Deep microbial colonization during impact-generated hydrothermal circulation at the Lappajärvi impact structure, Finland

Domain: earth_science
Published: 2025-09-17
Editorial outcome: minor_revision

Abstract
Deeply fractured rocks of meteorite impact structures have been hypothesized as hotspots for microbial colonization on Earth and other planetary bodies. Biosignatures of such colonization are rare, however, and most importantly, direct geochronological evidence linking the colonization to the impact-generated hydrothermal systems are completely lacking. Here we provide timing constraints to microbial colonization of the 77.85 ± 0.78 Ma old Lappajärvi impact structure, Finland, by using coupled microscale stable isotope biosignature detection and radioisotopic dating of vug- and fracture-filling assemblages in impactites. The first detected mineral precipitation at habitable temperatures for life (47.0 ± 7.1 °C) occurred at 73.6 ± 2.2 Ma and featured substantially 34S-depleted pyrite consistent with microbial sulfate reduction. Later stages of vug-mineral precipitation occurred more than 10 Myr later, at gradually lower temperatures, and featured δ13C values diagnostic for both anaerobic microbial consumption and production of methane. These insights confirm the capacity of medium-sized (and large) meteorite impacts to generate long-lasting hydrothermal systems, enabling microbial colonization as the crater cools to ambient conditions, an effect that may have important implications for the emergence of life on Earth and beyond.

Introduction
Meteorite impact craters occur on all planetary bodies in our Solar System. Once thought to be purely destructive events, there is a growing consensus that meteorite impacts may have played an important role in the origin and early evolution of life on Earth1,2. An impact event deposits a tremendous amount of energy in a planetary crust, which results in the fracturing and heating of a huge volume of rock that scales with crater size. Fractured rocks within impact structures are considered key candidates for deep microbial colonization on planetary bodies due to their sustained heat flow, the formation of impact-generated hydrothermal (IGH) systems with distinct geochemical and thermal gradients, and the presence of pore spaces that support microbial colonization1,2. However, biosignatures of microbial colonization have been reported from only a handful of the more than 200 confirmed impact structures on Earth3,4.

Impact events create hydrothermal systems that initially exceed temperatures that hyperthermophiles can survive (up to 122°C5), requiring cooling prior to any microbial colonization6,7. The time needed to reach habitable conditions depends on the size of the impact structure. For example, modeling suggests that the 4 km diameter Kärdla impact structure could become habitable for microbial communities within tens to a few hundreds of years post-impact8. Larger impact structures, which undergo significant melt production and deep heat sterilization, take longer to become habitable.

The scale of the impact, along with other factors2 determines the longevity of IGH systems. Large craters like the ~250 km Vredefort and Sudbury structures as well as in the ~200 km Chicxulub structure, may have hosted persistent IGH systems for hundreds of thousand of years, up to 1–2 million years9–11. In the latter structures, near-surface regions feature effective convective heat transport, while deeper levels are marked by dominantly conductive heat transport6. Mid sized to large structures can host prolonged IGH systems if they are added heat to near-surface parts by meteoric water and groundwaters that are heated at back-circulated from several kilometers depth after infiltration along faults and fractures6,9. High-precision U-Pb dating12 and 40Ar/39Ar-based thermal modeling13 of the 23 km diameter-sized and 77.85 ± 0.78 Ma old, Lappajärvi impact structure in Finland, suggest it has experienced hydrothermal circulation and post-impact cooling below 250°C as late as ~1 to 1.6 Myr after the impact. Duration of this magnitude is more extensive than thermal modeling predictions for two other impact structures: (1) the Ries impact structure (24 km diameter), where the IGH system ceased in less than 250,000 years14, and (2) the Haughton impact structure (23 km diameter), where temperatures enabling thermophiles and hyperthermophiles to thrive (50–120°C) lasted over 50,000 years15. The extended duration of the hydrothermal system at Lappajärvi would have provided ample time for microorganisms to colonize, making it a prime target for exploring biosignatures of ancient microbial life. However, no evidence of microbial colonization at Lappajärvi has previously been reported.

Putative body and/or trace fossils of microbes, some more ambiguous than others, have been reported in impact materials from a handful of impact structures: Ries16,17, Chesapeake Bay18, Siljan19 and Dellen20. Stable isotopic biosignatures indicating post-impact microbial activity have been identified in several terrestrial impact structures. At Haughton, pyrite and marcasite in impact breccias with δ34S values as low as −42 ± 2‰, suggest microbial sulfate reduction (MSR)21. Likewise, at Rochechouart, δ34S sulfide values down to −36‰ are consistent with MSR22. In the Siljan structure, fracture-coating calcite with δ13C values ranging from −52 to 22‰ is indicative of microbial methanotrophy and methanogenesis23, potentially in syntrophy with fungi19. Pyrite δ34S values in the same fractures were as low as −42‰, suggesting MSR23. At the Chicxulub impact crater, microscopic pyrite framboids with δ34S values as low as −35‰ and Δ34S sulfate-pyrite values up to 54‰ are also consistent with MSR24, and studies of extant microbiological communities show that the impact-induced geochemical boundary has shaped the modern-day deep biosphere in the granitic basement underlying this crater25.

Although these biosignatures have been reported from a limited number of impact structures, they provide valuable evidence of microbial activity following impact events. However, the timing of microbial colonization with respect to these impact events remains uncertain. In other words, there is a possibility that the previously reported biosignatures from impact structures are unrelated to the impact crater formation, and instead represent outcomes of long-term recolonization of the site.

Only a few studies have directly dated the materials hosting or associated with biosignatures. Using in situ Rb/Sr dating of calcite-albite-K feldspar assemblages occurring alongside fossilized fungi, that were initially attributed to colonization during impact-related hydrothermal activity26, Tillberg et al.27 found that fungal colonization of the 458 Ma Lockne impact structure occurred at least 100 million years after the impact. Similarly, micro-scale U-Pb dating of 13C-enriched methanogenesis-related calcite in fractures and breccias at Siljan yielded ages between 80 and 22 Ma, indicating colonization occurred 300 million years after the 380.9 ± 4.6 Ma impact event23. In the same structure, U-Pb carbonate geochronology provided a maximum age of 39.2 ± 1.4 Ma for coeval fossilized fungi19. These findings suggest that documenting biosignatures through petrographic relationships to impact rocks alone cannot confirm impact-related microbial activity. Radioisotopic dating, along with other contexts, such as petrographic relations, is essential to verify such colonization.

In this study, we investigated the Lappajärvi impact structure in Finland to detect ancient biosignatures of microbial colonization in the crater-fill impactites, and to establish timing and temperature constraints. Using micro-scale isotopic techniques, we provide direct evidence of microbial colonization during the waning stages of the IGH system of this terrestrial impact structure. This research has significant implications for the hypothesis that impact craters serve as hotspots for microbial colonization on Earth and, by analogy, on other planetary bodies. This is particularly relevant given that deep biosphere habitats in impact structures are considered favorable targets for Mars exploration for signs of extinct life28.

Results
Petrography and mineralogy in fractures and vugs
Impact melt rock (locally referred to as ‘kärnäite’), impact melt-bearing breccia, and lithic (i.e., melt-free) impact breccia, were sampled for vug- and fracture-filling calcite and pyrite, in two cored boreholes (impact melt rock-rich LA1 and impact melt-bearing breccia-rich LA4, Fig. 1). The impact melt rock contains abundant mineral-filled cm-sized vugs at 3–10 m, sporadically around 30 m, and at 144–145 m depth (just above the contact to impact melt-bearing breccia) as well as fractures at 110–140 m depth. The impact melt rock is underlain and surrounded by impact melt-bearing breccia and lithic impact breccia that gradually proceeds downward and outward from the central part of the crater into autochthonous impact breccia, and finally to the fractured bedrock29. Several intervals of the impact melt-bearing breccia contained a high proportion of mm-to cm-sized mineral-filled vugs within impact glass of various degrees of alteration (Supplementary Fig. 1, details in Methods). For simplicity, the impact melt rock in LA1 is grouped into upper (3–31 m) and lower (137–145 m) intervals; the impact melt-bearing breccia in LA4 is similarly divided into upper (23–82 m) and lower (142–153 m) intervals.

The vugs and fractures of the impactites have mm to cm thick coatings of secondary minerals (Supplementary Figs. 1, 2) identified via SEM-EDS (Supplementary Figs. 3–6) and X-Ray Diffraction (XRD, Supplementary Fig. 7). including zeolites (chabazite, erionite, and heulandite), siderite (Supplementary Fig. 7a) clay minerals (a poorly crystalline phase with a best XRD-fit to vermiculite, hereafter termed vermiculite* as no certain confirmation could be performed [Supplementary Fig. 7b]), quartz, and Fe(-Ti)-oxides, in addition to calcite and pyrite that are the focus of this study. Veins in the lower impact melt rock feature several generations of mineral precipitation. First, subhedral pyrite crystal aggregates intergrown with calcite (with ~3% Fe, 0.6% Mn, 0.4% Mg) occur in coatings lining the fracture walls (calcite group i, Fig. 2a and Supplementary Fig. 3, and EDS-maps in Supplementary Fig. 6). The calcite is overgrown by vermiculite* and siderite. A younger calcite filling with less Mn content than calcite-i occupies the central, most voluminous part of the vein (calcite ii, Supplementary Fig. 3). In impact melt rock vugs, subhedral Fe-poor calcite is frequent (calcite iii, Fig. 2b and Supplementary Fig. 4), occasionally intergrown with aggregates of subhedral pyrite (Fig. 2a), and occasionally with euhedral pyrite on the calcite crystal surfaces (Supplementary Fig. 2a). Calcite is frequent in vugs of the impact melt-bearing breccia, with scalenohedral crystals (calcite y) with overgrowths (calcite yy) being common in the lower interval, in particular (Fig. 2c and Supplementary Fig. 5, showing slightly higher Mn and Fe concentrations in the younger BSE-bright zone). Zeolites, such as erionite and chabazite (Supplementary Fig. 1a, c), as well as pyrite (Fig. 2d) and Fe-Ni-sulfide aggregates that may contain remnants of the impacting projectile according to previous studies30, are intergrown with impact glass in the impact melt-bearing breccia. There are also mm-sized euhedral pyrite crystals (Supplementary Fig. 2b) frequently occurring in vugs of the impact melt-bearing breccia and the lithic impact breccia. Occasionally, the latter pyrite occurs adjacent to mm-sized scalenohedral calcite crystals in the lower impact melt-bearing breccia (Supplementary Fig. 2c).

