimensions of the boundaries of these basins must match. The validity of this proposition is confirmed by Fig. 4. Six lines representing the distribution of the fractal dimension of the boundaries of the six basins coincide with almost full accuracy. Further analysis of Fig. 4 allows us to infer the conclusion that there is almost total chaos at the value db = 1.9021 (μ = 0.005). On the other hand, as space resistance increases to the value of μ = 0.22, there is a rapid decrease in the value of the fractal dimension of the boundary of each basin to the level of 1.2628; when μ = 0.34, then db = 1.2382. In that case, the value of the fractal dimension stabilizes, and at μ = 0.46, db = 1.2444 and finally for μ = 0.52, db reaches the value of 1.0412. The icons presented in Fig. 4 in lines 1 and 2 have slightly different structures than the icons in Fig. 3, due to different values of μ in certain cases.Figure 4The box dimension of the edges of the attraction basins depending on the μ coefficient (separately for each attractor). Legend: 1–boundaries of single attraction basins, 2–entire basins.Full size imageThe global dimension of the attraction basin of each city as an irregular geometric figureThe full symmetry of the basins of attraction of individual cities can be disturbed by the shape of the geometric figure on which the deterministic fractal is modeled. Such a situation occurs in the present case. Due to the fact that the fractal in Fig. 1 is formed on the surface of a square, the final basins of attraction of cities 1, 3, 4 and 6 are obviously larger than those of cities 2 and 5. Of course, these differences do not occur when considering the surface inside the hexagon.In Fig. 5, the line marked in black color represents the average value of the fractal dimension of the basins of attraction of individual cities, the value of which is (overline{{d }_{b}}=1.77). It can be seen that at very high values of the fractal dimension in the range (1.750, 1.775), there are db oscillations around this line. This is precisely the effect of modeling the fractal on the surface of the square, rather than the properties of this fractal. Therefore, (overline{{d }_{b}}=1.77) should be regarded as the global dimension of the basin of attraction (of each city) treated as an irregular figure.Figure 5Box dimension of the attraction basins as a geometric irregular figure in the gravitational fractal. Legend: 1-basins of the first city, 2-basins of the second city, 7-basins of all cities.Full size imageLocal dimensions of the boundary of the selected characteristic fragmentsFigure 6 presents fractal dimensions, with the Box and Ruler as functions of μ, and the boundaries of the attraction basins of individual cities occurring in all fragments A, …, E.Figure 6Distribution of the values of fractal dimensions of the boundaries of the attraction basins identified in selected fragments of a fractal; Legend: (A, D)-fragments marked in Fig. 1.Full size imageIt is evident that the structures of Fig. 6 (Box and Ruler) are almost identical. This means that, as has been stated earlier, when describing complex fractal objects, it does not really matter which type of dimension is used.Of interest here is the variability of the structure of both figures along with the increase in the value of the parameter μ. Fragments A, …, E (see Fig. 1) are characterized by high complexity, i.e. the intertwining attraction basins of the individual attractors (cities). This observation is confirmed by the numerical results of both fractal dimensions whose values are in the range (1.68–1.82). To illustrate the spatial complexity of these fragments, and thus their dimensions, by way of example, two fractal fragments are considered below: fragments A and D (see also Fig. 7).Figure 7Box dimension of the edge of each gravitation basin in A and D. Legend: The icons show the variability of the fragments A and D due to the share of the attraction basins of individual cities (3, 4 and 6).Full size imageFigure 6 offers important conclusions concerning the organization of social and economic life in the geographical area surrounding individual cities (attractors).

1.

Out of all the separated fragments, only in fragment A do we find the attraction basins of all the cities intertwined across the entire range of variation μ, i.e. (0.00–0.48). Hence, the graph of fractal dimension (db) (blue line) as a function of μ is continuous, and when the resistance of space is the greatest (μ = 0.48), the fractal dimension d = 1.00. This means that chaos has given way to total order, and fragment A has been symmetrically divided between cities 1 and 6. Hence, there are two colors left, namely red and purple.

2.

A similar situation occurs in the case of fragment D (yellow line), where the attraction basins of individual cities intertwine continuously within the range: 0.00 ≤ μ ≤ 0.46. Beyond the value of 0.46, the entire fragment D is filled with purple: the closest city 1 dominates it.

