Vitis18K SNP chip for linkage mapping.We present a high-density integrated map for grapevine, allowing refinement and improved understanding of the grapevine genome, while demonstrating the applicability of the Vitis18K SNP chip provides a useful genotyping tool for genome-wide marker analysis. Most linkage maps are based on single mapping populations, but an integrated map can increase marker density and show order conservation. Here we present an integrated map based on three mapping populations. The parents consist of the well-known wine cultivars ‘Cabernet Sauvignon’, ‘Corvina’ and ‘Rhine Riesling’, the lesser-known wine variety ‘Deckrot’, and a table grape selection, G1-7720. Three high-density population maps with an average inter-locus gap ranging from 0.74 to 0.99 cM were developed. These maps show high correlations (0.9965–0.9971) with the reference assembly, containing only 93 markers with large order discrepancies compared to expected physical positions, of which a third is consistent across multiple populations. Moreover, the genetic data aid the further refinement of the grapevine genome assembly, by anchoring 104 yet unanchored scaffolds. From these population maps, an integrated map was constructed which includes 6697 molecular markers and reduces the inter-locus gap distance to 0.60 cM, resulting in the densest integrated map for grapevine thus far. A small number of discrepancies, mainly of short distance, involve 88 markers that remain conflictual across maps. The integrated map shows similar collinearity to the reference assembly (0.9974) as the single maps. This high-density map increases our understanding of the grapevine genome and provides a useful tool for its further characterization and the dissection of complex traits.The improvement of grapevine through biotechnology requires identification of the molecular bases of target traits by studying marker-trait associations. The The online version contains supplementary material available at 10.1007/s00122-022-04225-6. Vitis vinifera, is a commercially important fruit crop since the berries are used for a wide range of products such as wine, table grapes, raisins and spirits. Each of these products requires specific berry attributes concerning color, composition and firmness; therefore, grapevine improvement strategies, including breeding programs, have been tailored for such traits. Grapevine cultivation is increasingly prone to the impacts of the changing climate  or to exploit all available markers as predictors of breeding value (genomic selection) . In addition to demonstrating the application of the Vitis18K SNP chip across various mapping populations, we confirmed by mapping, the positions for about 9500 of the included SNPs. By constructing a high-density integrated map a reliable order, validated in many cases across multiple maps, was released, while also providing a list of markers with less reliable positioning. This high-density integrated map represents a highly valuable tool that can now be exploited for QTL mapping of relevant traits segregating in the populations.This study aims to generate the first integrated map using the Vitis vinifera mapping populations were used in this study to build the single population genetic maps from which the high-density integrated grapevine map was constructed. The CSxC mapping population was derived from a cross made in 2015 by the University of Verona between the ‘Cabernet Sauvignon’ (VIVC 1929) and ‘Corvina Veronese’ (VIVC 2863) varieties, originally including 680 seedlings planted on their own roots in 2017. A population sub-portion including the 142 progenies here considered for mapping purposes was later propagated and planted in two replicates at Società Agricola Vivai Gozzo in Verona, Italy  on SO4 rootstock. The DRxG1 mapping population resulted from a cross made by the Agricultural Research Council (ARC) in 2009 between the wine grape ‘Deckrot’ (VIVC 3482) and a table grape selection, G1-7220, originating from a cross between ‘Black Rose’ (VIVC 1408) and ‘Muscat Seedless’ (VIVC 8251). The DRxG1 population, including 225 plants, was planted in 2011 in a single block at ARC Infruitec-Nietvoorbij in Stellenbosch, South Africa . A subset of 137 individuals was used for mapping purposes. The RRxCS mapping population was obtained from a cross made in 2005 between ‘Riesling Weiss’ (VIVC 10077) and ‘Cabernet Sauvignon’ (VIVC 1929) and was planted in 2008 in the experimental field “Giaroni” of the Fondazione Edmund Mach (FEM) in San Michele all’Adige, Trento, Italy . It included 300 vines maintained with standard viticultural practices among which 139 were here considered for mapping purposes. The DRxG1 and CSxC progenies were tested for trueness-to-parent using five SSR markers , whereas the RRxCS progeny was tested using the nine SSR markers recommended for characterization of regional cultivars by the European Project GrapeGen06  . For each of the populations, one individual was genotyped in duplicate to determine reproducibility of the genotyping. Genomic DNA was extracted from 100 mg fresh young green leaves with the QIAGEN ® DNeasy Plant Mini Kit as per the manufacturer’s instructions. Following DNA quantification, using the NanoDrop™ Spectrophotometer 2000 (Thermo Scientific™), 200 nanograms were used for the genotyping assay. Raw SNP data were visualized and clustered in GenomeStudio v2011.1  using a GenCall threshold of 0.20. Subsequently, SNPs were re-clustered using ASSiST v1.01 .The three mapping populations were genotyped with the Illumina A and 60 s at 72 °C; followed by a final extension step of 30 min at 60 °C. SSR genotyping in the CSxC and RRxCS populations was performed as described by Emanuelli et al. . For SSR genotyping, genomic DNA was independently extracted from 100 mg fresh young green leaves using a CTAB extraction  were used as queries for BLASTN (threshold 1xE−05) searches to identify their theoretical position on the ‘PN40024 12X.v2’ reference genome sequence  was selected. For SSR markers, expected physical positions were recovered as described in Delfino et al.  derived from NCBI (https://www.ncbi.nlm.nih.gov/nuccore) used as a query to recover the SSR physical position. The ‘PN40024 12X.v2’ theoretical physical positions for all markers used in this study are reported in Table S1 (SNPs) and Table S2 (SSRs) alongside the mapping information. Linkage disequilibrium (LD) was estimated in Plink v1.90 between all genotyped SNPs with MAF > 0.05 for each chromosome either within each population or considering individuals from all three populations. Average r2 in sequential bins of 20 Kbp was plotted against physical positions with R 4.2.1.SNP marker expected positions, when available, were recovered from the literature  mapping algorithm, which uses the Haldane mapping function Haldane . The ML The integrated map was developed from the three population maps based on the reduced datasets, as explained previously, which included only one representative marker per group of identical markers. This was implemented to discard redundant information for integration while maximizing shared markers.The marker orders of the three population maps were inspected visually for collinearity. For each chromosome, the map positions of each marker were compared against each other in MS Excel to identify marker conflicts between population maps. The conflict orders were forced in the other population(s) to test whether the alternative orders were also acceptable. If the forced order resulted in an acceptable map quality , the conflict across maps was considered as inconclusive, and the shared acceptable order was accepted for building the integrated map. Although this sometimes introduced conflicts to the assembly in the other populations, we gave priority to the marker order obtained by the genetic data. Alternatively, if the forced order resulted in poorer map quality or higher stress of markers, the conflict order was not accepted in the other population, resulting in a conclusive conflict between populations. In this case both orders were retained in the respective population for building the integrated map , was visually depicted by means of Circos diagrams  to construct three population maps. To develop linkage maps 142, 137 and 139 individuals for CSxC, DRxG1 and RRxCS populations, respectively, were used. Since markers were already filtered for segregation distortion in ASSiST, only 3 SNPs were filtered for distortion during map building. On average across the three mapping populations, 13% of the ASSIsT-recovered SNPs were subsequently removed during map building. Although this is higher than the proportion (2%) of poor fit markers in the rest of the robust dataset, which includes only successfully genotyped SNPs, the recovery of SNPs by ASSiST still resulted in the addition of 152–217 SNP markers in the population maps. Furthermore, most poor fit markers (80%) still originated from the robust dataset. In general, segregation was evenly spread between maternal (lmxll) and paternal (nnxnp) segregation, although the DRxG1 parents had fewer shared alleles (segregation types efxeg and hkxhk) compared to the other parental pairs (Table S4).For the CSxC population, 7133 polymorphic markers, which were represented by 3467 representative markers with fully matching genotypes of the same segregation kind, finally mapped at 1795 unique positions in 19 linkage groups corresponding to the 19 chromosome pairs of grapevine  were mapped only in a single population whereas 4382 markers (40.24%) and 2681 markers (24.62%) were shared by