Stable isotope compositions of pyrite and calcite
The δ34S values of all pyrite crystals analyzed by SIMS in this study range substantially, from −31.9 to 102.2‰ (Supplementary Data 1). The δ34S variability is largest in impact melt rock (−31.9–102.2‰). The most 34S-depleted pyrite (Fig. 3a and Supplementary Fig. 8a) occurs intergrown with calcite group i in the oldest parts of veins of the lower impact melt rock (Fig. 2a). In this sample, there is a trend of increasing δ34S values from the crystal cores to rims, by up to 25‰ in single crystals (Fig. 3a and Supplementary Fig. 8a). The most significant small scale δ34S-variability, ranging from −4.3 to 102.2‰ occurred in a polycrystalline pyrite aggregate in a chalcedony-rich interval of the upper impact melt rock (Fig. 3b). Most δ34S values in the impact melt-bearing breccia and lithic impact breccia show small variation, such as in the earliest coarse-grained pyrite with an average of 3.1 ± 2.1‰ (1 std. dev.). Two samples with high values, at depths of 29 m (83.2‰, Fig. 3c) and 54 m (76.8‰), make the range substantial. Highly 34S-enriched pyrite also exists in a lithic impact breccia sample at 130 m, showing increasing values from core to rim (δ34S of 60.4‰, Supplementary Fig. 8b).

The δ13C values ranged from significantly 13C-depleted, −50.9‰, to significantly 13C-enriched, 16.4‰, and the δ18O values from −19.8 to −2.7‰ (Fig. 4a and Supplementary Data 2). There is a distinct difference in δ13C values between the impact melt rock (1.7 to 16.4‰) and impact melt-bearing breccia (−52.3 to −14.7‰). Impact melt rock-hosted calcite exhibits homogeneous isotope composition within single crystals (Fig. 4b–d) but clusters into three distinct groups based on their δ13C/δ18O composition (Fig. 4a). The group with the highest δ18O values (group i: −6 to −3‰) is petrographically older (Figs. 2a and 4b, c) than the group (ii) with δ18O values of −13 to −8‰. The most 13C-enriched calcite (up to 16.4‰, Fig. 4d) in the impact melt rock is from a coating in a 4 cm diameter vug at 30 m in LA1 (Fig. 2b). The calcite in impact melt-bearing breccia can be divided into two groups, one (group yy) with δ13C values of −50.9 to −39.8‰ and one with higher values (y: −31.6 to −14.7‰) and with a larger span in δ18O. A large proportion of the impact melt-bearing breccia-hosted calcite exhibits δ18O values of around −11 to −8‰. The most 13C-depleted calcite group (yy) only occurs in the lower impact melt-bearing breccia of LA4, and only in the youngest overgrowths of scalenohedral calcite crystals in the vugs (Figs. 2c, 4e). The single lithic impact breccia-hosted calcite sample had stable isotope values between the impact melt rock and impact melt-bearing breccia groups (Fig. 4a).

Clumped isotope compositions of calcite
The clumped isotope analyses yielded Δ638 values which were used for temperature reconstructions (hereafter ‘temperatures’), the latter ranging from 8.0 ± 6.8°C to 47.0 ± 7.1°C (Supplementary Data 3, 4). Distinct patterns of temperatures are observed between calcite in different δ13C/δ18O groups (Fig. 5). The first group of calcite (i) in LA1;137 (cf. Figs. 2a, 4b), has the highest temperature at 47.0 ± 7.1°C and two other impact melt rock-hosted calcite group i samples show similar, but slightly lower temperatures (Fig. 5a). Two samples from impact melt rock-hosted calcite group ii and iii have temperatures around 30°C. The calcite samples from the impact melt-bearing breccia, LA4;40 and LA4;152, have temperatures of 8.0 ± 6.8 and 8.9 ± 5.3°C. The δ18O composition of the fluids (Supplementary Data 5), from which the calcite precipitated, were calculated based on clumped isotope temperature reconstructions and bulk δ18O values by applying temperature-dependent fractionation factors31, showing three distinct δ18Ofluid groups with gradually lower temperature (Fig. 5). The highest δ18Ofluid values occur in impact melt rock-hosted calcite group i (−2.6 ± 1.6 to −1.0 ± 1.4‰), followed by impact melt rock-hosted calcite group ii/iii (−8.6 ± 1.1 to −8.1 ± 2.5‰) and by the impact melt-bearing breccia-hosted calcite samples (−14.2 ± 1.9 to −12.7 ± 1.9‰).

U-Pb carbonate geochronology
The two calcite groups i and ii of sample LA1;137 (Figs. 2a and 4b, c) yielded robust ages when targeted for U-Pb geochronology via LA-ICP-MS in the same crystals as previous SIMS-analyses (Supplementary Figs. 3–5). Group i yielded an age of 73.6 ± 2.2 Ma (Fig. 6 and Supplementary Fig. 9) and ii 62.5 ± 3.0 Ma (Fig. 6 and Supplementary Fig. 9). These calcite groups show no discernable individual growth zonation, and their ages are interpreted to represent two single separate precipitation events. Impact melt rock-hosted calcite of the second generation, but with methanogenesis δ13C signature (group iii, LA1;30, Figs. 2c and 4d), yielded an age that is too imprecise for interpretation (74 ± 28 Ma, Supplementary Figs. 10, 11). The crystal cores (group y) of impact melt-bearing breccia-hosted calcite with AOM signatures in the overgrowths (Figs. 2d, 4d) yielded an age of 43.0 ± 9.6 Ma (Supplementary Figs. 9, 11), giving a rough maximum age of the AOM-related calcite.

Methods
Sample materials and site
The 23 km diameter Lappajärvi impact structure in central-western Finland is a well-preserved, albeit moderately eroded impact structure61. A concentric arrangement of impact-produced crater-fill deposits remains underneath Quaternary sediments (Fig. 1). Impact melt rock currently constitutes a 145 m thick coherent layer. The upper 24 m and lower 26 m of the impact melt rock have a crystalline matrix in the form of recrystallized glass, whereas the intermediate impact melt rock has a glassy (perlitic) matrix38 references therein. The impact melt rock is relatively homogeneous and can be reproduced by a mixture of the Paleoproterozoic (ca. 1.90–1.87 Ga) target rock: 76 vol.% mica schist, 11% granite pegmatite, and 13% amphibolite62. The impact melt rock is both underlain and surrounded by impact melt-bearing breccia and lithic impact breccia. In the impact melt rock, porosity is around 1%, whereas in impact melt-bearing breccia and lithic impact porosity is as high as 20%, which is significantly higher than in unweathered crystalline rocks, in which porosity is typically less than 0.5%63. Gradually, the impact-produced crater-fill deposits proceed downward and outward from the central part of the crater into autochthonous impact breccia, and finally to the fractured bedrock29. The outer margin of the crater is rimmed by a megablock zone38 of autochthonous breccia in which a circular depression (ring graben) is filled with allochthonous Mesoproterozoic sedimentary rocks29, which locally overlaid the crystalline bedrock at the time of the impact, at a thickness of less than 200 m38. Samples were collected from two cored boreholes (LA1 and LA4 in Fig. 1) drilled into the impact structure.

Impact melt rock in drill core LA1 was sampled in intervals with abundant mineral-filled cm-sized vugs at 3–10 m and 144–145 depth (just above the contact to impact melt-bearing breccia) and fractures at 110–140 m depth. The 10–110 m interval has few vugs and sealed fractures, but a calcedony interval occurs (sampled). Impact melt-bearing breccia in LA4 was sampled in intervals with abundant mineral-filled mm-to cm-sized vugs at 23–35 m (high proportion of vugs), at 35–66 m (moderate proportion of vugs), at 66–76 m (fragmented and heavily weathered interval, high proportion of vugs) at 76–82 m (high proportion of vugs) and at 142–152 m (moderate proportion of vugs). Lithic impact breccia in LA4 was sampled in intervals with abundant mineral-filled cm-sized vugs at 83–90 m (very high proportion of vugs), at 90–95 m (heavily weathered and partially dissolved interval, high proportion of vugs), at 95–106 m (heavily weathered interval, high proportion of vugs) and at 106–148 m (moderate proportion of vugs).

Sample preparation
Thirty-three rock core intervals containing open, partly sealed or completely sealed fractures and vugs were targeted and sampled for secondary coating minerals, focusing on calcite and pyrite, for stable isotopes, radioisotopic dating, X-ray diffraction of clay minerals (method in Supplementary Text 3, data in Supplementary Data 8) and organic molecules (too contaminated to be included, see Supplementary Text 4). Eight of the intervals are from the 218 m long and 42 mm diameter (plunge: 80°, trend: 030°, year: 1988)38 drill core K231388R301 (here denoted LA1). The remaining 25 intervals are from the 275 m long and 55 mm diameter (plunge: 80°, trend 030°, year 1990)38 drill core K231390R304 (LA4). The crystals were characterized under a stereomicroscope and identified directly on the uncoated surfaces of vugs, veins and fractures at low-vacuum mode and for carbon-coated thin sections at high-vacuum mode using a FEI QUANTA FEG 650 Scanning Electron Microscope (SEM) equipped with an integrated energy dispersive spectroscopy (EDS, Oxford T-Max 80 detector). The acceleration voltage was 20 kV, and the instrument was calibrated with a cobalt standard.

Secondary Ion Mass Spectrometry (SIMS)
Following sample characterization and mineral identification, single crystals or aggregates were separated from fractures and vugs with a chisel and/or tweezers, under a stereomicroscope, and mounted in epoxy. The epoxy-mounted grains were polished to expose crystal cross-sections and characterized in SEM once again to identify crystal zonation, cracks, mineral inclusions and impurities, and to optimize SIMS spot placement. Intra-crystal SIMS-analysis (10-μm lateral beam dimension, 1–2 μm depth dimension) of carbon, oxygen and sulfur isotopes was performed on a CAMECA IMS 1280 SIMS at NordSIMS, Swedish Museum of Natural History, Stockholm, Sweden. The thematic spatial correlation of different techniques used for the various mineral materials, are detailed in Supplementary Figs. 3–5.