The research conducted here also confirms the conclusions presented in previous works by Banaszak et al.15,16 concerning the transformation of chaos into spatial order, which means the stabilization of permanent dominance, usually of one attractor (city). Thus, with regard to fragments A and D, in fragment A there is a constant dominance (in half of the area) of cities 1 and 6, from the limit value of μ = 0.24 onward. In the case of fragment D, beginning with the value of μ = 0.36, only city 1 dominates (purple). That is, in the final phase of establishing the order in spatial interactions in the arrangement of areas A and D, the role of the dominant attractor (city) is played by city 1 (purple).Due to the symmetry of Fig. 1, similar effects can be observed in other parts of this fractal, located symmetrically in relation to A, …, E (see Supplementary Material).Figures 1 and 6 confirm the findings, known in the theory of city development, that urban (and other) centers rise in the hierarchy (or their rank decreases), depending on the external and internal factors conditioning their development. In the model used in this study, the parameter μ represents external factors (space resistance). If μ values are low, all cities are attractive from the point of view of spatial interactions and create their own but symmetrical basins of attraction. When the resistance of space increases, one city becomes the dominant center, and its basin of attraction is a uniform compact isotropic surface.However, this is not a simple mechanism, since, as has been demonstrated by simulation experiments described in this paper, within a certain range of μ values, another city (attractor) may dominate the others during chaotic interactions. The dynamic history of urban development confirms this observation, for example, in relation to historical capitals of some countries that have lost their functions as administrative capitals.Local dimension of the boundary of each attraction basin in a selected fragment of a fractalFragments A, …, E (Fig. 1 and the Supplementary Material) consist of mutually intertwined basins of attraction (six cities) whose boundaries with complicated courses have a fractal dimension, e.g. a box dimension.Figure 7(fragment A) shows the distribution of db as a function of μ in this fragment. In the case of total internal chaos, the fractal dimension of the boundaries of the attraction basins of all cities is identical and amounts to 1.9152. A clear differentiation of db is noticeable from μ = 0.1 onward. It should also be noted that orange and blue, red and purple, yellow and green lines mutually coincide. The red–purple line tend towards db = 1 as μ increases. However, orange, blue, yellow and green lines reach a value of db = 0.The fractal dimension db = 1.0 is most closely represented by the blue line (city 2), then the red line (city 6) and the purple line (city 1). Since these lines almost coincide, and the red and purple lines are the last to reach the value db = 1, at μ = 0.48, fragment A is symmetrically covered in red and purple. Therefore, with very high spatial resistance, fragment A is dominated by two cities, namely by 1 and 6.In turn, Fig. 7(fragment D) illustrates the variability of the fractal dimension of boundaries of the attraction basins in this fragment. This dimension depends on the complexity of the mosaic patterns formed in this fragment, with varying μ values. When the values of μ are close to zero, all cities contribute to filling the space of fragment D. When μ = 0.18, city 1 (purple color) falls out of the competition for space, but only up to the value of μ = 0.24, when it starts to compete again with other cities. From the point of view of spatial interactions, in the final phase of this process (μ = 0.44), city 2 (blue) and city 6 (red) dominate to a small extent, because cities 3, 4 and 6, starting from μ = 0.3, do not play any role in fragment D.Figure 7 shows that the value μ = 0.3 is a characteristic point. It is a locus where all the curves representing the attraction basins of individual cities meet. As has already been stated, three of them lose their influence over the space of fragment D.Local dimensions of parts of the attraction basins treated as an irregular geometric figureIn each of the selected fragments A, …, E, some of the boundaries of the attraction basins of individual cities are distributed differently. They create certain holes in the form of irregularly colored mosaic patterns that have a certain fractal dimension. To present its variability, fragments A and D were used again. Figure 8 shows the distribution of db values depending on the value of μ.Figure 8Local dimensions of parts of the attraction basins treated as an irregular geometric figure in (A) and (D). Legend: The icons illustrate the variability of the shape of some of the attraction basins of individual cities in fragment (A) and (D) for cities 3, 4 and 6.Full size imageThe function has several characteristic points. Up to the value of μ = 0.04, attraction basins show a jumble in which no predominant color or shape can be identified. The fractal dimension is then: db = 1.7697. From this value onwards, where μ = 0.042, the interior of fragment A becomes increasingly ordered. With a value of μ = 0.125, the city’s attraction basins 3 and 4 begin to disappear in fragment A. The same happens to the city attraction basins 2 and 5 for the value of μ = 0.24.The final effect of the increase in space resistance (with μ = 0.50) leads to the filling of fragment A with two colors, i.e., purple and red. This means that cities 1 and 6, have won the competition for the space of fragment A. In this case, the fractal dimensions db equal 1.90.Figure 8 presents the variability of the fractal dimension and the effects of the competition for space between cities in fragment D. As is the case in fragment A and all others, i.e. B, C and E (see the Annex with Supplementary Material), the intertwined attraction basins are represented by the area consisting of an endless number of differently colored dots. Hence, up to the value of μ = 0.042, fragment D is dominated by pure spatial chaos that extends over its entire area. It is characterized by the fractal dimension db = 1.7697. This means that with an increase in the value of μ, for the emergence of an irregular shape of a geometric figure, chaos must be accompanied by an increase in the value of the fractal dimension. Its limiting value is number 2. Then, spatial dominance is usually gained by one city and the examined fragment is filled with one color (‘the winner takes it all’).This is precisely the situation in Fig. 8 where city 1 (purple color) has apparently won the competition. Since this color fills area D completely, we find the plausible result db = 2.0. More