two and three maps, respectively. As expected, due to the common parent, the CSxC and RRxCS maps shared more markers (22.80%) than CSxC and DRxG1 (8.71%) or RRxCS and DRxG1 (8.73%). Similarly, population DRxG1 contained more unique markers (16.03%) than CSxC and RRxCS (9.38% and 9.73%) , but in all chromosomes the correlation was equal or higher than 0.9797, with an average value of at least 0.9965 Table . High covs physical distances , 1994 (32%) and 2731 (38%) markers were involved in conflictual ordering across the 19 chromosomes in the CSxC, DRxG1 and RRxCS maps, respectively. The collinearity between the population maps and reference genome assembly was also further investigated by plotting genetic The comparison to the reference genome assembly was then used to enquire specific inconsistencies. A detailed investigation identified a group, including only 66 markers in the three different populations , which were allocated, on the basis of linkage mapping, to a chromosome different from that of their expected physical position  27 additional markers located on chromosomes 3, 4, 10, 12, 13, 15 and 17 were identified . Most of these markers were mapped to regions of chromosomes 2, 7 and 10. For 193 markers mapping was confirmed in more than one population. Interestingly, by comparison to the ‘PN40024 12X.v2’ genome assembly we found that the genetic mapping was always consistent inside the different scaffolds. In detail, among 104 newly anchored scaffolds, 86 were anchored to the genome in at least two different populations . Eighteen scaffolds were anchored in only one of the populations.Population maps including only one representative marker per group of identical markers were considered for the purpose of building an integrated map. We considered 6697 representative informative markers, of which 3467, 3173 and 3459 were mapped in the CSxC, DRxG1 and RRxCS populations, respectively. Of these 2023 markers mapped in two populations, and 690 markers mapped in all three populations.The marker orders of the population maps were compared to identify non-collinear regions to be preliminary addressed to avoid integrated map inflation due to conflicts. Of these non-collinear regions, 65% (51 regions) could be solved since the alternative marker order was of acceptable quality in the other population(s), indicating that marker order between population maps was largely conserved. The remaining 35% (31 regions) resulted in a conclusive conflict, meaning that the alternative order was not acceptable in the other population map(s) (Table S10). This involved 88 markers distributed on 14 of the 19 grapevine chromosomes. The highest number of these conflicts was on chromosomes 7 (13 markers), 4 and 15 (10 markers), 3 (8 markers) and 13 and 10 (7 markers). These non-collinear markers were subsequently treated as independent markers and were mapped in more than one position in the integrated map .Despite the high over-all collinearity of the population maps to the ‘PN40024 12X.v2’ genome assembly described previously , in agreement with the well-recognized low marker transferability in Vitis . It is also possible that ascertainment bias during the SNP chip development reduced the number of informative markers in DRxG1. Most of the Vitis18K SNP chip . Three genetic maps have been built so far using data derived from the Vitis18K SNP chip , no mapping information had been reported. Most markers mapped showed a genetic position that was congruent to their expected theoretical position, which confirmed the SNP chromosomal assignment according to BLASTN of flanking sequences. We only identified a few SNPs which mapped to different chromosomes compared to their expected location (64 SNPs that are included in the 66 markers listed in Table S7) or which showed large inconsistencies (more than 10 cM) compared to the expected locations . Our SNP mapping results were highly congruent with the available published map data of Duchêne et al. (Vitis18K SNP chip in both works (Table S14). Moreover, among the 66 markers mapping to chromosomes different from those expected that were identified in the present study (Table S7), two (ae_9_7573501 and ae_5_12606386) were also mapped in ‘Riesling’ by Duchêne et al. . Out of these, only 15 markers had been genetically positioned in previous studies  suggests that their incorrect assignment might be related to short, repeated regions and consequent BLAST issues. Accordingly, our BLAST data show that more than one BLAST hit was found for 37 out of the 64 of the SNP flanking sequences. In more than half of these cases (21 out of 37), the alternative position established by genetic mapping was identified in the secondary BLAST hit(s) (in bold in Table S7b). Incorrect allocation by BLASTN mainly concerned SNP probes originating from species other than Vitis18K SNP chip already reported, no integrated map has been built so far to combine such information. To increase map marker density and provide a reliable shared marker order, we constructed an integrated reference map from three population maps. Two alternative approaches can be used in producing integrated genetic maps. The first, a “genetic merging approach”, makes use of recombination frequencies from genotypes to develop the integrated map in a similar fashion to bi-parental mapping, as is employed by the softwares Carthagene  was only observed for chromosome 9 . The parental genotype (provided in TOP format as extracted from GenomeStudio), the outcome of the analysis performed in ASSiST, the segregation pattern, the outcome after the analysis performed in JoinMap® and the map position (as chromosome_cM) are reported for each marker in each population. For the dataset reduction, where markers with similar genotypes were excluded, the representative marker that was retained in each population is shown. The position in the integrated map is also indicated for each representative marker (as chromosome_cM). Finally, each marker is classified as congruent if it was mapped to the same chromosome of the theoretical position assigned to that marker within a genetic interval of ±10 cM. Abbreviations: chr = chromosome, CS = ‘Cabernet Sauvignon’, C = ‘Corvina’, DR = ‘Deckrot’, G1 = G1-7720, RR = ‘Rhine Riesling’, NA = not analyzed. Table S2: List of SSR markers genotyped in this study. For each marker, the reference and primer sequences and the position on the ‘PN40024 12X.v2’ genome assembly are indicated. The allele sizes (in bp) of the parents in each population, as well as the segregation pattern, the outcome after the analysis in JoinMap® and the map position for each marker are included. The position in the integrated map is also indicated (as chromosome_cM). Abbreviations: chr = chromosome, CS = ‘Cabernet Sauvignon’, C = ‘Corvina’, DR = ‘Deckrot’, G1 = G1-7720, RR = ‘Rhine Riesling’, NA = not analyzed. Table S3: Genotypic information (.loc file) of 142 progeny of the ‘Cabernet Sauvignon’ × ‘Corvina’ (CSxC) population for 7 464 informative markers (a), 137 progeny of the ‘Deckrot’ × G1-7720 (DRxG1) population for 6732 informative markers (b) and 139 progeny of the ‘Rhine Riesling’ × ‘Cabernet Sauvignon’ (RRxCS) population for 7461 informative markers (c). Table S4: Segregation patterns of the SNP and SSR markers in the three populations , respectively. Table S5: Final population maps based on the whole dataset for CSxC (‘Cabernet Sauvignon’ × ‘Corvina’), DRxG1 (‘Deckrot’ × G1-7720) and RRxCS (‘Rhine Riesling’ × ‘Cabernet Sauvignon’). Abbreviation: chr = chromosome. The map position of each marker is indicated as chromosome_cM. Table S6: Evaluation of the population maps based on genetic data only (using the reduced dataset). Section (a) indicates the Spearman rank correlation coefficients between the population maps (obtained from genetic data only) and the ‘PN40024 12X.v2’ genome assembly. Section (b) refers to chromosome 8 from DRxG1 parental maps  before and after the integration of assembly information (fixed order). The table reports some map quality parameters  and Spearman rank correlation coefficients between the maps and the ‘PN40024 12X.v2’ assembly. In both sections, maps with poor correlation to the ‘PN40024 12X.v2’ assembly are indicated in bold. Abbreviations: chr = chromosome, CSxC = ‘Cabernet Sauvignon’ × ‘Corvina’, DRxG1 = ‘Deckrot’ × G1-7720 and RRxCS = ‘Rhine Riesling’ × ‘Cabernet Sauvignon’. Table S7: Comprehensive list of markers mapping to a different chromosome from that previously reported on the ‘PN40024 12X.v2’ genome assembly. Section a) indicates for each marker the expected physical position as well as the genetic position in the CSxC (‘Cabernet Sauvignon’ × ‘Corvina’), DRxG1 (‘Deckrot’ × G1-7720) and RRxCS (‘Rhine Riesling’ × ‘Cabernet Sauvignon’) maps, respectively. Markers where the alternative chromosome assignment is confirmed by mapping in two or more populations are shown in bold. Section (b) indicates the highest BLASTN result together with alternative BLASTN results. Secondary BLASTN hits supporting the alternative chromosome genetic assignments are indicated in bold. Abbreviations: chr = chromosome, pos = position . Table S8: Markers with inconsistencies larger than 10 cM in comparison with the reference genome assembly. Section a) lists the markers with the mapped position in the CSxC (‘Cabernet Sauvignon’ × ‘Corvina’), DRxG1 (‘Deckrot’ × G1-7720) and RRxCS (‘Rhine Riesling’ × ‘Cabernet Sauvignon’) maps, respectively. Marker inconsistencies, which were confirmed in more than one population, are indicated in bold. Section b) lists the expected physical position (sstart-send) of these markers on chromosomes (sseqid), according to BLASTN results. Bold indicates multiple hits in the same chromosome where the marker was mapped. Abbreviations: chr = chromosome, pos = position . Table S9: Markers physically assigned to the “ChrUn” grapevine chromosome (columns B-C) and now genetically assigned to chromosomes from 1 to 19 in one or more populations (columns D-I). Column J reports the anchoring of each marker to one of the 104 scaffolds located to the Unknown chromosome from the Vitisagp grapevine genome assembly . Abbreviations: chr = chromosome, pos = position , CSxC = ‘Cabernet Sauvignon’ × ‘Corvina’, DRxG1 = ‘Deckrot’ × G1-7720 and RRxCS = ‘Rhine Riesling’ × ‘Cabernet Sauvignon’. Table S10: List of markers that were non-collinear between population maps. The table reports for each marker the original name, the name with the suffix used in the integrated map (including the information on one or both the conflict(s), in letters as explained in Materials and Methods, and the number of population maps where it was mapped, within brackets), the physical position in the ‘PN40024 12X.v2’ genome assembly, the genetic position in the maps where it was mapped, the two genetic positions in the integrated map and the conflict type. Abbreviations: chr = chromosome, pos = position , CSxC = ‘Cabernet Sauvignon’ × ‘Corvina’, DRxG1 = ‘Deckrot’ × G1-7720 and RRxCS = ‘Rhine Riesling’ × ‘Cabernet Sauvignon’. Table S11: List of markers whose genetic position was in conflict with the assembly in at least one of the population maps, which were based on the reduced datasets of markers and were used to construct the integrated map. The table reports for each marker the original name, the name with the suffix used in the integrated map (including the information on one or both the conflict(s) type, in letters as explained in Materials and Methods, and the number of population maps where it was mapped, within brackets), the physical position in the ‘PN40024 12X.v2’ genome assembly, the number of population maps where it was mapped, the map where the conflict(s) occurred and the genetic position on the integrated map (indicated as chromosome_cM). Markers involved in conflicts to the assembly for which the conflict was originally revealed in more than one map (not introduced by the conflict solving step) are shown in dark blue, while conflictual markers affecting just one specific map, or the shared ones introduced by forcing marker orders during conflict solving are shown in light blue. Markers for which the support for the alternative marker order was introduced in more maps by forcing the orders during the conflict solving step and for which alternative orders are likely were not labeled. Abbreviations: chr = chromosome, pos = position . Table S12: Grapevine integrated map based on three mapping populations . Markers in the population genetic map(s) that are mapped in conflictual positions compared to their physical positions are represented in blue , while markers involved in conflicts across maps are coded in red. Priority is given to red labeling. Abbreviation: chr = chromosome. Numbers within brackets indicate the number of population maps in which the markers are mapped. The position of each marker in the integrated map is indicated as chromosome_cM. Table S13: Spearman rank correlation coefficients between the population maps and the integrated map. Abbreviations: chr = chromosome, CSxC = ‘Cabernet Sauvignon’ × ‘Corvina’, DRxG1 = ‘Deckrot’ × G1-7720 and RRxCS =‘Rhine Riesling’ × ‘Cabernet Sauvignon’. Table S14: Comparison of ‘Riesling’ maps obtained in Duchêne et al. (2020) and in this study. Section (a) reports chromosome assignment and map position (in cM) for 784 common markers. Markers mapping in both studies to the same alternative chromosome compared to that expected are shown in bold. Section (b) indicates the Spearman rank correlation coefficients between the two maps calculated separately for each chromosome and as an average value across chromosomes. Abbreviations: chr = chromosome.Supplementary file 1. Figure S1: Decay of LD estimated for each of the 19 grapevine chromosomes. A regression line for the average r2 value estimated in sequential bins of 20 Kb against the physical distances between SNP is shown for each of the three populations. Decay in CSxC  is shown in blue, in DRxG1  in red and in RRxCS  in green, respectively. The LD decay for the population built including individuals from each of the three populations (418 plants) is shown in yellow. Abbreviations: chr = chromosome. Figure S2: Detailed grapevine integrated map based on three mapping populations  indicating the names of all non-collinear (with the ‘PN40024 12X.v2’ assembly and between populations) markers and markers mapped in all three populations (bold). Markers that are in non-collinear position with the 'PN40024 12X.v2’ genome assembly order are indicated in blue . The population map involved in the conflict is indicated with suffix –A (CSxC), –B (DRxG1) or –C (RRxCS). Markers that are in non-collinear positions between population maps are indicated in red with the suffixes –AB (CSxC vs DRxG1), –AC (CSxC vs RRxCS) or –BC (DRxG1 vs RRxCS) to indicate conflictual populations. Priority is given to red labeling. The suffixes (1) (2) (3) indicate the number of population maps in which the markers were mapped. Markers that are collinear with the ‘PN40024 12X.v2’ assembly and between population maps and are not mapped in all three populations are only indicated with a line.Supplementary file 2. Below is the link to the electronic supplementary material.


This endogenous assemblage results from re-speciation and drying of cooling C–O–S–H–N fluids entrapped below 600 °C–2 kbars in rocks forming the present-day oceanic lithosphere. Serpentinization dries out the system toward macromolecular carbon condensation, while olivine pods keep ingredients trapped until they are remobilized for further reactions at shallower levels. Results greatly extend our understanding of the forms of abiotic organic carbon available in hydrothermal environments and open new pathways for organic synthesis encompassing the role of minerals and drying. Such processes are expected in other planetary bodies wherever olivine-rich magmatic systems get cooled down and hydrated.How simple abiotic organic compounds evolve toward more complex molecules of potentially prebiotic importance remains a missing key to establish where life possibly emerged. The limited variety of abiotic organics, their low concentrations and the possible pathways identified so far in hydrothermal fluids have long hampered a unifying theory of a hydrothermal origin for the emergence of life on Earth. Here we present an alternative road to abiotic organic synthesis and diversification in hydrothermal environments, which involves magmatic degassing and water-consuming mineral reactions occurring in mineral microcavities. This combination gathers key gases (N How complex organics form in a prebiotic world remains a missing key to establish where life emerged. The authors present a road to abiotic organic synthesis and diversification in hydrothermal contexts involving magmatism and rock hydration. Abiotic methane (CH4) is the most abundant of those compounds, and can be accompanied by short-chain hydrocarbons  or organic acids  in fluids8 occurring in molecular hydrogen (H2)-enriched hydrothermal systems where olivine-bearing rocks are altered via serpentinization reactions9 in various geological contexts . Because of this limited variety of small abiotic organic molecules and their strong dilution in hydrothermal fluids, prebiotic reactions cannot easily lead to more complex molecules of biological interest; thus, constituting a limiting factor for a unifying hypothesis for a hydrothermal origin of life on Earth. Without evidence for alternative abiotic organic molecules or pathways and based on an abundance of diverse organic molecules in comets and meteorites12  many have considered that life’s ingredients had an extraterrestrial origin.In nature, very few organic compounds are recognized as abiotic, i.e., formed by mechanisms that do not involve life13. Such a process requires a nitrogen source for amine formation and a polyaromatic precursor whose origin remains unknown, and suggests the availability of more diverse abiotic organic reactants than previously expected on Earth, notably in the subseafloor. The discovery of low-T formation of abiotic carbonaceous matter in ancient oceanic lithosphere14 also leads to the consideration of new paradigms for organic synthesis pathways within the rocks hosting hydrothermal fluid circulation15. Processes leading to such complex, condensed compounds, during rock alteration are unknown but must differ from the mineral-catalyzed Fischer-Tropsch Type (FTT) process that is the most invoked so far in hydrothermal fluids19 to explain the formation short-chained hydrocarbons. Understanding the variety and formation mechanisms of abiotic organic compounds on Earth, as well as their preservation, has important implications for the global carbon cycle, but also expands the inventory of the forms of carbon available for present-day ecosystems and prebiotic chemistry, and compliments data from extraterrestrial systems21.Recent studies of serpentinized rocks along the Mid-Atlantic Ridge (MAR) have highlighted low temperature (T), abiotic formation of aromatic amino acids via Friedel–Crafts reactions catalyzed by an iron-rich saponite clay22 where the discharge of abiotic H2, CH4 and formate