SIMS-Pyrite
Analytical transects were made from core to rim within 53 pyrite crystals from 24 drill core samples. In total, 344 analyses were made for δ34S. Sulfur was sputtered using a 133Cs+ primary beam with 20 kV incident energy (10 kV primary, −10 kV secondary) and a primary beam current of ~1.5 nA. A normal incidence electron gun was used for charge compensation. Analyses were performed in automated sequences, with each analysis comprising a 70 s pre-sputter to remove the gold coating over a rastered 15 × 15 μm area, centering of the secondary beam in the field aperture to correct for small variations in surface relief and data acquisition in sixteen four-second integration cycles. The magnetic field was locked at the beginning of the session using an NMR field sensor. Secondary ion signals for 32S and 34S were detected simultaneously using two Faraday detectors with a common mass resolution of 4860 (M/ΔM). Results are reported as ‰ δ34S based on the Canon Diablo Troilite (V-CDT)-standard value64. Full data, including reference materials, are in Supplementary Data 1.

SIMS-Calcite
Analytical transects were made in 56 calcite crystals collected from 15 drill core samples. In total, 449 analyses (settings follow46) were made, 220 for δ13C and 229 for δ18O. Influence of organic matter and inclusions of other minerals was avoided by careful spot placement to areas in the crystals without micro-fractures or inclusions. The uncertainty associated with potential organic inclusions and matrix composition is therefore considered to be insignificant compared to the isotopic variations. Calcite results are reported as ‰ δ13C and δ18O based on the Pee Dee Belemnite (V-PDB)-standard value. Analytical sessions were carried out, running blocks of six unknowns bracketed by two standards. Spot transects were made from core to rim within the crystals (full data, including values of reference materials in Supplementary Data 2). Corresponding analytical spots for C and O isotopes were closely placed within the crystals and analyzed at separate sessions. Isotope data were normalized using calcite reference material S0161, a granulite facies marble in the Adirondack Mountains, courtesy of R.A. Stern (Univ. of Alberta). The values used for IMF correction were determined by conventional stable isotope mass spectrometry at Stockholm University on ten separate pieces, yielding δ13C = −0.22 ± 0.11‰ V-PDB (1 std. dev.) and δ18O = −5.62 ± 0.11‰ V-PDB (1 std. dev.). Precision was δ18O: ± 0.2−0.3‰ and δ13C: ± 0.4−0.5‰. Calcite trace element concentrations may influence SIMS matrix effects, at least for δ18O ref. 65, but since 1) the 3−4 mass% of Fe+Mn measured with EDS in the calcite at Lappajärvi (Supplementary Fig. 3) may only influence the δ18O by a maximum of ~1‰ when applying a correction of Rollion-Bard and Marin-Carbonne65, and 2) the clumped isotope measurements and SIMS measurements generally overlap for same samples, we have not applied any correction based on trace element content to the SIMS data.

Carbonate clumped isotope analysis
About 2 mg of powdered carbonate was reacted with dehydrated phosphoric acid under vacuum at 70°C. The conventional isotope ratio measurement relative to V-PDB was calibrated based on repeated measurements of International Atomic Energy Agency (IAEA) reference materials. Clumped isotope values (Δ638) are reported in the Intercarb Carbon Dioxide Equilibrium Scale (I-CDES) reference frame defined by ETH 1–4 carbonate standards and Carbon Dioxide Equilibrium Scale (CDES) reference frame defined by water-equilibrated gases and heated gases66,67. The Δ638 notation refers to measurements using a tunable infrared laser differential spectrometer (TILDAS), performed at the Environmental Isotope Laboratory at the University of Arizona. The precisions for these measurements are ± 0.021‰ for Δ638, ± 0.04‰ for δ18Ocarb, and ± 0.03‰ for δ13Ccarb (pooled reproducibility68, 1σ), respectively. The laser-based Δ638–temperature relationship for carbonates at relatively low temperatures is based on 44 synthetic calcites that equilibrated at temperatures from 6 to 70°C69: Δ638CDES = (0.0405 ± 0.0006) × (106/T2) + (0.1822 ± 0.0061), R2 = 0.985 (3)

Temperature estimates using laser-based Δ638 and mass spectrometry-based Δ47 measurements have been shown to be statistically equivalent69. Equation (3) is within error of recent mass spectrometry-based Δ47-temperature relationships and correctly predicted precipitation temperatures for a suite of 17 natural carbonates for both calcite and aragonite69. Completed details of this method and the calibration of the Δ638–temperature relationships are described in 70,71. For sample LA1;137, a mini drill was used to separate the oldest generation (group i) of calcite for clumped isotope analysis. All clumped isotope samples were analyzed for three to six replicates. Each replicate measurement is based on four comparisons (sub-cycles) of the sample and reference gas. We exclude replicates with fewer than two sub-cycles to calculate the mean value of each sample. The reported clumped isotope values were averaged from the three to six accepted replicates. Supplementary Data 3 and 4 provide full clumped isotope data, including excluded replicates.