Differences in mucus physicochemical factors and nutrient elements among electrical stimuliPhysical and chemical factorsMucus contains electrolytes, such as potassium and multivalent calcium and magnesium ions, which participate in the osmoregulation of the earthworm body to maintain the metabolic balance of the organism7,23. When earthworms are subjected to different stimuli, the mucus composition changes10. As shown in Fig. 1a, earthworms produced mucus with significant (P  More

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This study provides quantitative data for Ocyropsis spp. feeding mechanisms and in situ data for gut contents during both day and night to begin assessing their trophic role in oceanic waters. Previous studies qualitatively described the feeding pattern of Ocyropsis spp.15 whereby this animal uses a unique capture mechanism among lobate ctenophores: direct transfer from lobe to mouth and encounters involving the mouth actively grabbing copepod prey24. These previous observations are confirmed as Ocyropsis spp. is able to deploy its dexterous, prehensile mouth to effectively capture prey within the lobes (Figs. 2, 3) and quantitative assessments of predation are also provided. It should be noted that while Ocyropsis spp. are known to occasionally consume a wide variety of prey types and sizes15, this study focuses only on copepod prey because our field data showed recognizable prey in Ocyropsis spp. guts was almost exclusively copepods.For example, mean speed of the mouth is less than 6 mm s−1 during predation events on copepods. Thus, while it may look rapid to the human eye, this is far below the escape swimming speeds exhibited by many copepods which are capable of moving at speeds of up to 500 mm s−125,26. Our observations show that the mechanism of capture is thus not reliant on grabbing copepods from the water between the ctenophore lobes with the mouth, but rather aided by copepod contact with the ctenophore lobes. Copepods between the lobes swam only with a speed of 7.94 mm s−1 (S.D. 7.25), to which the average mouth speed (5.83 mm s−1 (S.D. 1.68)) is comparable (Table 1). This suggests that Ocyropsis is able to reduce copepod swimming activity either by trapping them against the lobes (lobes respond to contact by prey) and/or the use of some form of adhesion or chemical that acts to reduce copepod activity. This unusual form of predation using a prehensile mouth allows Ocyropsis to be highly effective predators without the use of prey capturing tentillae seen in other lobate species.The presence of multiple prey has the potential to disrupt a raptorial type feeder such as Ocyropsis spp. more so than other lobates, since they lack tentillae, which would allow them to capture multiple prey simultaneously. Instead Ocyropsis spp. transfer one prey at a time directly from lobe to mouth15,27. So how is this ctenophore able to maintain such a high overall capture rate? The answer appears to be that Ocyropsis will modulate the number of attempts with the prehensile mouth depending on the number of prey present. For example, we did not observe any captures on the first attempt with the mouth with multiple prey, but the animals made up to 8 attempts at capturing the nearest copepod. This is in contrast to single copepod encounters in which ctenophores captured copepods on the first attempt 61% of the time and rarely made over 2 attempts, never exceeding 3 attempts (Figs. 3a, 5a, Table 1). This demonstrates Ocyropsis spp. can adjust its behavior to maintain high overall capture success when presented with multiple simultaneous prey. It is also interesting to note that the resulting increase in handling time due to making more attempts during multiple prey encounters is still lower than the handling time for most other lobates dealing with single prey27,28. It is not clear how often Ocyropsis spp. need to deal with multiple copepods simultaneously in nature, as oceanic waters contain characteristically low ctenophore prey densities compared to coastal zones9,29, however prey can be highly patchy and it appears that the unique prey capture mechanism of Ocyropsis spp. is still able to operate effectively in high density patches by increasing the number of attempts before aborting the attack which could serve as a means to maintain similar ingestion rates to single prey encounters.Typically, the feeding sequence of a ctenophore involves capture of prey in sticky colloblast cells and retraction of tentillae and/or ciliary transport of prey to the mouth15,27,30. These feeding mechanisms result in a range of handling times ranging from 2.5 s for Bolinopsis. infundibulum28 to nearly 22 min for Pleurobrachia bachei27. Capture rates can also be quite high, with overall capture success rates up to 74% for Mnemiopsis leidyi2,3. We found Ocyropsis has a relatively fast mean handling time of 6.3 s when a single copepod was present between the lobes, but handling time increased by approximately 2.5-fold if multiple prey were present. Overall capture success rates were comparable to the highly effective coastal ctenophore, M. leidyi, with a 71% success rate with single