have been observed in fluids23. Within the shallow rock substrate of Hole 1309D (~170 m.b.s.f), abiotic amino acids were identified13. At deeper levels (1100–1200 m.b.s.f), olivine-rich igneous rocks such as troctolites are particularly fresh and rich in FIs where they form linear trails of various orientations within olivine grains 26. During cooling, rocks were progressively exhumed below an extensive fault zone up to their present-day position (P < 0.3 kbar and T~100 °C27).Here we demonstrate how deep mid-ocean ridge processes can provide an unexpected diversity of abiotic organics as gaseous and condensed phases thanks to water-consuming mineral reactions. Our study focuses on the investigation of olivine mineral microcavities (secondary fluid inclusions (FI)) aligned along ancient fracture planes where circulating fluids were trapped within one of the deepest igneous-rock sections drilled along the MAR, i.e., IODP Hole 1309D, 1400 meter-depth below seafloor – m.b.s.f., at the Atlantis Massif  and/or CH4(g) as well as secondary minerals serpentine (lizardite ± antigorite), brucite, magnetite, or carbonates , as previously observed in similar or ancient rocks32 , methanethiol (CH3SH(g)), and variably disordered polyaromatic carbonaceous materials (PACMs) closely associated with secondary minerals in FIs  were made on 36 closed FIs in olivine grains forming three troctolites cored at IODP Hole 1309D . The samples were very fresh and only displayed a localized alteration along thin serpentinized veinlets, underlined by magnetite grains Fig. . All FIsFIs Fig. .Fig. 2ReTo further investigate the nature of the PACMs, high-resolution 3D Raman mapping was carried out on two FIs from one olivine grain of sample 1309D-228R2 Fig.  and were36. Previous investigations established that the trend followed by terrestrial kerogens and meteoritic carbonaceous matter in such diagrams reflects an increase in thermal maturation during prograde metamorphism. Thermal maturation globally involves organic matter carbonization characterized by a decrease of the full-width at half maximum of the disorder (D) band (FWHM (D))36. It is materialized by a decrease in heteroatom-bearing groups  and aliphatic units, and an increase in the degree of aromaticity. It can be followed by graphitization during which the structural order of the graphitic material increases. This corresponds to a decrease of defects in aromatic planes, characterized by the decrease of the relative intensities R1 of the D and graphite (G) bands (R1 = ID/IG). While such a metamorphic history does not apply in the present context of cooling and exhumation of deep-seated rocks at the Atlantis Massif, this trend is used here to chemically and structurally describe the observed material based on its comparable spectroscopic characteristics. The PACMs contained in our two 3D-imaged FIs cover an unusually large range depicted in Fig. Quantitative parameters were extracted from 3D hyperspectral Raman data collected on FI3 and FI5 (see Methods) and compared to those of graphite, terrestrial biologically-derived kerogens, as well as carbonaceous matter in meteorites Fig. 33–36. Pr−1 is characteristic of the stretching mode of the carbonyl functional group (C = O), and the shoulder around 1100 cm−1 fits well with stretching vibrations of C–O/C–O–C in ether or carboxylic ester functional groups37. In PACM1, a shoulder is also visible near 1200 cm−1. A similar component has been described in natural and synthetic functionalized carbon systems, while lacking in more carbonized or graphitic materials38. Its origin is not well understood, but it was previously attributed to vibrations of C–H/C–Calkyl in aromatic rings39. 3D data indicate that PACM1 is spatially well distributed in the FI and is primarily associated with phyllosilicates, corresponding to the gel-like phase wetting serpentine and brucite fibers in FI5  and graphite (G) bands, two additional contributions are detectable Fig. . The banFI5 Fig. . HR-TEM FI5 Fig. . This agFI5 Fig. .Raman and SEM imaging shows that PACM2, observed in both FIs, is localized on olivine walls where it forms a mesoporous texture made of nanofilaments Fig.  of ~20 nd~0.20 nm that corresponds to the d111 of cubic nano-diamond (nD). Raman signals of nD strongly depends on their structure, purity, crystal size and surface chemistry41, but the smallest ones (<few tens of nm) commonly display a downshift and broadening of the D band due to phonon confinement effects44 and an additional G band attesting to residual defects and graphitic domains within a surrounding carbon shell43. PACM3, which plots in the lower-left end of Fig. −1, a FWHM-D of 54–70 cm−1 and a G band near 1550 cm−1. 3D Raman data of nD (PACM3) are also co-localized with PACM2 that shows a dense and spotted texture made of particles 5–50 nm in diameter, hence attributed to nD . This confirms previous observations of the dominance of a macromolecular structure with H- and O-bearing functional groups. The remaining contributions correspond to carbon in the form of carbonate . The latter was not clearly located but it most probably contributes to the nano-particles observed in the C-rich PACM2 . A minimum concentration of 143 µmol of CH4 per kg rock was measured on this sample. 431 and are close to the compositions of CH4 venting in Lost City hydrothermal chimneys nearby on the same massif19.Carbon and hydrogen isotopic composition of the CH2, CH4, CH3SH, PACMs, carbonates, carbide, and nD) requires an aqueous fluid enriched in C, N and S, to be trapped as olivine-hosted inclusions. At MOR, such a fluid can be magmatic or seawater-derived, or a mix of both. The fresh character and the Sr isotopic compositions of deep magmatic rocks from the same hole attest of their very limited interaction with seawater48 that resulted in late serpentine veinlets, formed after the FIs  and depth conditions of fluid trapping26 since seawater-derived fluids would have also undergone boiling and phase separation51. Hence, a dominant magmatic origin, resulting from magma degassing, is favored for the fluid trapped in our inclusions as previously proposed for olivine under similar deep crustal conditions53. Such fluids, exsolved from melts, are dominated by CO2-rich vapors that can evolve to more H2O-enriched compositions with progressive fractionation52. Indeed, at MOR, the source of magma is located in the shallow upper mantle where equilibrium thermodynamic speciation for fluids in the C–O–H–N system strongly favors N2 and CO2 relative to NH3 and to other carbon species considered (CO and CH4), respectively54. This supports the abiotic, dominantly mantle-derived, origin of the N2 and the carbon involved in the various organic compounds observed in the FIs. The absence of CO2 and the occurrence of H2 and CH4 suggests a complete reduction of initial CO2 to CH4 during fluid-olivine reactions inside the inclusions, at a temperature corresponding to H2 production by serpentinization (<350–400 °C), rather than a CO2–CH4 equilibration at higher temperature. This is consistent with the clumped isotopologue data on CH4 from seafloor hydrothermal sites, including Lost City, which imply a formation of CH4 at ~250–350 °C55. The complete reduction of CO2 to CH4 also fits the 13C of magmatic CO2.The nature of the original fluid can be inferred from the current phases found in the FI that attest to in situ reactions with olivine walls. The occurrence of hydrated secondary minerals , is usually between the log fO2,g set by the fayalite-magnetite-quartz (FMQ) mineral buffer FMQ-1 (1 log unit below FMQ) and the nickel-nickel oxide (NiNiO) mineral buffer NiNiO+2 (2 log unit higher than NiNiO), and their pH is acidic with trace amount of HCl (see Methods).Accordingly, we argue that the aqueous fluid initially trapped in the FIs was dominantly composed of NA two-step abiotic process of fluid cooling and subsequent fluid-mineral reactions (serpentinization) is proposed to account for our observations in FI as described below and in Fig. Stage 1–Trapping and cooling of a magmatic-dominated fluid, T ~ 400–600 °C.fH2 ≥ FMQ) favor CH4, CH3SH, and graphite below ~550 °C, and NH3 below ~450 °C , possibly associated with carbides, can be initiated by a similar surface-controlled process61 18. Since PACM, CH3SH or N2 were not reported, these species were not included in the previous models, but increasing levels of H2 were predicted between 400° and 300 °C, shifting the system by more than 2 logfH2,g units to highly reducing conditions, allowing CH4 formation via reaction (2). Reaction (2) is thermodynamically favored with decreasing T and water activity (aH2O) but its slow kinetic at T < 400 °C16 needs to be overcome by long residence times (thousand years) of the fluids in FIs53.Stage 2 – Serpentinization and formation of various metastable organic compounds. Once T falls below 400 °C, fluid water becomes both liquid and gaseous and olivine is prone to serpentinization therefore leading to the formation of serpentine, brucite, magnetite and H2 Fig. , stage 262. At this short time scale, the kinetic inhibition of methane formation prevails and metastable organic compounds are predicted to form, including aliphatic and polyaromatic hydrocarbons (PAHs), organic and amino acids or condensed carbon64. Suppressing CH4, we have modeled the redox evolution of the fluids in our FIs during serpentinization . The increase of fH2,g and pH due to serpentinization can progressively shift the carbon speciation in solution toward the fields of organic acids  and orange arrow on Fig. 123. These