LA-ICP-MS U-Pb Geochronology
U-Pb geochronology via the in situ LA-ICP-MS method was conducted at the Geochronology & Tracers Facility, British Geological Survey (Nottingham, UK). The method utilizes an Elemental Scientific Lasers ImageGeo excimer laser ablation system, coupled to a Nu Instruments Attom single-collector ICP-MS. The method is previously described71 and involves a standard-sample bracketing with normalization to either 91500 zircon72 or NIST 614 silicate glass for Pb-Pb ratios73 and WC-1 carbonate for U-Pb ratios74. The laser parameters comprised either a 60 or 120 μm static spot, fired at 10 Hz, with a ~4 J/cm2 fluence, for 30 s of ablation. No common lead correction is made; ages are determined by Model 1 regression and the lower intercept on a Tera-Wasserburg plot (using IsoplotR75). Duff Brown, a carbonate previously measured by Isotope Dilution mass spectrometry, was used as a validation and yielded pooled ages of 62.6 ± 0.78 Ma (unanchored regression) or 66.76 ± 0.72 Ma (anchored to 207Pb/206Pb = 0.7385), overlapping the published age of 64.04 ± 0.67 Ma76. All ages are plotted and quoted at 2σ and include propagation of overdispersion and systematic uncertainties according to recommended guidelines72. Data are screened for very low Pb and U counts below detection (<20 cps), and anomalously high ratio uncertainties (>50%). Eight samples were analyzed in total, with only three providing robust age constraints. Analyses were conducted on individual crystals that were previously used for in situ stable isotope analysis via SIMS. Full analytical data are listed in Supplementary Data 6; analytical conditions in Supplementary Data 7, and further details on the analytical sessions are given in Supplementary Text 5.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dear Dr Rosch,\nthank you for choosing me as a reviewer of the manuscript \"Deep microbial colonization during impact-generated\nhydrothermal circulation at the Lappajärvi impact structure, Finland\", submitted to Nature Communications by Drake et al.\nOverall, this is a well-written, but arguably quite technical, manuscript. Its actual core message, which is to highlight medium-\nsize (and large) terrestrial impact structures as long-lasting hotspots for the emergence and evolution of microbial life in a\nhydrothermal setting as the crater cools down to ambient conditions, could be conveyed a little more clearly especially in the\nabstract. The study presents some fascinating (and, as far as I can tell, reliable) U-Pb age results gained from calcite that\nsuggest the crater cooling process lasts, in fact, several million years even in the case of a ~20 km crater – which may be\nsurprising to some (but less so to others). This would make Lappajärvi the single most intensely studied impact crater in\nterms of geochronology, using different geochronometers and dating minerals, regarding the crater cooling question. It also\nmakes me wonder – if Lappajärvi stays warm for this long – how long will much larger, Chicxulub-size impact craters, take to\nfully cool down?\nIf the authors manage to set out their core message and its relevance in a somewhat more striking way, I think publication in\nNat. Comm. will be justified. However, looking at the present manuscript I’d rather recommend a more specialized journal,\ne.g., “Astrobiology”, “Earth and Planetary Science Letters”, or “Geochimica et Cosmochimica Acta”. I also encourage the\nauthors to add a brief discussion of potential mechanisms that could explain the surprising longevity of the heat source or\npockets within the crater.\nFor the manuscript itself (regardless where it will be published) I recommend minor revision.\nSpecific comments:\nAbstract, line 24: I’d write “10 Myr later”, since this describes a time span, and not an age. (I acknowledge some journals only\naccept Ga/Ma/ka as geologic time units and sometimes change those within the course of the proofing and typesetting\nprocess.)\nLine 35: Two other suitable references would be Schmieder & Kring (2020, Astrobiology, with a focus on impact ages) and\nKenkmann (2021, MAPS).\nL 52: ~1 to 1.6 Myr after… see comment above. “1.6 Ma” is a point in time within the Pleistocene.\nL 200/201: Here and in the following lines, the authors report a number of “temperature reconstructions” and choose to label\nthem “temperatures”. This sounds a bit like isotope jargon slang and might be misleading to some readers. Perhaps add “…\nyielded temperature reconstructions (henceforth ‘temperatures’), ranging from…”. Alternatively, those temperatures could be\ncalled paleotemperatures, mineralization temperatures, or simply reconstructed temperatures.\nL 326-328: There is something wrong with the sentence “Thermal modelling suggests that cooling below 250 °C took place\nup to ~1.6 Ma after the impact event12, although11 estimated slightly faster cooling of >200ºC, locally lasting for >1 Myr.” Are\nthe authors talking about post-impact cooling rates? Also see comment above regarding Ma vs. Myr.\nL 333: The authors touch on the geothermal gradient here. This is an interesting aspect. The authors should, at least briefly,\ndiscuss the anatomy and role of the central uplift at Lappajärvi, which is not prominent morphologically/topographically as it\nis concealed by the impact melt sheet. Nevertheless, an impact crater the size of Lappajärvi should be characterized by\nsome ~2.x km of structural uplift within the crater basement at its center. This uplift may also affect the original geothermal\nstratification within the target rock, i.e., leading to an upward ‘heat bulge’ (compare Naumov, 2005; Schmieder & Jourdan,\n2013). Would there be any measurable effect of a ~2 km uplift of the geothermal isoline pattern at Lappajärvi, and could this\nhelp explain the ~73 Ma low-temperature ‘warm oasis’ that we see in the calcite U/Pb results? Is Lappajärvi associated with\na resolvable geothermal anomaly (compared to the surrounding bedrock) in geophysical maps today?\nL 343: check “a three-stages of evolution”\nFig. 2d: are the two roundish pyrite grains near the bottom of the right image framboidal pyrite? Or just round crystalline\ngrains? Just as a remark: there are also Fe-Ni-rich metal and sulfide particles in the Lappajärvi melt lithologies that may\ncontain remnants of the impacting projectile (see Fregerslev 1976). Maybe this is worth mentioning.\nSupplementary file and main text: If possible and data exist, the authors could elaborate in a little more detail in which way\n(if at all) the calcite isotope results are influenced by any internal mineral zonation, e.g., with Fe-, Mn-, and/or Mg-rich zones\nwithin the calcite domains (the authors already mention ankerite in the text). At Chicxulub we see an interesting zonation\npattern both in SEM-BEI and CL between a Mn-poor and a Mn-rich variety in post-impact hydrothermal sparry carbonate.\nTable S6, Excel file: It might be useful to see, in addition to the raw/corrected U/Pb data, individual calculated spot ages with\nerrors, as well.\nApparent age values should be rounded appropriately (I don't think it is useful to report a number as \"0.032 +/- 567.610 Ma\").", "reviewer_2": "Congratulations on your excellent contribution to enhancing our understanding of when deep subsurface microbial life was\nable to recolonize impact-deformed geological formations and their role in deep subsurface elemental cycling (i.e., microbial\nsulfate reduction and anaerobic methane oxidation).\nI have only one comment: Many studies have shown that these microbial processes are ongoing even in nutrient-deprived\nextreme deep biosphere environments, such as described here. A recent survey from Quraish et al. (2024) in Geobiology\n(not cited) showed that extant microbial communities associated with mineral veins and contact zones between breccia and\ngranites, etc., in the granitic basement below the Chicxulub impact crater contain the genetic machinery for\nchemolithoautotrophic carbon fixation and organoheterotrophy plus cycling of S, Mn, and Fe.\nTo what extent can the precise dating, identification of minerals, and isotopic analysis on a microscale exclude the\npossibility that modern activities caused bias of the isotopic results? For example, extant chemoautotrophic activities could\nresult in the deposition of microbial carbonates enriched in d13C (overshadowing d13C depleted signals indicative of the\nonset and extent of methane cycling?). I suggest the authors discuss this so that microbiologists interested in this paper\nknow that you have considered it even if it is perhaps a no-brainer for specialist readers that the microscale analysis can rule\nout that some of the analyzed material was deposited more recently and has no impact on the data or outcomes of the study.", "reviewer_3": "This is a very interesting paper that I thoroughly enjoyed reading. I appreciate the coordination between stable isotope and\nradiometric analyses to understand the timing of microbial colonization as it is linked (or not linked) to impact-generated\nhydrothermal systems. This specific type of work in the field of terrestrial impact cratering, and particularly at the intersection\nof astrobiology, is sorely needed and is highly relevant as we continue to explore craters on Earth and the ancient Martian\nsurface (and potentially subsurface, in the future!)\nThe work presented here addresses a key question related to impact-generated hydrothermal (IGH) systems which is timing,\nas it relates to post-impact colonization by microbial communities. Previous studies on sulfide mineral isotopes, for example,\nin impact craters have identified biosignatures in the form of highly reduced (isotopically light) δ34S, implying a biogenic\nrather than (abiotic) thermochemical sulfate reduction origin, but the timing of this activity has remained ambiguous and often\nlinked directly to the hydrothermal system without more definitive (e.g., radiogenic isotope) constraints.\nI have a few comments on general observations of the manuscript as well as line-by-line suggestions, below.\nSections of the methods and results related to mineral identification and lithologic context – and by extension the\ncorresponding isotopic analyses - are unclear and would benefit from better organization and clarification. I find it difficult to\ninterpret the isotope data without a better understanding of the context and what exactly has been analyzed. Are many of\nthese generational relationships implied based solely on BSE images of rough sample surfaces? Is there supporting\nchemical data (e.g., something that clearly shows different generations of calcite) or possibly thin section images to support\nthese implied relationships? If that data is available it would be immensely helpful for the reader and solidify some of these\ninterpretations. I understand this paper represents a lot of coordination between in-situ and bulk analyses, and care should\nbe taken to make it clear what has been analyzed, actually identified and what it represents.\n- For example, to examine the identification of a “serpentine-group clay” (Lines 122, 126, 144) I would expect XRD data: I\nsee one XRD pattern - supplemental Fig. 9 - with no labeled peaks or mineral ID’s, and S8_XRD representing only one\nanalysis, with a list of potential library matches, but I see no labeled clay (or other mineral) d-spacings indicative of\nserpentine, vermiculite or corrensite 001’s, 060’s. Nor do the methods describe the careful analyses to identify corrensite,\nserpentine or vermiculite, a high charge smectite, were undertaken. The caption also states this was the clay coating on the\nvug, in the hand sample, but it’s unclear how this context was determined - if this is a bulk, crushed sample of a melt rock it\ncould have been a secondary clay in the matrix, or something else as clays can form from altering many of the primary\naluminosilicate phases under hydrothermal conditions.\n- As another example, I see lots of beautiful photographs and BSE images of rough sample surfaces but no thin sections or\nchemical (EDS) data to support these interpretations. If this is available (Lines 461-464 suggest EDS analyses should exist),\nplease add a few figures (e.g., chemical maps, spot analyses, something to help place the isotopic analyses and mineral,\nlithologic relationships into context).