prey present and 81% capture rates if multiple prey were present between the lobes. Thus, Ocyropsis spp. are able to capture prey with high efficiency despite the differences in feeding mechanics compared to coastal lobate ctenophores. Additionally, since encounter rates of planktivores are directly related to the time spent searching for prey and time spent handling prey27, the relatively short handling time of Ocyropsis spp. and their direct feeding mechanism may allow them to sample more water and encounter a larger proportion of the available prey population than other species.Diel patterns of prey consumptionMany planktivorous species exhibit higher gut fullness at night31,32, due to higher prey availability in surface waters as a result of a diel vertical migration33,34. In situ gut content images showed that Ocyropsis spp. had a significantly higher gut fullness at night (12.4%) compared to during the day (4.2%) (Fig. 7). Ocyropsis spp. also had higher numbers of prey per individual gut at night, although overall biomass was not significantly different between night and day (Fig. 7). This can be explained by differences in prey characteristics; prey observed in the gut during the day were significantly larger (Table 2). This may be due to an ability to feed more selectively during the day since overall prey densities are lower. It should also be considered that turbulence in surface waters is, on average, much lower at night compared to daytime35 and that even small amounts of turbulence can negatively impact ctenophore feeding36,37. Therefore, smaller prey may have a higher likelihood of evading detection of Ocyropsis during the day compared to night, especially since these animals are most frequently observed in the upper 15 m of oceanic waters.Kremer, et al.38 estimates that O. crystallina requires 252 prey items to sustain itself. On average, Ocyropsis spp. in this study consume over 500 prey d−1. This exceeds their metabolic demands and suggests the observed population, on the western edge of the Gulf Stream, are likely to be actively growing and reproducing. The time required to digest prey items averaged 44 min for Ocyropsis which is faster than many, but not all, gelatinous zooplankton39,40,41. Digestion times of other gelatinous taxa span a range of times from 15 min to over 7 h at 20 °C40 and are impacted by size and number of prey per gut as well as temperature39,42,43. Digestion observations were performed at an ambient temperature of 25 °C and thus, these numbers represent a conservative estimate because the temperature of the water from which the animals were collected was 26.7–27.4 °C. Ocyropsis spp. would likely experience an increase in digestion rate with increased temperature.Digestion time was not impacted by the number of prey in the gut or by ctenophore body length. This differs from trends seen in other gelatinous taxa, such as A. aurita, M. leidyi, and B. infundibulum, where increasing body size resulted in faster digestion time39,40 and where increasing number of prey in the gut leads to longer digestion times39,40,41. In this study however, ctenophores were offered only a few copepods to ingest, thus it is likely they were not fed enough prey to satiate and slow the digestion process. Also worth considering is that the metabolic rate of O. crystallina does not appear to be affected by body size38. Though metabolic rates were not measured, this aligns with our finding that body size had no significant effect on digestion time. Analysis of in situ gut contents showed a significant positive logarithmic relationship between ctenophore length and total prey biomass per gut (Fig. 8). Individuals smaller than 20 mm in this study typically had fewer than the average number of copepods per gut (19), and larger individuals were the main driver of this relationship. This suggests that small Ocyropsis ( More

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The ecological baseline in TirezThe geology and the climate of the Tirez region favored the generation and maintenance of a type of hypersaline habitat characterized by extreme seasonality: the sulfate-chlorine waters, with sodium and magnesium cations, showed significant seasonal variations15. The alkaline pH, the low oxidant value for the redox potential of the water column and the highly reduced sediments imposed extreme conditions (see Table 1 and Supplementary Information for details). This extreme seasonality requires to define a valid representative ecological baseline to compare the ecology of the lagoon between 2002 and 2021 and, in this way, set the basis to proposing our model of ecological succession with increasing dryness as a “time-analog” for early Mars. Taxonomic data from 2002 is a snapshot of the community during one season, so we include in our discussion the results presented by Montoya et al. (2013) from a sample campaign carried out in 2005, because they16 analyzed both water and sediment and during both the wet and dry seasons.We consider here only the results obtained by Montoya et al.16 by gene cloning, since those obtained by isolation and sequencing are not comparable. At the level of large groups, no major seasonal differences were observed: Pseudomonadota, followed by Bacteroidetes, were the dominant phyla, in both water and sediments, and both in the dry and the rainy seasons; although Alphaproteobacteria was the dominant class in water, while Gammaproteobacteria was dominant in sediments (in both dry and rainy seasons). With respect to the archaeal domain, all the identified sequences were affiliated to Halobacteriales order, mainly Halorubrum (water) and Halobacterium (sediment), both within Halobacteriaceae family. We can consider these results presented in Montoya et al.16 as the “ecological baseline” for Tirez, however taken with a grain of salt, because only 43 bacterial and 35 archaeal sequences, including rainy and dry seasons and water and sediments, were considered for