fields widen with decreasing water activity (aH2O) and T Occurrence of thioester functions is also possible through condensation of available thiols and carboxylic acids according to reaction (5):67, in addition to organic functional group transformation reactions68, which both considerably enlarge the range of organic compounds that can be formed. The absence of liquid water in the FI today attests to the full consumption of water during serpentinization of the olivine walls that should have progressively enhanced reactions (1) to (4) and condensation reactions ). Based on the structural and chemical characteristics of PACM1 71, as well as experimentally72. It has also been predicted by thermodynamic models73. Our results first suggest that nDs formation in such low P–T environments (≤2 kbars–400 °C) possibly occurs via an intermediate, amorphous, organic material. CH4 and possibly other hydrocarbons28 can also form later in FIs from reaction (2) or from further dehydration74 and hydrogenation75 reactions of PACMs simplified by pyrene (C16H10) in the following reaction.Metastable phases such as PACM1 and PAMC2 are prone to evolve after their formation. Here, they seem to serve as organic precursors for nD nucleation under the low P–T conditions of modern oceanic setting, similarly to higher P–T processes in subduction zones (>3 GPa)2 and CH4 in fossil and active oceanic lithosphere53, but the discovery of the new compounds has further implications. The likelihood that fluid inclusions can be opened during lower-T alteration processes at shallower levels in the oceanic crust, render the components trapped in the inclusions available for further diversification and complexification that can benefit prebiotic reactions. Ingredients, gathered and preserved in olivine pods, can suddenly be released in an environment that is far from the original equilibrium conditions. Such a high degree of disequilibria favors the production of many additional organic compounds and promotes the development of chemotrophic microbial metabolisms77. As an example, similar FIs could have provided nitrogen and aromatic precursors required for the local synthesis of abiotic amino acids that are described in the shallower part of the same drill hole13. PACMs may also be the locus of further precipitation of carbonaceous material assisted by mineral reactions at low-T14. The studied FIs also provide the first evidence for an abiotic source of CH3SH in present-day oceanic rocks where a thermogenic origin was favored up to now66. Availability of CH3SH and organosulfur compounds such as thioesters may be crucial to initiate proto-metabolisms in primitive hydrothermal systems66. In modern systems, such FIs may also provide nutrients for hydrocarbon degrading micro-organisms that have been revealed by genomic studies in magmatic rocks at various depths in IODP Hole 1309D78.Considering the geological context of such systems, our observations indicate that the timely interplay between magmatic degassing and progressive serpentinization is an ideal combination for the abiotic synthesis of varied gaseous and condensed organic compounds. Fluid inclusions have long been recognized as a major source of H2 and CH4 enriched alkaline environments created by low T serpentinization have been recognized as providing some of the most propitious conditions for the emergence of life81. Our results strengthen this hypothesis by highlighting new reaction routes that encompass the progressive time-line of geologic events in such rock systems. Unexplored prebiotic reaction pathways based on similar processes may have occurred in the primitive Earth and on Mars where hydrothermal environments rooted on olivine-rich magmatic rocks  are thought to be widespread85. The more reduced state of the mantle on early planets should have favored reduced species87 in the percolating magmatic fluids. Some studies of Martian meteorites have already suggested synthesis of PACM on Mars in relation to combined magmatic and hydrothermal processes12. This may be extrapolated to other planetary bodies such as icy moons where serpentinization has become a focus of attention89.HOptical imaging of rock thin sections (30 µm thick) have been done under plane-polarized and cross-polarized light using a Leica transmitted light microscosope.−1 and from 2500 to 3800 cm−1. The first one is associated to the Raman fingerprint of minerals and covers the first order region of PACM with D and G bands. The second one presents hydroxyl’ stretching bands of phyllosilicates, hydrated oxides, CH4 stretching modes and the second order of PACM.We acquired all the individual Raman spectra and 3D hyper-spectral Raman images (3D HSR images) with a LabRam HR evolution from Horiba™ manufacturer and a 532 nm DPSS laser. The laser beam was focused onto the sample with an Olympus ×100 objective. The probe spot has a diameter of around 0.9 μm. We used 600 grooves/mm grating to collect Raman spectra in two wavelength ranges, from 120 to 1800 cmWith four acquisitions of 500 ms per spectrum, the recording of a 3D HSR image is up to 39 h per spectral range. That means, twice for a full 3D acquisition of IF3 and almost 32 h for IF5. We minimized this time by scanning laser beam instead of shifting the position of the sample with the holding stage. We retain the true confocal performance of the microscope by using the DuoScan® hardware module in stepping mode. The laser was stepped across the sample in X and Y direction by two piezoelectric mirrors. The surface map has a small and high accuracy step, down to 250 nm in our case. Then stacking 2D HSR images from the surface and down in the hosting olivine with Z steps of 250 nm, we composed a 3D image of the fluid inclusion. After data preprocessing of the Raman spectra (see below explanations), we powered these images and 3D animations with the 3D Surface and Volume Rendering (3D SVR) application for LabSpec6®. Assuming minerals and gases are here transparent and our microscope is confocal, 3D shapes can be rendered by association of a color channel to a Raman signature. We used filters to remove voxels which have low color intensity and thresholds to control the transparency.The preprocessing data treatment differs between the 3D HSR images and the Raman signature of the PACM. In regards to the images, we operated the following sequence of preprocessing: (1) extraction of the relevant wavenumber range, (2) removal of extremely low and high signal corresponding either to low Raman diffusion or to high luminescence, (3) correction of the background with a polynomial base line.90. We applied the same procedure as in Quirico et al. (2014)91. The two Raman bands D and G were fitted with the so-called Lorentzian-Breit-Wigner-Fano (LBWF) spectral model36. Raman spectral parameters characterizing the PACM were extracted: width at half maximum , peak position  and ratio of peak intensity R1 (ID/IG) with a GOF (Goodness of fit). This parameter was used to remove fits with low RMS fitting error and R-squared. Eventually, we kept on working with typical batches of 600 up to 10,000 spectra with spectral parameters associated. The following table provides characteristic parameters of the averaged PACM end-members Table The first order Raman signature of the PACM was extracted from the whole spectra  acquired during 3D HSR images recording. We used a homemade algorithm with the Matlab® software to extract this Raman signal from each spectra and perform an iterative data fitting with the PeakFit Matlab® application tool peakfit.met al. 20491. The 92. We concatenated the Raman spectral parameters of the two IF to plot the diagram representing FWHM-D as a function of R1. We selected 3 groups of data as endmembers in this diagram to discuss spectral properties of each one and localization between each other in the inclusion.Data mining and visualization of spectral parameters in a workflow were powered with the software OrangeAfter being located by transmitted light microscopy and analyzed by Raman spectroscopy, fluid inclusions within the thin section sample were opened by using a FIB - SEM workstation  coupling a SIINT zeta ionic column  with a Zeiss Gemini I electronic column. For FIB operation, the thin section was coated with a carbon layer of about 20 nm by a carbon coater (Leica EM ACE600) to prevent electrostatic charging.First, a platinium coating was deposited with the in-situ gas injection system to define the interest region and to protect the surface from ion beam damage. Prior to milling and imaging, a coarse trench was milled around the region of interest to a depth of 30 µm. The inclusions were closed and not visible on the surface of the sample. The abrasion was therefore done progressively with FIB parameters adjusted to 30 kV and 10 nA until breaking through and obtaining a cross-section of the inclusion.Subsequently, the observations were performed using backscattered electrons with the so-called Energy and angle selective BSE detector (EsB) and secondary electrons with the Secondary Electrons Secondary Ions detector (SESI). These experiments were operated at 15 kV and in high vacuum. Chemical composition of solids in fluid inclusions was obtained simultaneously by EDX analyses using an Aztec Oxford system (EDS Oxford Instruments Aztec-DDI detector X MAXN 50).The studied cross sections were then extracted and thinned to a thickness of 100 nm by the ion beam following the lift-out method.2 system (Oxford Instruments) allowed the acquisition of images in annular bright field and a precise chemical analysis of the