\n- As a final example, the context for many of these minerals used for isotopic analysis is unclear. E.g., It appears there are\nmore than one occurrences of pyrite in these samples (Fig. 2a vs 2d). Where are the pyrite crystals shown in Supplemental\nFig. 3 (and by extension, the longer list of sample ID’s in the excel sheet) from, other than “the melt rock” or “the lithic impact\nbreccia?” Are these fracture coatings or vug-fillings (e.g., Fig. 2)? Are they potentially hand-chiseled out (Lines 466-468)\nfrom the melt or breccia matrix – some of these are 100 microns or less, as low as 10 microns (Line 134) in size? These\nrelationships really matter when it comes to interpreting their isotopes, particularly as there is such an unusually large range\nof data here. I have no doubt that with the amount of work performed here, the data confirming these mineral identifications\nand lithologic, contextual relationships is available to show the reader?\nThis is exciting work and I hope the authors can take the steps to organize, present, coordinate and clarify their methods and\nresults, especially the implied mineral identifications, relationships and context. This is a really interesting study and I would\nlook forward to seeing some of this information clarified to better piece together the story presented here!\nLine by line comments:\nLines 49-52: As written it is unclear whether this statement implies that hydrothermal circulation and cooling below 250\ndegrees began at 1-1.6 Ma or lasted this long. Please clarify.\nLine 53: Minor typo: “…modeling have predicted…” to “…modeling has predicted…”\nLines 75-78: I would argue here that Chicxulub is an exception - those framboids are intergrown with the dachiardite and\nanalcime - these zeolites are high(er) temperature hydrothermal minerals, supporting these LIKELY formed during a period\nof elevated temperatures. The analcime samples in particular - the images in Kring et al. (2021), and the mineralogical, in-\nsitu context of those crystals - it’s hard to make the argument that the framboids formed much later, during diagenesis/far\nbelow and outside IGH conditions.\nLines 80-85: Very minor comment – I agree with the overall statement – that there is a need for timing constraints with the\nbiosignature examples described in the previous section, but the biosignatures described in this specific section are very\ndifferent from the ones listed in the previous paragraph, which is a tad misleading (e.g., 34S of sulfides attributed to bacterial\nsulfate reduction at elevated temperatures). I would also suggest adding a couple words here that indicate these\nbiosignatures were initially attributed directly to impact hydrothermal activity. When I first read this I thought “well yeah\nthese old rocks are probably covered in some sort of fungus” but went to the reference (and was surprised) to see someone initially\nclaimed these were hydrothermal system-hosted fungi! Similar comment for calcite, which can easily be reset and even\ninitially form at surface diagenetic conditions.\nLines 97-98: The comment on methane seepage is a bit out of place. I understand it’s relevance but I would suggest\nremoving this entirely or elaborating on how this is tied to the rest of the sentence and section (e.g., are the authors implying\nthe methane seepage was directly linked to IGH system-hosted microbes?) I would err on the side of caution here, as the\nsource of Martian methane detections are a highly contested topic, and very unlikely to be IGH system related.\nLine 131-133: “Clay minerals, zeolite and pyrite were among the first mineral coatings to appear intergrown with impact\nglass (Supplementary Fig. 1a,c)” Clay minerals and pyrite are not the first secondary minerals to form in these systems –\nthey are often later stage, especially clays. What is the evidence for this, based only on the BSE images? I also see no clays\nlabeled or identified here. Is there EDS data to go with these images?\nLine 132: Which zeolite? Or change to zeolites.\nLine 140, Figure 2: Is there a difference in composition between these different types of calcite?\nLine 143: Where is the data indicating this is ankerite? I see no labeled XRD patterns or in-situ EDS data on thin sections?\nLines 149-150: What pyrite is being analyzing here exactly?\nLines 163-170, Figure 3: what is the context for these pyrite grains (e.g., matrix, fracture or vug coating?) Additionally, what is\nthe other mineral in 3b?\nLines 192-193: How are these petrographically identified as chronologically older? Based on BSE images from rough\nsample surfaces? Are there thin sections (and corresponding EDS analyses) of these samples?\nLines 271-275: These are very unusual and heavy values for sulfides – a very interesting and exciting find! I see examples\nof these isotopically heavy sulfides in Fig. 3b and c – any chance you also have corresponding EDS data on these? Or the\nother mineral intergrown with the pyrite in 3b. I’m curious to know if there’s a chemical difference or zoning – anything – that\nmight help interpret these unexpected results.\nLines 452-464: I see methods listed here for low vacuum analyses on rough sample surfaces. Is this what the chronological\nrelationships and mineral identification, context is based on? Other than a single sample bulk XRD analysis and the second\nset of “SEM analysis” of hand-chiseled and epoxy mounted minerals listed 466-471? Once the samples are hand chiseled\nyou lose a lot of contextual information. I’m curious why no thin section petrographic work was completed. If it was (e.g., EDS\nmapping at high vacuum, or spot analyses and imaging to understand the in-situ mineralogical, chemical context), please\nadd it to the manuscript.\nSupplemental Fig. 1,2: I see several BSE images showing beautiful mineral surfaces, and vugs but no chemical (e.g., EDS,\nWDS) or crystallographic data (e.g. XRD, outside the unlabeled Supp. Fig. 9).\nSupplemental Fig. 9: Please label mineral peaks here (e.g., mineral ID and their respective, indicative 001, 060 d-spacings).\nHow was vermiculite characterized? Clay minerals are notoriously difficult to identify – vermiculite is a high-charge smectite.\nIs there additional information that shows this is indeed vermiculite? What about the other clays listed in the manuscript –\nserpentine and corrensite?"}, "author_response": "*Response: We thank the reviewer for the insightful comments and the overall positive assessment\nregarding our study. We agree with the reviewer that our study contributes new conclusions in line\nwith previous studies regarding the longevity the postimpact hydrothermal activity and relatively slow\ncooling of the Lappajärvi impact crater, and that we go beyond these by tying microbial colonization\nto the IHG system. Our study thus builds upon and take a significant leap from previous studies rather\nthan contradicts them.\nRegarding the general comments on 1) a more striking core message, and 2) the longevity of the heat\nsource, we have made the following amendments, that we believe are in agreement with what the\nreviewer aimed to achieve by the excellent remarks:\n1) In the abstract we changed the following (changes are in bold):\nL 24-29: Later stages of vug-mineral precipitation occurred more than 10 Myr later, at gradually lower\ntemperatures, and featured δ13C values diagnostic for both anaerobic microbial consumption and\ncalcite\nproduction of methane. These new insights confirm a capacity of medium-sized (and large)\nmeteorite impacts in generating long-lasting hydrothermal systems enabling related microbial\ncolonization as the crater cools down to ambient conditions, that may have important implications\nfor the emergence of life on Earth and beyond.\nIn the introduction we added a more striking start of the introduction, to even more justify the\nimportant context of this work. L32-34: “Meteorite impact craters occur on all planetary bodies in our\nSolar System. Once thought to be purely destructive events, there is a growing consensus that\nmeteorite impacts may have played an important role in the origin and early evolution of life on\nEarth1,2. An impact event deposits a tremendous amount of energy in a planetary crust, which results in\nthe fracturing and heating of a huge volume or rock that scales with crater size.”\n1 (19)\n2) The longevity of the heat source is discussed significantly, by including a new paragraph in the text\nand by adding a long discussion in the Supplementary information, see a response below, to a specific\ncomment.\nFor the manuscript itself (regardless where it will be published) I recommend minor revision.\nSpecific comments:\nAbstract, line 24: I’d write “10 Myr later”, since this describes a time span, and not an age. (I\nacknowledge some journals only accept Ga/Ma/ka as geologic time units and sometimes change those\nwithin the course of the proofing and typesetting process.)\n*Response: Thanks, we agree that it should be “10 Myr later”, since this describes a time span.\nLine 35: Two other suitable references would be Schmieder & Kring (2020, Astrobiology, with a focus\non impact ages) and Kenkmann (2021, MAPS).\n*Response: Thanks for suggesting these two suitable references. We have added here Kenkmann\n(2021, MAPS) as it is newer.\nL 52: ~1 to 1.6 Myr after… see comment above. “1.6 Ma” is a point in time within the Pleistocene.\n*Response: We agree and have changed to “~1 to 1.6 Myr”\nL 200/201: Here and in the following lines, the authors report a number of “temperature\nreconstructions” and choose to label them “temperatures”. This sounds a bit like isotope jargon slang\nand might be misleading to some readers. Perhaps add “…yielded temperature reconstructions\n(henceforth ‘temperatures’), ranging from…”. Alternatively, those temperatures could be called\npaleotemperatures, mineralization temperatures, or simply reconstructed temperatures.\n*Response: Thanks for these constructive suggestions, we agree that it might be misleading to only\nlabel the temperature reconstructions as “temperatures”. In the same paragraph as L200/201, between\nL217-220, we have changed the following (changed are in bold):\n” The clumped isotope analyses yielded Δ638 values which were used for temperature\nreconstructions (hereafter ‘temperatures’), the latter ranging from 8.0 ± 6.8 °C to 47.0 ± 7.1 °C\n(Supplementary Tables 3, 4). Distinct patterns of temperatures are observed between calcite in\ndifferent δ13C/δ18O groups (Fig. 5).\nL225-229: The δ18O composition of the fluids (Supplementary Table 5), from which the calcite\nprecipitated, were calculated based on clumped isotope temperature reconstructions and bulk\nδ18O values by applying temperature-dependent fractionation factors29, showing three distinct\ncalcite\nδ18O groups with gradually lower temperature (Fig. 5).”\nfluid\nL 326-328: There is something wrong with the sentence “Thermal modelling suggests that cooling\nbelow 250 °C took place up to ~1.6 Ma after the impact event12, although11 estimated slightly faster\ncooling of >200ºC, locally lasting for >1 Myr.” Are the authors talking about post-impact cooling\nrates? Also see comment above regarding Ma vs. Myr.\n2 (19)\n*Response: Thanks for pointing this out, we agree and have changed to:\nL347-350: “Thermal modelling of the post-impact cooling scenario suggests that temperatures\nbelow 250 °C were sustained for up to ~1.6 Myr after the impact event13, although Kenny et\nal.12 estimated slightly faster cooling rates resulting in temperatures >200ºC, locally lasting\nfor more than 1 Myr.”\nL 333: The authors touch on the geothermal gradient here. This is an interesting aspect. The authors\nshould, at least briefly, discuss the anatomy and role of the central uplift at Lappajärvi, which is not\nprominent morphologically/topographically as it is concealed by the impact melt sheet. Nevertheless,\nan impact crater the size of Lappajärvi should be characterized by some ~2.x km of structural uplift\nwithin the crater basement at its center. This uplift may also affect the original geothermal\nstratification within the target rock, i.e., leading to an upward ‘heat bulge’ (compare Naumov, 2005;\nSchmieder & Jourdan, 2013). Would there be any measurable effect of a ~2 km uplift of the\ngeothermal isoline pattern at Lappajärvi, and could this help explain the ~73 Ma low-temperature\n‘warm oasis’ that we see in the calcite U/Pb results? Is Lappajärvi associated with a resolvable\ngeothermal anomaly (compared to the surrounding bedrock) in geophysical maps today?