analysis.Prokaryotic diversity in 2002As can be expected for an extreme environment, the bacterial diversity detected in 2002 was low, although we cannot exclude the possibility that this may reflect the limitation of DNA sequencing techniques at the time. 59% of the obtained clones in the then-wet sediments corresponded to the Malaciobacter genus. Malaciobacter (previously named17 Arcobacter) is an aerotolerant Epsilonproteobacteria. Species within this genus are moderately halophilic, e.g., M. halophilus, capable to grow in up to 4% NaCl. Even though the role that Malaciobacter can play in the environment is not known, it seems to thrive in aquatic systems, like sewage, with a high organic matter content17: e.g., M. canalis, M. cloacae, or M. defluvii.After Malaciobacter-like clones, the next most numerous group belongs to the phylum Bacillota (27% of the sequenced clones; Table 2). Under stressful environmental conditions, members of the genus Virgibacillus produce endospores, a very useful property in an extreme and variable environment (ionic strength, temperature, light intensity), easy to compare with early Mars. Endospores facilitate species survival, allowing them to overcome drastic negative changes, like dry periods, and to germinate when the conditions are favorable again. The closest identified species was the halotolerant V. halodenitrificans, but with low homology, not far from other halotolerant (e.g., V. dokdonensis) or halophilic (e.g., V. marismortui) species within the same genus. The other Gram-positive clones belong to the order Clostridiales. These clones cluster in two taxonomic units related with the strictly anaerobic genus Tissierella.Despite the abundance of Pseudomonadota, their biodiversity was very low, reduced to only two genera within the Epsilon- and Delta-proteobacteria. Six sequences affiliated to Deltaproteobacteria, and clustered in one OTU (salB38, similarity 96.6% with Desulfotignum), were retrieved. Its presence in anaerobic media rich in sulfates (Table 1) seems reasonable. In fact, sulfate-reducing activity was detected using a specific enrichment assay.Finally, one taxon belonging to the phylum Spirochaetota (previously named Spirochaetes) was identified. The presence of Spirochaetota in this system is not strange because members of the genus Spirochaeta are very often found in mud and anaerobic marine environments rich in sulfates18. Moreover, the closest species to SalB63, although with a low similarity of 87%, was Spirochaeta bajacaliforniensis, a spirochete isolated19 from a microbial mat in Laguna Figueroa (Baja California), an extensive hypersaline lagoon with high gypsum content, very similar, although much bigger, than Tirez lagoon.The diversity within the domain Archaea was very low in 2002. The phylogenetic analysis of 96 clones indicate that they correspond to one specie belonging to the obligate halophile genus Methanohalophilus. Their high similarity (99.3%) with several species of Methanohalophilus, such as M. portucalensis (isolated from sediments of a solar saltern in Portugal), M. mahii (isolated from sediments of the Great Salt Lake), or M. halophilus (isolated20 from a cyanobacterial mat at Hamelin Pool, Australia), makes impossible its adscription to any particular species level. Methanohalophilus is strictly methylotrophic, which is consistent with this environment, given that the methylotrophic methanogenesis pathway, non-competitive at low-salt conditions, is predominant at high saline concentrations21. We further confirmed methanogenic activity in Tirez by the measurement of methane by gas chromatography in enrichment cultures.Prokaryotic diversity in context of other studies between 2002 and 2021It was challenging to establish a timeline for the succession of the populations involved, because the scarcity of data harvested and published so far from Tirez. However, combining our results with the few data available in Montoya et al.16 and Preston et al.22 on samplings carried out on 2005 and 2017, respectively, we can see a clear predominance of the phylum Pseudomonadota: Epsilonproteobacteria, i.e. Arcobacter-like, and Deltaproteobacteria, mainly sulfate-reducing bacteria (this work, sampling 2002), and Gammaproteobacteria16 when Tirez maintained a water film, to eventually a final predominance of Gammaproteobacteria, e.g. Chromatiales and Pseudomonadales, in the dry Tirez (this work, 2020 sampling). The Bacillales order has remained widely represented both in the wet and dry Tirez.Regarding the archaeal domain, the few references available (Refs.16,22; this work) confirm that the members of the Halobacteriaceae family are well adapted to both the humid and dry ecosystems of Tirez, being predominant in both conditions. Preston et al.22 found that the second most abundant group of archaea in the dry sediments of Tirez was the Methermicoccaceae family, within the Methanosarcinales order, Methanomicrobia class. Taking into account the results obtained in the dry Tirez (Preston et al.22; and this work, sample 2020), the methanogenic archaea have decreased drastically through time, probably due to salt stress and the competition with sulfate-reducing bacteria.Prokaryotic diversity in 2021From a metabolic point of view, most of the bacteria present today in the sediment are chemoorganotrophs, anaerobes, and halophilic or halotolerant. Scarce information is available about the predominant OTU, Candidate Division OP1. The OP1 division was one of the main bacterial phyla in a sulfur-rich sample in the deepest analyzed samples from the Red Sea sediments under brine pools23. In addition, the phylogenetically related Candidate division KB1 has been observed in deep-sea hypersaline anoxic basins at Orca Basin (Gulf of Mexico), and other hypersaline environments24. Eight of the nine genera identified show coverage greater than 1% of the sequences: i.e., Rubinisphaera, Halothiobacillus, Thiohalophilus, Anaerobacillus/Halolactibacillus, Halomonas, Halothermothrix, and