solid and condensed organic phases in the fluid inclusions.The structural organization of these thin foils was investigated by TEM. A 2100 JEOL operating at 200 kV was used to study precisely the carbon-rich regions within the fluid inclusions. A STEM (scanning transmission electron microscopy) module coupled with a EDX XMAX 80 mmFast Fourier Transform analysis of high-resolution images of nano-diamond-rich areas were used to determine cell parameter of the ~5 nm-size particles using Digital Micrograph software©.s peak at 284.8 eV. Before acquisition of the spectra, a short Ar ion etching was performed  to limit the presence of adventitious carbon on the surface. C1s spectra were obtained using a pass energy of 23.5 eV. All the peaks were fitted with Multipak software using a Shirley background. Quantification was carried out using the transmission function of the apparatus and angular distribution correction for a 45° angle. Sensitivity factors were extracted from Wagner et al., (1981)93 in which they integrate cross section and escape depth correction.XPS analyses were carried out ate the Ecole Centrale de Lyon (France) on a PHI 5000 Versaprobe II apparatus from ULVAC-PHI Inc. A monochromatized AlKα source (1486.6 eV) was used with a spot size of 10 µm. A charge neutralization system was used to limit charge effect. The remaining charge effect was corrected fixing the C–C bond contribution of C194 and the crusher activated several times until the CH4 signal approached that of the blank. The gas released by crushing was focused on a Porapak Q filled quartz capillary trap held at liquid nitrogen temperature. Gases were released from the trap by moving it out of the liquid nitrogen and into a 150 °C heating block.A portion of the studied rock sample was initially crushed with a stainless steel mortar and pestle and sieved to collect 1–2 mm chips. These chips were then heated at 60 °C under vacuum to remove surficial water. Approximately 0.23 g of these chips were placed into a hydraulic rock crusher with a continuous He stream similar to that of Potter and Longstaffe (2007)2. Following the oxidation oven, the gases entered a Thermo Fischer Delta V isotope ratio mass spectrometer (IRMS). Data reduction was performed by comparing an in house CH4 isotope standard to Indiana University Biogeochemical Laboratory CH4 standards #1, #2, #5, and #7.The released gases were separated on a HP 6890 gas chromatograph fitted with an Agilent Poraplot Q column  temperature programmed from −30 to 80 °C. The column effluent was fed into an oxidation oven containing NiO, CuO and Pt catalysts where the reduced gases were converted to CO97. The calculations were conducted with the Deep Earth Water (DEW) Model98. The Gibbs free energies of formation of minerals and solid condensed carbons at high temperatures and pressures were calculated using the SUPCRT92b code, an adaption of SUPCRT9299.Equilibrium reaction constants at elevated temperatures and pressures are used to construct the equilibrium speciation diagram Fig.  and 4. F96, and minerals from Berman (1988)100, Berman and Aranovich (1996)101, and Sverjensky et al. (1991)102. We adopted the thermodynamic properties of CH3SH,aq from Schutle and Rogers (2004)103, which are consistent with Shock et al. (1997)97. We also included the thermodynamic data of condensed aromatic organic carbons of Richard and Helgeson (1998)104, which are consistent with Berman (1988)100.Thermodynamic data files used in the calculations were built using data for aqueous species from Shock et al. (1997)105 to simulate reactions between a cooling magmatic-dominated fluid and olivine. We consider the system as progressive alteration of olivine in a closed system in which there was always the reaction affinity for the alteration of olivine by water. We set 30 moles of olivine (Fa15Fo85) reacting with 1 kg water, so the approximate water:rock = 1:4.5. It represents a low W/R ratio relevant with the geological settings observed here (very limited fluid captured as olivine inclusions). The dissolved salts of Ca, Mg, Fe, C, Si, N, S were considered in the calculations as well as all available minerals. All the calculations were carried out with the aqueous speciation, solubility, and chemical mass transfer codes EQ3 & EQ6 which have been recompiled from a traditional version106 for the purpose of simulating temperature and pressures higher than water saturation conditions, using thermodynamic data files prepared as described above. The codes are accessible freely to the public through the Deep Earth Water community (http://www.dewcommunity.org/). We first simulated the volcanic gas and starting fluid using EQ3 code. We then let the gas cool down to 400 °C before reacting with olivine in a continuous cooling (<400 °C) and enclosed system (2000 bars), mimicking the high-temperature and low-pressure environment where the fluid inclusions formed. It is within the T range of fluids when they are trapped in the inclusions.To simulate fluid-rock reactions, we applied purely chemical irreversible mass transfer models0 represents the initial temperature in °C; ξ represents the reaction extent; tk1, tk2, and tk3 are three parameters. Here, we set tk1 = −200 for the two cooling calculations: 600–400 °C (without olivine) and <400 °C (with olivine); we used the first cooled fluid (at 400 °C) as the starting fluid to react with olivine for the second stage cooling calculation.The cooling rate is set by the following equation in the model input:2O, CO2, and H2, with other trace gases108. The composition of the volcanic gas varies depending on several geological factors, including the extent of degassing of the magma, redox state, and temperature and cooling history108. Under the circumstances of this study, the simulation used volcanic CO2,g as the only carbon source. Provided the reported CO2/H2O ratio in volcanic gases, we set the starting CO2/H2O ratio as 0.3 in our starting fluid.Volcanic gas is mainly composed of steam HfO2,g values set by fayalite-magnetite-quartz (FMQ) mineral buffer minus one log unit (FMQ-1) and nickel-nickel oxide (Ni/NiO) mineral buffer plus two log unit (Ni/NiO+2) . In our simulation, we set log fO2,g of the starting fluid equal to these two values, representing two boundary cases (Supplementary Fig. 2,g and trace amounts of HCl and S gases108, the starting pH would be acidic. The neutral pH at 600 °C and 2 kbars is 5.3. Therefore, in our simulation, we set the initial pH as 4 to represent an acidic condition.Compilation of the volcanic gas indicated that the redox state of volcanic gas is between the log Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1


Nature Communications 10.1038/s41467-020-20573-7, published online 22 January 2021.Correction to: In the original version of this Article, it was incorrectly stated that the GTEx dataset version V7 aligned with STAR was used. Throughout, the GTEx dataset version V6p aligned with TopHat was used. In addition, it was stated incorrectly that we filtered out splice sites and introns with zero coverage in 95% of the sample. Instead, we kept only splice sites and introns with at least one read coverage in 95% of the samples. These errors do not affect any of the results or conclusions of the study. Corrections to the text are listed below.24.’ The correct version states ‘V6p’ in place of ‘V7’.The original version of this Article contained an error in the first sentence of the second paragraph of the Results section, which incorrectly read ‘To establish FRASER, we considered the GTEx project dataset (V7)24 (hereafter the GTEx dataset).’ The correct version states ‘V6p’ in place of ‘V7’.The first sentence of the Methods subsection ‘Datasets’ originally incorrectly read ‘7842 RNA-seq samples from 48 tissues of 543 assumed healthy individuals of the Genotype-Tissue Expression Project V749 based on the GENCODE v19 annotation28.’ The correct version replaces these sentences with ‘For GTEx, we obtained the BAM files from dbGaP (phs000424.v6.p1), which were already aligned by the GTEx consortium with TopHat (version v1.4) against the GRCh37 genome assembly49 based on the GENCODE v19 annotation28.’The fifth and sixth sentences of the Methods subsection ‘Datasets’ originally incorrectly read ‘For GTEx, we obtained the BAM files from dbGaP (phs000424.v7.p2), which were already aligned with STAR (version 2.4.2a). The GTEx consortia used the same parameters, with the exception of mapping against the GRCh37 genome assemblyThe last sentence of the Methods subsection 'Read counting and splicing metrics' originally incorrectly read 'Further, we filtered out splice sites and introns where more than 95% of the samples had zero coverage.' The correct version replaces this sentence with 'Further, we kept only splice sites and introns with at least one read coverage in 95% of the samples.’24.’ The correct version states ‘V6p’ in place of ‘V7’.The first sentence of the Methods subsection ‘Enrichment analysis’ originally incorrectly read ‘from the GTEx whole-genome sequencing genetic variant data (V7)The first sentence of the Data Availability statement originally incorrectly read ‘The GTEx dataset is available through dbGaP (accession number: phs000424.v7.p2).’ The correct version states ‘phs000424.v6.p1’ in place of ‘phs000424.v7.p2’.The final sentence of the Acknowledgements section originally incorrectly read ‘under accession number dbGaP:phs000424.v7.p2.’ The correct version states ‘phs000424.v6.p1’ in place of ‘phs000424.v7.p2’.These errors have been corrected in both the PDF and the HTML versions of the Article.