\n*Response: These are relevant comments that will be important to address when discussing the\nprolonged “heat”. We included a discussion about this in the main text:\nL369-384: “The longevity of the hydrothermal system at Lappajärvi, or at least pockets of prolonged\nfluid flow, compared to the similarly-sized Haughton (~50,000 years) and Ries (~250,000 years), may\nhave several explanations. Given the similar size of these three structures, it is unlikely that the\nelevated geothermal gradient due to impact-induced uplift or residual heat generated by shock and\nfriction within the central uplift would be that different at Lappajärvi. We suggest that the simplest\nexplanation is due to the predominance of crystalline metamorphic rocks in the target rocks at\nLappajärvi. This led to the generation of a thick (currently 145 m but originally likely much thicker)\nlayer of silicate impact melt rock, which is the major heat source for hydrothermal systems in mid-size\nimpact structures. In contrast, no coherent bodies of silicate melt rock are known at Haughton and only\nvery small (few m) examples at Ries due to the presence of a thick layer of sedimentary cover rocks at\nthe time of impact. The relatively “dry” crystalline target rocks at Lappajärvi may have also led to\nrelatively slow conductive heat transfer from the crater center, and the low permeability of the target\nrock could also have prolonged hydrothermal activity (see Supplementary Text 2 for an extended\ndiscussion on mechanisms that could explain the longevity of the heat source or pockets within the\ncrater).”\nAnd a significantly longer and more detailed text about this matter in the Supplementary information\n(too long to include here).\nRegarding question 2), we have explored a map of geothermal heat production of the Finnish bedrock\n(available at Geological Survey of Finland’s map service, https://gtkdata.gtk.fi/maankamara/).\nAccording to the map, the bedrock at Lappajärvi does not have any anomalous high heat generation. In\naddition, according to Veikkolainen and Kukkonen (2019), the bedrock itself at Lappajärvi, does not\nproduce anomalous radioactive heat at present. We did not add information about this in the revised\nmanuscript.\nL 343: check “a three-stages of evolution”\n3 (19)\n*Response: Thanks for the comment, we have checked the sentence. “(2005)” was deleted from the\nreference and we also improved the wording of the sentence.\nIn addition, Osinski et al.55 used fluid inclusions to describe three-stages of evolution of the IGH\nsystem at Haughton impact structure, which is approximately the same size as Lappajärvi.\nFig. 2d: are the two roundish pyrite grains near the bottom of the right image framboidal pyrite? Or\njust round crystalline grains? Just as a remark: there are also Fe-Ni-rich metal and sulfide particles in\nthe Lappajärvi melt lithologies that may contain remnants of the impacting projectile (see Fregerslev\n1976). Maybe this is worth mentioning.\n*Response: Thanks for the interesting remark regarding the pyrite grains, which are actually not\nframboidal as can been seen in the attached images below. The pyrite fills tiny 10-100 µm-sized,\nspherical pits at the inner wall of a larger, ~0.1-1mm-sized spherical vesicles within melt glass of the\nimpact melt-bearing breccia. The pyrite grains might appear to be framboidal-like because they fill and\nare confined by the spherical pits, and could therefore be described to have a spherical shape. In image\nx, a pyrite grains with probable dodecahedral crystal habit is seen in the upper right corner. In the other\nimage, the pyrite grows in ringlike structures in the pits. We don’t have any δ34S values of these pyrite,\nbut it would be interesting to measure in the future. Another possibility is that the pyrite could be\ninclusions of sulfide melt droplets from the impact melt, which makes them out of the scope of the\ncurrent study, but nevertheless of large scientific interest for future work.\nFig x. pyrite filling tiny 10-100 µm-sized, spherical pits at the inner wall of a larger, ~0.1-1mm-sized\nspherical vesicles within melt glass of the impact melt-bearing breccia.\nRegarding Fe-Ni-rich metal and sulfide mentioned in Fregerslev (1976), we have found a similar\nspherical grain in the impact melt rock (locally known as “kärnäite”). The composition of this spherule\nclearly differs from the pyrites of which we measured for sulfur isotope composition of. These sulfide\ninclusions are a topic of a follow-up study.\nWe added the following text (ref 30 being Fregerslev, 1976, now added):\nL145-148 Zeolites, such as erionite and chabazite (Supplementary Fig. 1a,c), as well as pyrite\n(Fig. 2d) and Fe-Ni-sulfide aggregates that may contain remnants of the impacting projectile\naccording to previous studies30, are intergrown with impact glass in the impact melt-bearing\nbreccia.\n4 (19)\nImages of the inclusions with EDS data, for information here only.\nSupplementary file and main text: If possible and data exist, the authors could elaborate in a little more\ndetail in which way (if at all) the calcite isotope results are influenced by any internal mineral\nzonation, e.g., with Fe-, Mn-, and/or Mg-rich zones within the calcite domains (the authors already\nmention ankerite in the text). At Chicxulub we see an interesting zonation pattern both in SEM-BEI\nand CL between a Mn-poor and a Mn-rich variety in post-impact hydrothermal sparry carbonate.\n*Response: Thanks for suggesting this improvement. Zonation occurs as seen in our BSE-SEM\nimages (Supplementary Fig. 4), of mainly Mn and Fe, and in the CL-images (Mn variability indicated\nbetween growth zones). We have added EDS-derived information of the different calcite generations\nin new figures of the Supplementary information, as well as added some details to the main text\nregarding compositional differences of the growth zones.\nAdded figures with compositional data given for different calcite generations:\nCalcite i and ii:\n5 (19)\nCalcite iii:\n6 (19)\nCalcite y and yy:\n7 (19)\nA compositional dependence of SIMS δ18O and δ13C bias might arise from the Fe concentrations, as\nFe has been shown to give such an effect, at least for other carbonates (by perhaps 1 permil for δ18O at\nthese concentrations, see Rollion-Bard and Marin-Carbonne, 2011), and probably even less for the less\nstudied C-isotope system (not studied for calcite, and hence not discussed in the paper, see e.g.\nŚliwiński, M.G., Kitajima, K., Kozdon, R., Spicuzza, M.J., Fournelle, J.H., Denny, A. and Valley, J.W.\n(2016), Secondary Ion Mass Spectrometry Bias on Isotope Ratios in Dolomite–Ankerite, Part II: δ13C\nMatrix Effects. Geostand Geoanal Res, 40: 173-184. https://doi.org/10.1111/j.1751-\n908X.2015.00380.x for comparison). We have a secondary control in the form of δ18O/δ13C from\nclumped isotope measurements that show overlapping values with the SIMS data (a slight tendency\ntowards higher δ13C in the 13C-rich LA1:30 sample is seen in the clumped isotopes, which shows, if\nanything, that the strong 13C-enrichment is rather understated than overstated). This shows that any\ncompositional dependence on the SIMS data has not affected the values significantly, and certainly not\nto the level where they affect the process interpretations. For the interpretations of δ18O-fluid, we have\ngenerally used the δ18O from the clumped isotope measurements, so that any minor SIMS-IMF effect\nis not propagated to those calculations. We added to the method section L543-548: “Calcite trace\nelement concentrations may influence SIMS matrix effects, at least for δ18Oref.65, but since 1)\nthe 3-4 mass% of Fe+Mn measured with EDS in the calcite at Lappajärvi (Supplementary\nFigure 3) may only influence the δ18O by a maximum of ~1‰ when applying a correction of\nRollion-Bard and Marin-Carbonne65, and 2) the clumped isotope measurements and SIMS\nmeasurements generally overlap for same samples, we have not applied any correction based\non trace element content to the SIMS data.”\nTable S6, Excel file: It might be useful to see, in addition to the raw/corrected U/Pb data, individual\ncalculated spot ages with errors, as well.\nApparent age values should be rounded appropriately (I don't think it is useful to report a number as\n\"0.032 +/- 567.610 Ma\").\n*Response: Although individual ages can be calculated from the ratios, they are meaningless by\nthemselves, and therefore open to possible misinterpretation. We prefer to let readers calculate these if\nthey wish.\nThe table only includes ratios and not ages. It is standard practice to present the raw isotopic data as\nthey are, even if they seem meaningless. In this case, the quoted number reflects data that is below\ndetection, but that we still tabulate for completeness.\n*** End of review ***\nKind regards\nMany thanks again for a constructive review\n8 (19)\nReviewer #2 (Remarks to the Author):\nCongratulations on your excellent contribution to enhancing our understanding of when deep\nsubsurface microbial life was able to recolonize impact-deformed geological formations and their role\nin deep subsurface elemental cycling (i.e., microbial sulfate reduction and anaerobic methane\noxidation).\nI have only one comment: Many studies have shown that these microbial processes are ongoing even\nin nutrient-deprived extreme deep biosphere environments, such as described here. A recent survey\nfrom Quraish et al. (2024) in Geobiology (not cited) showed that extant microbial communities\nassociated with mineral veins and contact zones between breccia and granites, etc., in the granitic\nbasement below the Chicxulub impact crater contain the genetic machinery for\nchemolithoautotrophic carbon fixation and organoheterotrophy plus cycling of S, Mn, and Fe.\nTo what extent can the precise dating, identification of minerals, and isotopic analysis on a microscale\nexclude the possibility that modern activities caused bias of the isotopic results? For example,\nextant chemoautotrophic activities could result in the deposition of microbial carbonates enriched in\nd13C (overshadowing d13C depleted signals indicative of the onset and extent of methane cycling?). I\nsuggest the authors discuss this so that microbiologists interested in this paper know that you have\nconsidered it even if it is perhaps a no-brainer for specialist readers that the microscale analysis can\nrule out that some of the analyzed material was deposited more recently and has no impact on the data\nor outcomes of the study.\nResponse:\nWe thank the reviewer the overall very positive assessment and for the important and insightful\ncomments regarding the potential for extant microbial biosignatures (chemolithoautotrophic carbon\nfixation and organoheterotrophy plus cycling of S, Mn, and Fe) to be associated with mineral veins\nand contact zones between impact breccia and target rocks.\nWe have reasons why we are confident that modern activities have not caused any bias of our isotopic\nresults. First, we made sure to measure the isotopic ratios with SIMS well within the mineral crystals.\nSecondly, we have robust U-Pb dating on some of the calcite crystals, which exclude that possibility\nthat modern activities caused bias of the isotopic results…In some calcite crystals AOM-signatures\nwere measured in the outermost part of calcite crystals. Since no robust U-Pb dating were made on the\noutermost part or the latter crystals, modern activities cannot be ruled out as being responsible for the\nAOM-signatures, although not highly likely even in that case, as that growth zone show evidence of\nprecipitation (sharp border to earlier growth phase) from solution and not dissolution-reprecipitation\nthat is more likely if there is a modern influence on any of the mineral coatings. We have added some\ntext on this matter to highlight that we use mineral interiors to detect ancient biosignatures\n(introduction: “Using micro-scale isotopic techniques of mineral interiors, we provide direct\nevidence of ancient microbial colonization during the waning stages of the IGH system of this\nterrestrial impact structure”, and we have added the reference, as we believe that it is an important\nstudy to mention in the introduction, and we thank the reviewer for notifying us about this study. We\nadded this: “At the Chicxulub impact crater, microscopic pyrite framboids with δ34S values as\nlow as -35‰ and ΔS values up to 54‰, are also consistent with MSR24, and studies\nsulfate-pyrite\nof extant microbiological communities show that the impact-induced geochemical boundaries\nhave shaped the modern-day deep biosphere in the granitic basement underlying this crater25.”