Aliifodinibius are halophilic or halotolerant genera13,25.Regarding archaea, our analyses reveal archaeal groups that seem to thrive in sediments from extreme environments, e.g., marine brine pools/deep water anoxic basins or hypersaline lakes. The most abundant OTU, Thermoplasmata KTK4A, was found prominent and active in the sediment of Lake Strawbridge, a hypersaline lake in Western Australia26, and in soda-saline lakes in China27. The creation of a Candidatus Haloplasmatales, a novel order to include KTK4A-related Thermoplasmata, has been proposed27. On the other hand, both in the aforementioned soda-saline lakes in China27 and in a sulfur-rich section of the sediments from below the Red Sea brine pools23, retrieved sequences were assigned to Marine Benthic Groups B, D, and E. Finally, in the section of nitrogen-rich sediments from the aforementioned Red Sea brine pools, the unclassified lineage ST-12K10A represented the most abundant archaeal group. In the Tirez Lagoon sediment after desiccation, all Methanomicrobia readings belonged to this group.The significance and implications of an ecosystem characterized in 2021 by high diversity, high inequality, and lack of isolated representatives, resides in that Tirez is today an ecosystem in which many (most) of the species/OTUs present are dormant, and they do not play any metabolic role. Hence the high percentage of raretons, greater than 80% for both bacteria and archaea, which are actually present in the lagoon but with only one or two copies each. Only those species adapted to the conditions imposed by the extreme environment are able to actually thrive, and consequently only a few species carry out all the metabolic activity. We conclude that the microbiota in Tirez today represents an ecosystem with a high resilience capacity in the face of environmental changes that may occur.We want to clearly highlight that the technique available in 2002 to study the microbiota of the Tirez lagoon only allowed to obtain a low-resolution image, but that was the state-of-the-art procedure at the time, and the Tirez lagoon cannot be sampled again with the conditions back in 2002, which no longer exist and are not expected to return. Although we have kept in storage several samples of water and sediment from the 2002 Tirez lagoon, it is reasonable to assume that those laboratory microcosms would have chemically and microbiologically changed during the last 20 years, and as such no longer represent reliable replicas of the original lagoon, so we cannot use them for the purposes of this work. Therefore, we are aware that any comparisons of the 2002 laboratory results with the much more robust results obtained by Illumina in 2021 need to be taken with a grain of salt. With all the precautions required, in a high-level, first-order comparison, the most noticeable difference between 2002 and 2021 is a drastic change in the microbial Tirez population. Only some OTUs within Bacillales (Virgibacillus/Anaerobacillus), sulfate-reducing Deltaproteobacteria (Desulfotignum/Desulfobacteraceae-Desulfovibrio), and Spirochaetes are shared among the 2002 and 2021 samples. This comparison is enough for the purposes of this work, as we are interested in the evolution of the lagoon system as a whole to establish a “time-analog” with the wet-to-dry transition on early Mars, and not in the particular outcome of each and every OTU in Tirez. With the results at hand, we conclude that, since 2002, the lacustrine microbiota has shifted to one more adapted to the extreme conditions in the dry sediments, derived from the gradual and persistent desiccation concluding ca. 7 years ago (i.e., completely desiccated in 2015), such as lack of light, absence of oxygen, and lack of water availability. This shift has likely been triggered because organisms that were originally in the lagoon but at low abundance in 2002 became dominant as they were better adapted to desiccation, and because the incoming of new microorganisms transported by birds or wind28.Lipid biomarkers analysis of the desiccated lake sedimentsThe analysis of cell membrane-derived lipid compounds on the dry lake sediments at present allow to provide another perspective of the microbial communities inhabiting the Tirez lagoon, by contributing additional information about the ecosystem and depositional environment. It is important to note that, analyzing only the 2021 lake sediments, we cannot differentiate between lipidic biomarkers of the microorganisms inhabiting Tirez in 2002 and before from those left behind by the microorganisms living in the dried sediments today. Instead, the analyses of lipid biomarkers provide clues about the different microorganisms that have populated Tirez through time, including both older communities inhabiting the former aqueous system and also younger communities better adapted to the present dry conditions. Thus, the lipid biomarkers analysis can be considered as a time-integrative record of the microbial community inhabiting Tirez during the last decades.Based on the molecular distribution of lipid biomarkers, the presence of gram-positive bacteria was inferred from the relative abundance of the monounsaturated alkanoic acid C18:1[ω9], or iso/anteiso pairs of alkanoic acids from 12 to 17 carbons29 with dominance of i/a-C15:0 and i/a-C17:0 (Fig. 3B). In contrast, generally ubiquitous alkanoic acids such as C16:1[ω7], C18:1[ω7], or C18:2[ω6] suggested a provenance rather related to gram-negative bacteria30. The combined detection of the i/a-C15:0 and i/a-C17:0 acids, with dominance of the iso over the anteiso congeners, together with other biomarkers such as the mid-chain branched 10Me16:0, the monounsaturated C17:1, or the cyclopropyl Cy17:0 and Cy19:0 acids, may be associated with a community of SRB31 in today´s dry sediments of Tirez. Specifically, most of those alkanoic acids have been found in a variety of Deltaproteobacteria and/or Bacteroidota (previously named