Here we characterised the mechanisms and clinical impact of induced silver tolerance in Klebsiella pneumoniae.Silver ions have potent broad-spectrum antimicrobial activity and are widely incorporated into a variety of products to limit bacterial growth. In Enterobacteriaceae, decreased silver susceptibility has been mapped to two homologous operons; the chromosomally located K. pneumoniae carriage of the sil operon alone does not give elevated silver tolerance. However, when exposed to increasing concentrations of silver nitrate (AgNO3), K. pneumoniae strains which contain the sil operon, will preferentially mutate SilS, resulting in overexpression of the genes encoding the RND efflux pump silCBA. Those strains which do not carry the sil operon also adapt upon exposure to increasing silver concentrations through mutations in another two-component regulator CusS. Secondary mutations leading to disruption of the outer membrane porin OmpC were also detected. Both routes result in a high level of silver tolerance with MIC’s of >512 mg/L. When exposed to a high concentration of AgNO3 (400 mg/L), only strains that contained the sil operon were able to survive, again through mutations in SilS. The AgNO3 adapted strains were also resistant to killing by challenge with several clinical and commercial silver containing dressings.In K. pneumoniae has two possible pathways for development of increased silver tolerance but that the sil operon is preferentially mutated. This operon is essential when K. pneumoniae is exposed to high concentrations of silver. The potential clinical impact on wound management is shown by the increased survivability of these adapted strains when exposed to several silver impregnated dressings. This would make infections with these strains more difficult to treat and further limits our therapeutic options.This study shows that The online version contains supplementary material available at 10.1186/s12866-022-02532-y. With the exponential rise of antimicrobial resistance, alternative treatments like heavy metals are experiencing an increased interest in their potential to treat infections . Silver +) have potent broad-spectrum bactericidal activity [+ ions and incorporation of silver into the membrane structure [Silver is not required for bacterial metabolism, therefore silver ions  regulated by a divergently expressed two-component regulator (CusRS) [cus operon is also linked to decreased membrane permeability [Cus-mediated active efflux of silver ions was synergistically enhanced following the loss of one or both of the major porins OmpC and OmpF [Increased tolerance to silver has been shown for several Gram-negative organisms –12 and t (CusRS) . Increaseability . Cus-medand OmpF .sil operon whose function is predicted based on homology to the cus operon [sil RND efflux pump component (SilCFBA) shares a >80% homology to CusCFBA and is regulated by a two-component regulator SilRS [cus homologue are also found in the sil operon: SilP, a putative P-type ATPase transporter, and SilE and SilG, periplasmic sequestration proteins and metallochaperones [silR and silS have been shown to increase expression of the sil operon coupled with a decrease in silver susceptibility [The second is a plasmid based encoded s operon . The silor SilRS , 17. Thraperones , 18, 19.tibility .Klebsiella pneumoniae is an opportunistic pathogen belonging to the Enterobacteriaceae family. The species is prominent in causing wound infections as well as hospital and community-acquired urinary and respiratory tract infections [K. pneumoniae has an antimicrobial resistance profile with multi- and extensively drug-resistant strains (MDR and XDR) regularly isolated from patients [fections . Moreovepatients .K. pneumoniae the sil operon is essential for survival following direct exposure to high levels of silver ions and that the primary mechanism of adaptation to increased concentrations of silver is through mutations in the sensor kinase SilS leading to increased expression of the silCFBA operon. For strains that do not contain a sil operon, survival at low silver concentrations was through alterations in another sensor kinase CusS. A secondary mutation in the outer membrane porin OmpC was essential in these strains for survival at high silver concentrations. The potential clinical impact of this decreased silver susceptibility in treatment of infections was addressed using a range of prescribed and commercially available silver-containing dressings and this showed that the silver-adapted strains are resistant to killing by all dressings.In this study, we show that in K. pneumoniae that possessed the sil operon were more intrinsically resistant to silver, the MIC/MBC levels to silver nitrate (AgNO3) and silver sulfadiazine (AgSD) from a range of strains were assessed. These included strains from clinically important sequence types ST14/15, ST23, ST101 and ST258 (Supplementary Table Ssil operon (40/61) ranged from 8- >512 mg/L with a mode of 16 mg/L for AgNO3 and a range of 32- >512 mg/L with a mode of 32 mg/L for AgSD. For those strains lacking a sil operon (21/61) the MIC range was 8–64 mg/L with a mode of 32 mg/L for AgNO3 and a range of 32–256 mg/L with a mode of 128 mg/L for AgSD. When the genome sequences of the two strains (MGH 78578 and CFI_123_NDM1OXA232) which had high MIC values (>512 mg/L) were interrogated they were found to have unique mutations in SilS.To evaluate whether strains of sil operon carriage in adaption to silver, seven genetically similar K. pneumoniae strains from the clonal complex 15 (CC15) consisting of a mixture of ST14/15 isolates, and including isolates which lacked the sil operon, were exposed to increasing stepwise (SW) concentrations of AgNO3. All strains were able to adapt and survive in the highest concentration of AgNO3 tested; subsequent MIC analysis showed a significant increase to >512 mg/L for both AgNO3 and AgSD  were the only isolates which lacked the sil operon. Again, those isolates which survived showed a substantial MIC increase to >512 mg/L for both AgNO3 and AgSD  concentration of AgNOsilS  had greatly increased expression of silA (over 250-fold) and a smaller fold-increase for silS (18 fold). For cusA, one strain (13443 Ag) showed a large increase in expression (41.6-fold) but in the other strain (KPUK04a Ag) no increase was observed. Strains which contained mutations in cusS and ompC (CFI_124_NDM1OXA232 and CFI_134_NDM1) predictably correlated with a substantial increase in cusA expression (over 250-fold) and a decrease in ompC expression (over 30-fold)  mutations in SilS were detected at exposure to AgNO3 concentrations at the respective wildtype MIC level and above (16 mg/L and upwards). When these SilS containing mutants were tested, the subsequent MIC values were >512 mg/L for both AgNO3 and AgSD  mutations in CusS were observed at exposure levels below the MIC value (16 mg/L) and gave rise to a slight increase in tolerance to AgNO3 (fourfold increase). OmpC mutations were only found at higher AgNO3 challenge concentrations . Strains containing mutations in CusS and OmpC showed MIC levels for AgNO3 and AgSD of >512 mg/L  and phenylalanine-arginine β-naphthylamide (PaβN) affected silver tolerance in K. pneumoniae. Results showed that whilst the addition of PaβN had no effect on the MIC levels, CCCP was able to reduce the MIC to AgNO3 and AgSD for all parental strains by at least eightfold  in antimicrobial susceptibility with KPTR8 Ag showing increased susceptibility to doxycycline, chlorhexidine and DDAB. . This suggests that OmpF does not play a significant role in silver ion uptake into cells whilst OmpC mutations presumably restrict the access of silver through the outer membrane and can mediate high level tolerance in conjunction with the CusS mutations. Mutations in OmpC alone are not sufficient for high level silver tolerance and K. pneumoniae organisms with mutations in one or both major porins alone do not appear to be intrinsically more silver tolerant than those with intact porins. Indeed, strains with mutations in SilS do not appear to alter their ompC expression levels suggesting that only silCBA overexpression is required for high level silver tolerance. This data suggests that SilCBA is a more efficient mechanism for reducing the intracellular silver ion concentration. This could be because the rate of efflux through CusCBA is reduced relative to SilCBA, either due to an increased copy number of the plasmid based SilCBA or that SilCBA has an increased affinity for Ag+ relative to CusCBA and is thus a higher affinity silver transporter. There is a precedence to mutate SilS where this option is available but the presence of the sil operon alone does not appear to result in strains with high intrinsic silver tolerance relative to strains lacking this operon. The clear advantage of sil operon carriage was when K. pneumoniae was challenged with a supra-MIC dose of AgNO3, presumably because it is far more probable to survive after one mutational event rather than having to mutate two genes. The indication that the sensor kinase (CusS) is mutated before the porin (OmpC) was interesting and again suggests that CusCBA is a lower affinity silver transporter and that increased efflux through this pump will act as an intermediate for development of higher tolerance. This is often the case with other antimicrobials such as fluoroquinolone resistance in S. aureus [K. pneumoniae [In this study we have characterised the acquisition of increased silver tolerance in o silver , 12 and o silver . Those fo silver . For str. aureus  or increeumoniae , where m3 to see if this was simply due to higher tolerance but still saw no effect at the highest concentration tested (4096 mg/L). Above this concentration the solubility of AgNO3 becomes an issue. However, CCCP is a proton motive force disrupter and therefore has the potential to prevent influx of silver ions as well as blocking efflux. It also can make cells metabolically inactive and thus the synergy between silver and CCCP may be due to decreased metabolic activity rather than decreased efflux alone. Why two of the adapted strains (KPTR8 Ag and CFI_014_VIM4 Ag) were affected is unknown and does not appear to be reflected in the observed mutations. The effects of EPI’s upon silver tolerance are perhaps a little difficult to measure because one of the modes of action of silver ions is through disrupting membrane integrity [sil operon are better able to survive exposure to lethal silver concentrations. Unfortunately, we do not possess such a strain in our collection.CusCBA and SilCBA are RND type efflux pumps but the addition of the efflux pump inhibitor PaβN did not have an effect on the tolerance to silver in either the wild-type strains or the silver-adapted strains indicated that it does not appear to interact with these efflux pumps. The addition of CCCP did reduce the silver tolerance for the pre-adapted strains but not for most adapted strains, irrespective of the causative silver tolerant mutation(s). We did attempt to increase the concentration of AgNOntegrity  which mantegrity  particulntegrity  and has ntegrity . It woulK. pneumoniae strains, regardless of the concentration and composition of silver within the proprietary antimicrobial dressing. Silver tolerant bacteria may be readily isolated following treatment with silver [Silver coated dressings are often used in wound care such as burns , 32 and h silver , 38 suggK. pneumoniae regardless of sil operon carriage and that this level of susceptibility could significantly compromise silver-based treatment in the clinic. With increasing numbers of MDR infections, the emergence of wide-spread increased silver resistance could have severe implications for the use of infection prevention and control procedures and the long-term treatment of such infections.Our findings evidence that endogenous silver tolerance can be readily induced in K. pneumoniae strains used in this study are listed in Table All relevant characteristics of all 600 was measured after 16–20 h using a FLUOstar Omega plate reader . MBC determination was measured by plating out onto TSA plates 10 µls of MIC dilutions from and including the MIC level and the subsequent three further higher biocide concentrations (where applicable). The efflux pump inhibitors Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP) and phenyl-arginine-b-naphthylamide (PaβN) with 1 mg/L MgSO4 final concentration were added at concentrations of 10 mg/L and 25 mg/L respectively. Polymyxin B Nonapeptide (PMBN) was added at a final concentration of 30 mg/L.MIC assays were carried out by a broth microdilution method using a 96-well plate  according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as previously described . The OD6K. pneumoniae strains were selected for adaptation to AgNO3 and consisted of a mixture of isolates with and without the sil operon. All cultures were grown in MH2 overnight where they were diluted down to a starting OD600 of 0.01 in MH2 supplemented with AgNO3 at a concentration of quarter the Modal MIC value (4 mg/L) and incubated shaking at 37 °C and at 250 RPM. Every two days 30 µls of culture was subsequently transferred to 3 mls fresh MH2 containing double the previous AgNO3 concentration, up to a final experimental concentration of 128 µg/ml (8 × MIC). Surviving cultures were then plated out on TSA and then passaged on TSA 10 times in the absence of AgNO3 before being tested. To control against changes in AgNO3 tolerance potentially caused by repeated passaging all strains were passaged every two days in MH2 without AgNO3 alongside the adaptation experiment.Seven ST14/15 3 at a concentration of 400 mg/L . They were incubated at 37 °C with shaking at 250 RPM for two days. Surviving cultures were again plated out on TSA and passaged on TSA 10 times in the absence of AgNO3. For time kill experiments a starting OD600 of 0.01 in MH2 supplemented with AgNO3 at 64 mg/L was used. Viable counts were performed by plating on TSA plates at 1, 2, 4, 6 and 24 h post AgNO3 challenge.Cultures were set up as described in the previous section except that the starting culture contained AgNO600 of 0.1 and grown until an OD600 of 0.5 was reached at 37 °C with shaking. Cultures were then harvested using RNA protect (Qiagen) and RNA was extracted using the RNAeasy minikit (Qiagen) according to manufacturer’s instructions. cDNA was synthesized and real-time PCR was carried out using a StepOnePlus real-time PCR system and Fast SYBR green master mix (Life Technologies) as previously described [gapA as an internal control. Primers used were cusA , cusS , silA , silS  ompC .Overnight cultures in MH2 were backdiluted to an ODescribed . Data weK. pneumoniae silver adapted strains to five silver-containing wound dressings  was compared with a generic non-antimicrobial wound dressing. These dressings were available on the NHS supply chain and are listed in the British National Formulary (https://bnf.nice.org.uk). The dressings contained differing forms and concentrations of silver. Briefly, a culture at OD600 of 0.1 culture was streaked onto TSA plates using a sterile swab to give a lawn of confluent growth and 1 × 1 cm2 piece of each dressing was placed onto the plate. Plates were incubated at 37 °C for 20 h. Corrected zones of reduced growth (CZRG) were then determined by measuring the diameter of the zone of reduced growth around the area of the dressing. A mean CZRG was obtained from three distinct replicate experiments. An unpaired t-test was used to determine the significance of the difference between wildtype and adapted strains.The susceptibility of K. pneumoniae whole genome MLST database [This was carried out as previously described  and PHE database .Addtional file 1.