\nReference 25 being Quraish et al. (2024) in Geobiology, now cited.\n9 (19)\n10 (19)\nReviewer #3 (Remarks to the Author):\nThis is a very interesting paper that I thoroughly enjoyed reading. I appreciate the coordination\nbetween stable isotope and radiometric analyses to understand the timing of microbial colonization as\nit is linked (or not linked) to impact-generated hydrothermal systems. This specific type of work in the\nfield of terrestrial impact cratering, and particularly at the intersection of astrobiology, is sorely needed\nand is highly relevant as we continue to explore craters on Earth and the ancient Martian surface (and\npotentially subsurface, in the future!)\nThe work presented here addresses a key question related to impact-generated hydrothermal (IGH)\nsystems which is timing, as it relates to post-impact colonization by microbial communities. Previous\nstudies on sulfide mineral isotopes, for example, in impact craters have identified biosignatures in the\nform of highly reduced (isotopically light) δ34S, implying a biogenic rather than (abiotic)\nthermochemical sulfate reduction origin, but the timing of this activity has remained ambiguous and\noften linked directly to the hydrothermal system without more definitive (e.g., radiogenic isotope)\nconstraints.\n*Response: We acknowledge the reviewer for an overall positive assessment and for highlighting the\ngreater implications of our work. The detailed comments below are all addressed, and have improved\nthe revised version of the manuscript significantly.\nI have a few comments on general observations of the manuscript as well as line-by-line suggestions,\nbelow.\nSections of the methods and results related to mineral identification and lithologic context – and by\nextension the corresponding isotopic analyses - are unclear and would benefit from better organization\nand clarification. I find it difficult to interpret the isotope data without a better understanding of the\ncontext and what exactly has been analyzed. Are many of these generational relationships implied\nbased solely on BSE images of rough sample surfaces? Is there supporting chemical data (e.g.,\nsomething that clearly shows different generations of calcite) or possibly thin section images to\nsupport these implied relationships? If that data is available it would be immensely helpful for the\nreader and solidify some of these interpretations. I understand this paper represents a lot of\ncoordination between in-situ and bulk analyses, and care should be taken to make it clear what has\nbeen analyzed, actually identified and what it represents.\n*Response: Indeed, the paper summarizes a plethora of different techniques and materials, and we\nhave been very careful to control how these are interconnected. We agree that some of this information\ncould be better emphasized and more thoroughly presented in the manuscript, and we have made a\ncomprehensive effort include these in the revised manuscript. We acknowledge the reviewer for these\nimportant constructive remarks, that we believe have significantly improved the scientific\npresentation, and we believe that we have now reached a level of clarity that the reviewer aimed to\nachieve. For a detailed representation see responses to comments below. For instance, calcite\ngenerations are interpreted based on CL-images and BSE-images of polished crystal interiors of\nminerals on fracture surfaces and indeed also in thin sections and polished rock blocks analyzed by\nSEM (with EDS compositions for different minerals and generations, as well as EDS-maps for key\nfeatures, now added). These crystals are of various homogeneity and were targeted by micro analysis\nfor stable isotopes (SIMS) and radiogenic isotopes (LA-ICP-MS) and analysed in bulk or in\nsubgenerations (micro-drilled) for clumped isotopes. The relationships of these analyses are now better\ndefined, see below.\n11 (19)\nAre many of these generational relationships implied based solely on BSE images of rough sample surfaces- For example, to examine the identification of a “serpentine-group clay” (Lines 122, 126,\n144) I would expect XRD data: I see one XRD pattern - supplemental Fig. 9 - with no labeled peaks or\nmineral ID’s, and S8_XRD representing only one analysis, with a list of potential library matches, but\nI see no labeled clay (or other mineral) d-spacings indicative of serpentine, vermiculite or corrensite\n001’s, 060’s. Nor do the methods describe the careful analyses to identify corrensite, serpentine or\nvermiculite, a high charge smectite, were undertaken. The caption also states this was the clay coating\non the vug, in the hand sample, but it’s unclear how this context was determined - if this is a bulk,\ncrushed sample of a melt rock it could have been a secondary clay in the matrix, or something else as\nclays can form from altering many of the primary aluminosilicate phases under hydrothermal\nconditions.\n*Response: We have analyzed thin sections, polished rock blocks and uncoated open vugs under\npetrographic and stereomicroscope as well as SEM-EDS, and hand-picked minerals, that were\npolished to reveal cross-sections, and these cross-sections (calcite and pyrite) were also analyzed in\ndetail with CL and SEM-EDS to reveal growth zones, that were then targeted with SIMS spot analysis\nand LA-ICP-MS. We used a combination of petrographic observations, SEM-EDS, CL and SIMS data\nto define the generations of calcite presented. We have added a series of highly detailed images that\ncombines petrographic and EDS data with sketches of how minerals were sampled and later prepared\nand analyzed in microscale (and in bulk of subsamples) in Suppl. Figures 3-6 (3-5 enclosed below as\nwell). For the XRD, we have toned down some of these interpretations, because we cannot distinguish\nbetween corrensite and smectite with conventional methods as the reviewer notes. These observations\nare not critical for the main scope of this paper, but will be described more in depth in a future study\nfocused on clay mineralogy, lead by G. Osinski’s team. The XRD-analyses of the current manuscript\nare done on mineral separates, scraped of the fracture- and vug surfaces, and hand-drilled sample from\na vein. We have further added an XRD-spectrum of siderite and calcite. We have added a description\nof how the minerals were sampled for XRD to the method section in the Suppl.information. For\nvermiculite and for the siderite/calcite XRD data, we have updated the peaks in the supplementary\ninformation (S8_XRD) so that it is clear how peaks are labeled and assigned, and references to\naccompany them. We agree that vermiculite can only be identified with certainty using a more detailed\nclay mineral-specific XRD protocol, that can determine swelling components etc, and therefore (in\naddition to that the vermiculite seems to be of low crystallinity), we have termed it vermiculite*\nthroughout the manuscript and described the uncertainty of this identification.\nAdded figures with compositional data given for different calcite generations:\n12 (19)\nCalcite i and ii:\n13 (19)\nCalcite iii:\n14 (19)\nCalcite y and yy:\n- As another example, I see lots of beautiful photographs and BSE images of rough sample surfaces\nbut no thin sections or chemical (EDS) data to support these interpretations. If this is available (Lines\n461-464 suggest EDS analyses should exist), please add a few figures (e.g., chemical maps, spot\nanalyses, something to help place the isotopic analyses and mineral, lithologic relationships into\ncontext).\n*Response: As described in the comment above, we do not base our interpretations solely on\nphotographs and BSE images of rough sample surfaces, but also on BSE images and EDS data of\npolished rock surfaces (showing trace element differences between calcite generations for instance),\npolished thin sections, and EDS and SIMS data of polished pyrite- and calcite crystals in epoxy\nmounts. For SIMS, we need to use epoxy mounts with a large number of individual calcite and pyrite\ncrystals that are analyzed in an automated routine. Hence, we were able to gather a substantial number\nof δ13C, δ18O and δ34S values, which would not have been possible if using thin sections (in fact thin\nsections cannot be used easily in SIMS, and although it may have made the petrographic provenance\nmore robust, it would have decreased the number of spots by 90%, so it was an easy choice and we\nmade sure to have strong petrographic documentation of the mounted, hand-chiseled grains). We have\nadded more information (Suppl. Information Fig 3-6) and added EDS spectra and EDS-maps for key\npetrographic relation features.\n- As a final example, the context for many of these minerals used for isotopic analysis is unclear. E.g.,\nIt appears there are more than one occurrences of pyrite in these samples (Fig. 2a vs 2d). Where are\nthe pyrite crystals shown in Supplemental Fig. 3 (and by extension, the longer list of sample ID’s in\nthe excel sheet) from, other than “the melt rock” or “the lithic impact breccia?” Are these fracture\ncoatings or vug-fillings (e.g., Fig. 2)? Are they potentially hand-chiseled out (Lines 466-468) from the\nmelt or breccia matrix – some of these are 100 microns or less, as low as 10 microns (Line 134) in\nsize? These relationships really matter when it comes to interpreting their isotopes, particularly as\nthere is such an unusually large range of data here. I have no doubt that with the amount of work\n15 (19)\nperformed here, the data confirming these mineral identifications and lithologic, contextual\nrelationships is available to show the reader?\n*Response: We have replied to most of this to the comments above. But, in addition, we have added\nmore context. Like adding information to caption of Supplemental Figure 3, “from pyrite intergrown\nwith the first generation of calcite in this sample (i.e. with calcite group i in Fig. 2a of the main text).\nFor 3a”. And “petrographic context shown in Supplementary Fig 2b” for 3b. We have also added the\ndetailed figures on how samples are analyzed, from drill core to microscale, and added EDS spectra to\nthem as well. The samples are from both veins and vugs, as shown in the images. We have added host\nrock/type of sample to the Suppl lists of analyzes.\nThis is exciting work and I hope the authors can take the steps to organize, present, coordinate and\nclarify their methods and results, especially the implied mineral identifications, relationships and\ncontext. This is a really interesting study and I would look forward to seeing some of this information\nclarified to better piece together the story presented here!\n*Response: Once again, thanks again for the overall positive assessment and constructive comments\nthat will improve the paper significantly. We have made a substantial effort to comply with these\ncomments in our revised manuscript.\nLine by line comments:\nLines 49-52: As written it is unclear whether this statement implies that hydrothermal circulation and\ncooling below 250 degrees began at 1-1.6 Ma or lasted this long. Please clarify.\n*Response: corrected, as also replied to reviewer 1.\nLine 53: Minor typo: “…modeling have predicted…” to “…modeling has predicted…”\n*Response: Changed from“…modeling have predicted…” to “…thermal models have predicted…”\nLines 75-78: I would argue here that Chicxulub is an exception - those framboids are intergrown with\nthe dachiardite and analcime - these zeolites are high(er) temperature hydrothermal minerals,\nsupporting these LIKELY formed during a period of elevated temperatures. The analcime samples in\nparticular - the images in Kring et al. (2021), and the mineralogical, in-situ context of those crystals -\nit’s hard to make the argument that the framboids formed much later, during diagenesis/far below and\noutside IGH conditions.\n*Response: Although we don’t argue with that interpretation, the point we make here is that no\ngeochronological dating presented for the Chicxulub mineral assemblages. The point made in the\nintroduction is that in situ dating is required, especially since we show ourselves that mineral filled-\nvugs that are completely sealed and residing in impact glass may have ages being far younger than the\nimpact. Without any geochronology evidence, we would probably have interpreted them as primary\nand impact related. Hence, we did not change the text, the reader can go back to the original paper and\nmake their own assessment on the Chicxulub mineral assemblages.