Bacteroidetes). The presence of archaea was deduced from the detection of prominent peaks of archaeol in the polar fraction32 (Fig. 3C), as well as squalene and relatives (dihydrosqualene and tetrahydrosqualene) in the apolar fraction33 (Fig. 3A). Squalene and a variety of unsaturated derivatives are present in the neutral lipid fractions of many archaea with high abundances in saline lakes34. The relative abundance of autotrophs over heterotrophs35 can be estimated by the ratio of the autotrophically-related pristane and phytane over the both autotrophically- and heterotrophically-produced n-C17 and n-C18 alkanes ([Pr + Ph]/[n-C17 + n-C18]). A ratio of 0.56 in the Tirez sediments suggest the presence of a relevant proportion of heterotrophs in the ancient lacustrine system.Furthermore, the lipid biomarkers analysis was able to detect compounds specific of additional microbial sources, such as cyanobacteria36 (n-C17, C17:1, or 7Me-C15 and 7Me-C17), microalgae and/or diatoms (phytosterols37; or C20:5, and C22:6 alkanoic acids30), and other photoautotrophs (phytol and potentially degradative compounds such as pristane and phytane31). A relatively higher preservation of the cell-membrane remnants (i.e., lipids) compared to the DNA-composing nucleic acids may contribute to explain the lack of detection of cyanobacteria, diatoms and microalgae, and other phototrophs by DNA analysis (a deficit in our results shared with Montoya et al.16, and Preston et al.22). Although abundant in higher plants38, sterols such as those detected here (i.e., the sterols campesterol, stigmasterol, and β-sitosterol, as well as ergosterol) are also major sterols in some microalgal classes37 (such as Bacillariophyceae, Chrysophyceae, Euglenophyceae, Eustigmatophyceae, Raphidophyceae, Xanthophyceae, and Chlorophyceae), cyanobacteria (β-sitosterol), and fungi (ergosterol39).The carbon isotopic composition of lipid biomarkers provides a rapid screening of the carbon metabolism in a system, by recognizing the principal carbon fixation pathways used by autotrophs. The range of δ13C values measured in the Tirez sediments (from − 33.9 to − 16.1‰) denotes a mixed use of different carbon assimilation pathways, involving mostly the reductive pentose phosphate (a.k.a. Calvin–Benson–Bassham or just Calvin) cycle (from − 19 to − 30‰), and in lesser extent the reductive acetyl-CoA (a.k.a. Wood–Ljungdahl) pathway (from − 28 to − 44‰), and/or the reverse tricarboxylic acid (rTCA) cycle (from − 12 to − 21‰).The lipids synthesized by microorganisms using the Calvin or reductive acetyl-CoA pathway are typically depleted relative to the bulk biomass, particularly those produced via de latter pathway. In the dry Tirez sediments, the majority of the lipid compounds are more depleted in 13C than the bulk biomass (Fig. 4). In particular, the branched alkane DiMeC18 (Fig. 4A) and the SRB-indicative 10Me16:0 acid (Fig. 4B) showed the most depleted δ13C values and suggested the use of the reductive acetyl-CoA pathway. The rest of lipid compounds showed isotopic signatures (from − 16.1 to − 31.4‰) compatible with the prevalence of the Calvin pathway. These values may directly reflect the autotrophic activity of microorganisms fixing carbon via the Calvin cycle or heterotrophic activity of microorganisms growing on their remnants. Thus, the saturated and linear alkyl chains of lipids (i.e., n-alkanes, n-alkanoic acids, and n-alkanols) showing the most negative δ13C values (e.g., alkanes n-C17 and C17:1; or acid C18:1[ω7]) reflect prokaryotic sources of Calvin-users autotrophs (e.g., cyanobacteria or purple sulfur bacteria), while the rest of compounds with slightly less negative δ13C values instead stem from the autotrophic activity of eukaryotes also users of the Calvin cycle (unsaturated fatty acids and sterols) or from the metabolism of heterotrophs such as SRB (iso/anteiso-, other branched, and cyclopropyl fatty acids) and haloarchaea (isoprenoids, phytanol, and archaeol). All in all, the compound-specific isotope composition of the dry sediments in the today´s Tirez lagoon may indirectly reflect the prevailing autotrophic mechanisms in the present lacustrine system of Tirez, by showing isotopic signatures of secondary lipids similar to their carbon source40.In addition, the use of a number of lipid molecular ratios or proxies allow further characterization of the lacustrine ecosystem and depositional environment. For example, the average chain length of the n-alkanes (24.1) suggests a relevant presence of eukaryotic biomass in the lacustrine sediments, since long-chained alkanes ( > C20) are known to originate from epicuticular leaf waxes in higher plants41. Highlighting the relevance of eukaryotes and their ecological roles is one of the major contributions of this work, because previous studies on the microbial ecology of hypersaline environments have been focused primarily on prokaryotes42.The proportion of odd n-alkanes of high molecular chain (i.e., n-C27, n-C29, and n-C31) over even n-alkanes of low molecular chain (i.e., n-C15, n-C17, and n-C19) provides an estimate of the relative abundance of terrigenous over aqueous biomass43, which in Tirez is TAR = 1.8. The Paq index may also be used to differentiate the proportion of terrigenous versus aquatic (emergent and submerged) plant biomass44. A Paq of 0.3 in the Tirez sediments from 2021 supported the relative abundance of land plants. Finally, the depositional environment in the lacustrine system of Tirez may be also characterized analyzing the ratio of pristane over phytane (Pr/Ph), which is higher than 1 when phytol degrades to pristane under oxic conditions45. Assuming that both isoprenoids in the Tirez sediments derived from phytol31, according to their similarly depleted δ13C (Fig. 4A), we can conclude that the sediments in the Tirez lagoon were deposited under predominantly oxic conditions (i.e., Pr/Ph ratio of 1.1).In summary, the lipid biomarkers study revealed useful information about