\nLines 80-85: Very minor comment – I agree with the overall statement – that there is a need for timing\nconstraints with the biosignature examples described in the previous section, but the biosignatures\ndescribed in this specific section are very different from the ones listed in the previous paragraph,\nwhich is a tad misleading (e.g., 34S of sulfides attributed to bacterial sulfate reduction at elevated\n16 (19)\ntemperatures). I would also suggest adding a couple words here that indicate these biosignatures were\ninitially attributed directly to impact hydrothermal activity. When I first read this I thought “well yeah\nthese old rocks are probably covered in some sort of fungus” but went to the reference (and was\nsurprised) to see someone initially claimed these were hydrothermal system-hosted fungi! Similar\ncomment for calcite, which can easily be reset and even initially form at surface diagenetic conditions.\n*Response: Yes, agreed it needs more context, which we added (bold), L88-91: “Using in situ Rb/Sr\ndating of calcite-albite-K feldspar assemblages occurring alongside fossilized fungi, that were\ninitially attributed to colonization during impact-related hydrothermal activity26, Tillberg et\nal.27 found that fungal colonization of the 458 Ma Lockne impact structure occurred at least\n100 million years after the impact” Ref 26 being added Ivarsson et al.\nLines 97-98: The comment on methane seepage is a bit out of place. I understand it’s relevance but I\nwould suggest removing this entirely or elaborating on how this is tied to the rest of the sentence and\nsection (e.g., are the authors implying the methane seepage was directly linked to IGH system-hosted\nmicrobes?) I would err on the side of caution here, as the source of Martian methane detections are a\nhighly contested topic, and very unlikely to be IGH system related.\n*Response: Agreed, and if we were to elaborate on this, it would perhaps be too extraneous. We\nremoved that part of the text L106: “methane seepage has been detected in the Gale crater on Mars26.”\nLine 131-133: “Clay minerals, zeolite and pyrite were among the first mineral coatings to appear\nintergrown with impact glass (Supplementary Fig. 1a,c)” Clay minerals and pyrite are not the first\nsecondary minerals to form in these systems – they are often later stage, especially clays. What is the\nevidence for this, based only on the BSE images? I also see no clays labeled or identified here. Is there\nEDS data to go with these images?\n*Response: Agreed with respect to clay minerals. There is a clay mineral, plausibly vermiculite in Fig.\n2b, but we don’t know how old this mineral is but it is likely not one the first secondary minerals to\nform in these systems. “Clay” is erased from the sentence. The sentence has been changed to L145-\n148: “Zeolites, such as erionite and chabazite (Supplementary Fig. 1a,c), as well as pyrite (Fig.\n2d) and Fe-Ni-sulfide aggregates that may contain remnants of the impacting projectile\naccording to previous studies30, are intergrown with impact glass in the impact melt-bearing\nbreccia.” We also have added EDS data to the supplementary information, as detailed above. And we\nhave given details on the “vermiculite*” naming, previously and at other place in the manuscript.\nLine 132: Which zeolite? Or change to zeolites.\n*Response: Change to “zeolites.”\nLine 140, Figure 2: Is there a difference in composition between these different types of calcite?\n*Response: We have given some more information on this in the text and added a lot of information in\nthe supplementary information. Calcite I and ii are more Fe rich (albeit low %) than y and yy for\ninstance.\nLine 143: Where is the data indicating this is ankerite? I see no labeled XRD patterns or in-situ EDS\ndata on thin sections?\n17 (19)\n*Response: We have done a complete overhaul of this following these detailed comments (thanks for\nasking!). We have done substantially more SEM-EDS-work and found out that ankerite is very minor\nand siderite is much more abundant. Hence, we are not presenting ankerite as a certain identification\nany longer, but have instead added siderite (confirmed by EDS spectrum and XRD spectrum). We\nhave also added EDS data for the different calcites, and pyrite.\nLines 149-150: What pyrite is being analyzing here exactly?\n*Response: This text was added: L162-163: “of all pyrite crystals analyzed by SIMS in this study”\nLines 163-170, Figure 3: what is the context for these pyrite grains (e.g., matrix, fracture or vug\ncoating?) Additionally, what is the other mineral in 3b?\n*Response: 3a: fracture/vein, 3b: matrix, 3c: vug. We have added this information to the caption.\nThe other mineral is calcedony. We have EDS spectra for it, below. And have added this information to\nthe caption. “showing pyrite along with calcedony (BSE-dark mineral)“\nLines 192-193: How are these petrographically identified as chronologically older? Based on BSE\nimages from rough sample surfaces? Are there thin sections (and corresponding EDS analyses) of\nthese samples?\n*Response: These groups are visible in CL, BSE and/or indicated by SIMS data. From thin section and\nmainly from polished cross sections of individual grains targeted by SIMS spots. We added this\ninformation to the caption.\nLines 271-275: These are very unusual and heavy values for sulfides – a very interesting and exciting\nfind! I see examples of these isotopically heavy sulfides in Fig. 3b and c – any chance you also have\ncorresponding EDS data on these? Or the other mineral intergrown with the pyrite in 3b. I’m curious\nto know if there’s a chemical difference or zoning – anything – that might help interpret these\nunexpected results.\n*Response: We do not see any compositional difference distinguishable with EDS. All spectra show\njust Fe and S. However, high resolution methods like LA-ICP-MS might be able to distinguish such\nvariations. We aim to explore this in a future study.\nLines 452-464: I see methods listed here for low vacuum analyses on rough sample surfaces. Is this\nwhat the chronological relationships and mineral identification, context is based on? Other than a\nsingle sample bulk XRD analysis and the second set of “SEM analysis” of hand-chiseled and epoxy\n18 (19)\nmounted minerals listed 466-471? Once the samples are hand chiseled you lose a lot of contextual\ninformation. I’m curious why no thin section petrographic work was completed. If it was (e.g., EDS\nmapping at high vacuum, or spot analyses and imaging to understand the in-situ mineralogical,\nchemical context), please add it to the manuscript.\n*Response: As stated above we did use thin sections as well, and have added this information to the\nmethod section, and to the Supplementary information (including EDS mapping).\nSupplemental Fig. 1,2: I see several BSE images showing beautiful mineral surfaces, and vugs but no\nchemical (e.g., EDS, WDS) or crystallographic data (e.g. XRD, outside the unlabeled Supp. Fig. 9).\nSupplemental Fig. 9: Please label mineral peaks here (e.g., mineral ID and their respective, indicative\n001, 060 d-spacings). How was vermiculite characterized? Clay minerals are notoriously difficult to\nidentify – vermiculite is a high-charge smectite. Is there additional information that shows this is\nindeed vermiculite? What about the other clays listed in the manuscript – serpentine and corrensite?\n*Response: This is a very relevant comment, and we have added more information, as outlined in\ncomments above, and added peaks to the XRD spectrum, and added another XRD spectrum, as well as\nmany EDS spectra and maps. We have also toned done the clay mineral identifications, to being more\n“indications” than identifications.\n19 (19)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Dear Editor,\nhaving reviewed the second round of this manuscript submission I think the paper is now good to go, with a few final suggestions listed below.\nL 29. I’d write „…, which may have…”\nL66/L67: 2 x “have been reported”\nL72: …are also…\nL97: Yes, but there may be other contexts, e.g., hydrothermal mineral assemblages and sequences with traces of\ncolonization linked to specific minerals using purely petrographic methods, providing a relative timeline of colonization.\nL121: maybe remove “unshocked” and replace by “fractured” – this unit was presumably also shocked, but at low levels not\nimmediately recognizable in terms of shock metamorphism.\nGeneral: Please proofread a final time for the filling word ‘also’ – there is (locally) an overabundance. Perhaps replace by\n“Moreover,” or “as well” where appropriate.\nL195: 4 cm-long or 4 cm-diameter vug?\nL251: comma after parentheses\nL300: are consumed?\nL491: Sentences should not start with a number. Perhaps write out “Thirty-three..:”\nKind regards,\nMartin Schmieder", "reviewer_2": "Deeply fractured rocks within meteorite impact structures have been hypothesised as hotspots for microbial colonisation on\nEarth and potentially on extraterrestrial planetary bodies. Biosignatures have been identified from a limited number of impact\nstructures (notably the Chicxulub impact crater) and have offered valuable evidence of microbial activity following impact\nevents. However, the timing of microbial colonisation in relation to these impact events remains uncertain. Consequently, it\nis possible that the biosignatures previously reported from impact structures are unrelated to the impact crater formation and\ninstead reflect outcomes of long-term recolonisation of the site.\nTo test this possibility, this study employed microscale stable isotope biosignature detection, combined with radioisotopic\ndating, to precisely determine the timing and temperature constraints of post-impact microbial recolonisation of vug- and\nfracture-filling assemblages in impactites of the 77.85±0.78 Myr-old Lappajärvi impact crater (Finland).\nThe earliest evidence of microbial recolonisation was detected through the presence of 34S-depleted pyrite, consistent with\nsulfate-reducing bacterial (SRB) activity, in minerals precipitated approximately four million years after the impact (73.6±2.2\nMa ago). Clumped isotope analysis showed that the precipitation temperature was about 47 ± 7 degrees Celsius,\nsignificantly below the threshold for microbial life. δ13Ccalcite values indicated active methane cycling between\nmethanogens and anaerobic methane-oxidising archaea in symbiosis with SRB during vug mineral formation, which\noccurred over 10 million years later at progressively lower temperatures.\nAs concluded by the authors, it would be important to (re)consider previous studies from the Haughton and Chicxulub large\nimpact structures – that suggested 34S-depleted sulfides to be proof of microbial colonization of impact structures within the\nlifetime of the impact-generated hydrothermal system as these studies did not provide geochronological data and\ntemperature constraints were presented from veins at Haughton only.\nThe sampling and analytical approaches are sound and I did not find any flaws in the data analysis, interpretation, and\nconclusions."}, "author_response": "We thank the reviewer for the positive assessment of the second round of this manuscript submission. We also thanks\nthe reviewer for the final suggestions, which we have corrected:\nL 29. We added “an effect” before “that” in the sentence, so that it is grammatically correct:\n“These insights confirm the capacity of medium-sized (and large) meteorite impacts to generate long-lasting hydrothermal\nsystems, enabling microbial colonization as the crater cools to ambient conditions, an effect that may have important\nimplications for the emergence of life on Earth and beyond.”\nL66/L67: “has” is grammatically correct, so we did not change anything here.\nL72: We added “are” and improved to sentence to: “Likewise, at Rochechouart, δ34S sulfide values down to -36‰ are consistent\nwith MSR22”\nL 97: We changed to: “Radioisotopic dating, along with other contexts, such as petrographic relations, is essential to verify\nsuch colonization.”\nL121/L472: We agree, “unshocked” has been replaced by “fractured”\nGeneral comments about the filling word “also”:\nWe have replaced “also” by “moreover” or “as well” etc. where appropriate.\nL195: We changed to “4 cm diameter vug”\nL251: We added a comma after the parentheses\nL300: “is” is grammatically correct\nL491: We agree and changed to “Thirty-three..:”"}]

Decision letter