the depositional environment and lacustrine ecosystem, including the presence of active or past autotrophic metabolisms involving prokaryotes (e.g., cyanobacteria and purple sulfur bacteria) and eukaryotes (plants, diatoms and other microalgae), as well as heterotrophic metabolisms of likely SRB and haloarchaea growing on Calvin-users exudates. These results are quite in agreement with the microbial community previously reported16 in sediments from the wet and dry seasons: abundant Gammaproteobacteria and Alphaproteobacteria, together with Algae and Cyanobacteria, dinoflagellates and filamentous fungi, Bacillota, Actinomycetota (previously named Actinomycetes), and a halophilic sulfate-reducing Deltaproteobacteria.Tirez as the first astrobiological “time-analog” for early MarsEarly Mars most likely had a diversity of environments in terms of pH, redox conditions, geochemistry, temperature, and so on. Field research in terrestrial analog environments contribute to understand the habitability of this diversity of environments on Mars in the past, because terrestrial analogues are places on Earth characterized by environmental, mineralogical, geomorphological, or geochemical conditions similar to those observed on present or past Mars9. Therefore, so far analogs have been referred to terrestrial locations closely similar to any of the geochemical environments that have been inferred on Mars, i.e., they are “site-analogs” that represent snapshots in time: one specific environmental condition at a very specific place and a very specific time. Because of this, each individual field analog site cannot be considered an adequate representation of the changing martian environmental conditions through time. Here we introduce the concept of astrobiological “time-analog”, referred to terrestrial analogs that may help understand environmental transitions and the related possible ecological successions on early Mars. In this sense, they should be “time-resolved analogs”: dynamic analog environments where we can analyze changes over time. To the best of our knowledge, this is the first study that looks at the environmental microbiology of a Mars astrobiological analog site over a significant and long period of change, and try to understand the ecological successions to put them in the context of martian environmental evolution.As Mars lost most of its surface water at the end of the Hesperian5,9,12, this wet-to-dry global transition can be considered the major environmental perturbation in the geological history of Mars, and therefore merits to be the first one to be assigned a “time-analog” for its better understanding and characterization. The drying of Mars was probably a stepwise process, characterized by multiple transitions between drier and wetter environments12,47, and therefore the seasonal fluctuations and eventual full desiccation of Tirez represent a suitable analog to better understand possible ecological transitions during the global desiccation of most of the Mars’s surface before the Amazonian (beginning 3.2 Ga).To introduce Tirez as the first Mars astrobiological “time-analog” of the wet-to-dry transition on early Mars, the objective of this study was threefold: first, we wanted to identify the dominant prokaryotic microorganisms in the active Tirez lagoon 20 years ago, a unique hypersaline ecosystem with an ionic composition different from that of marine environments, and therefore potentially analogous to ancient saline lacustrine environments on Mars during the Noachian and into the Hesperian46,47. Our results provide a preliminary basis to hypothesize how the microbial communities on the Noachian Mars could have developed in salty environments with dramatically fluctuating water availability. The requirement to deal with important variations in ionic strength and water availability, involving at times the complete evaporation of the water, could have represented additional constraints48 for microorganisms on early Mars.The second objective of this investigation was the identification of the microbial community inhabiting the desiccated Tirez sediments today, after all the water was lost, as a potential analog to desiccated basins on Mars at the end of the Hesperian1,3,4,47. Our results suggest that hypothetical early microbial communities on early Mars, living with relative abundance of liquid water during the Noachian, would have been forced to adapt to increasingly desiccating surface environments, characterized by extreme conditions derived from the persistent dryness and lack of water availability. Our investigation in Tirez suggest that hypothetical microorganisms at the end of the Hesperian would have needed to evolve strategies similar to those of microorganisms on Earth adapted to living at very low water activity49, to thrive in the progressively desiccating sediments.And the third objective of this investigation was the identification of the lipidic biomarkers left behind by the microbial communities in Tirez, as a guide to searching and identifying the potential leftovers of a hypothetical ancient biosphere on Mars. Lipids (i.e., fatty acids and other biosynthesized hydrocarbons) are structural components of cell membranes bearing recognized higher resistance to degradation relative to other biomolecules, thus with potential to reconstruct paleobiology in a broader temporal scale than more labile molecules50. Our results reinforce the notion that lipidic biomarkers should be preferred targets in the search for extinct and/or extant life on Mars precisely because they are so recalcitrant. More

Biodiversity on Earth is threatened by land-use changes, overexploitation of resources, pollution, biological invasions, and current and projected climate change. Understanding how species will respond to these stressors is difficult, in part because stressors don’t occur in isolation, and because responses can trickle through ecological networks due to interactions among species. More