By examining the pressure threshold of the elastic recovery regime across n = 1–4 RPPs, we obtained molecular insights into the relationship between structure and deformation resistance in hybrid organic-inorganic perovskites.Multilayers consisting of alternating soft and hard layers offer enhanced toughness compared to all-hard structures. However, shear instability usually exists in physically sputtered multilayers because of deformation incompatibility among hard and soft layers. Here, we demonstrate that 2D hybrid organic-inorganic perovskites (HOIP) provide an interesting platform to study the stress–strain behavior of hard and soft layers undulating with molecular scale periodicity. We investigate the phonon vibrations and photoluminescence properties of Ruddlesden–Popper perovskites (RPPs) under compression using a diamond anvil cell. The organic spacer due to C4 alkyl chain in RPP buffers compressive stress by tilting ( Multilayers comprising alternating soft and hard layers offer enhanced toughness compared to all-hard structures. Here authors reveal how the hard and soft components in Ruddlesden–Popper perovskites work cooperatively to resist deformation under pressure, informing the design of alternating superlattices for engineering applications. The organic layers provide the dielectric and quantum confinement of the inorganic layers, giving rise to large exciton binding energies and high oscillator strength5. From a structural mechanics point of view, the inorganic layers serve as mechanical brace to support spring-like molecular layers, forming a “natural” undulating hard-soft system. In the realm of mechanical engineering, such a hard-soft multilayer system is highly sought after for its high plastic deformation resistance and increased fracture hardness, but requires a complex fabrication process. The undulating layers create bands of tensile and compressive stress that is different from that of single-component layers, thus research efforts on inorganic hard-soft multilayers are centered on engineering structure and compositional gradient to enhance the mechanical properties of materials. The general chemical formula for lead halide 2D RPPs is (RNH3)2Mn−1PbnX3n+1 (n represents the number of inorganic layers), where PbX42- is an inorganic perovskite layer of corner-sharing metal halide octahedra, RNH3+ is a long-chain alkylammonium organic molecular layer, such as butyl ammonium (BA), and M is a smaller cation filled into the 12-fold coordinated holes wrapped by the PbX6 octahedra, such as CH3NH3+ (MA+)8. The long-range structure of the organic array is a result of balancing local strain induced by methyl ammonium ions on the inorganic cage, hydrogen-bonding and electrostatic forces between the cationic head group of the organic molecule and the metal halide framework10. Thanks to the rigid-/soft-layer alternated stacking structure of 2D layered perovskites, the rigid inorganic framework can template the conformation of the soft organic molecule confined in the organic-inorganic atomic interface11. In the ground state of C4 alkyl chain, each CH2 moiety adopts the trans-trans (tt) conformation to avoid steric repulsion. Under compression, it can be expected that the lamellar layers will contract and the strain in the metal halide bonds will affect optical and electronic properties21. However, little is known about the conformational change of the organic cations and the plastic deformation or strain hardening process when the organic molecules re-orientate under pressure, this is because X-ray diffraction is mostly sensitive to the heavier inorganic components.Two-dimensional (2D) Ruddlesden-Popper perovskites (RPPs) consist of alternatively stacked layers of soft organic layers and rigid inorganic layers with highly tunable optoelectronic properties2MAn−1PbnI3n+1 (n = 1-4) during compression. Combining experiments and density functional theory (DFT), we observed that n = 1 and n > 1 exhibit markedly different mechanisms of compressive strain under pressure. In n = 2 RPP, under compression, the trans-trans (tt) structure of the butyl ammonium cation gradually transforms into the trans-gauche (tg) and gauche-gauche (g+g- and g+g+) conformations via C-C and C-N bond rotations, along with tensile strain of the inorganic slabs in both a and b axial directions. In contrast, for n = 1 RPP, tilting of the linear organic cation is along with in-plane shift in one direction (b axis), resulting in orthorhombic-to-monoclinic phase change under compression. n = 1 RPP shows remarkable elastic recovery ability due to the flexibility of organic cation that acts synergistically with the octahedral tilt of the inorganic cages. Our study shows how differing alternation of soft and hard layers allows compressive and tensile stress to evolve differently across n = 1 to n = 4 RPPs, which provides the essential experimental basis for understanding stress-strain behavior in nanometer scale multilayer hard-soft superlattices.Here, using a combination of in situ Raman and Photoluminescence (PL) spectroscopy, we investigate how compressive and tensile strain develops in the organic and inorganic components in (BA)To lay the groundwork for understanding the complex structural changes in the organic and inorganic parts of the hybrid perovskites upon hydrostatic compression, it is beneficial to organize the paper by discussing the changes revealed by first-principles calculations first, followed by comparison with what was observed in the experiments.n = 2 RPP. Two layers of interdigitated linear organic BA molecules alternately stack with two layers of Pb2I73- inorganic slabs. The unit cell height of ~ 2.1 nm is determined by atomic force microscopy (AFM)22. BA organic molecules in tt conformation dominate the large interlayer spaces, while MA ions occupy the inter-octahedral voids  conformers on the surface of RPP, which can be accomplished by rotating along the C-C/C-N chain  was adopted with the bi-layer RPP sample being sandwiched between two diamond slabs Fig. . Under cain Fig. . The rotain Fig.  shows thol Table , therefon = 2 RPP along - a and + b axes, as represented by the blue and grey arrows in Fig. 23. Such an interlayer shift maintains n = 2 RPP in the orthorhombic symmetry, accompanied by the conformational change of BA molecules without tilting under pressure.Compression results in the layer-to-layer shift of the bi-layer inorganic slabs in n = 1 RPP compared to its higher homologues, as illustrated in Fig. n = 1 RPP, the linear BA molecules tilt without undergoing conformational change till 7.36 GPa . The detailed structural evolution of n = 1 and n = 2 RPP with pressure is presented in the Supplementary Fig. Interestingly, we observed pronounced differences in the pressure response of c direction of 2D RPPs allows an in-plane layer shift between the adjacent inorganic layer, which can be evaluated by a layer shift factor (LSF)25. Here, using the lower inorganic layer as the reference, the relative coordinate deviation of the upper layer along the a axis is calculated as:LSF value of 0.25 on both a and b directions25, referring to the Supplementary Fig. n = 1 and 2 RPPs in the low-pressure region . The calculated LSF are represented by the green filled circles for n = 1 in Fig. n = 2 in Fig. n = 1 RPP has a maximum LSFb value of ~ 0.34 at 0.24 GPa, while n = 2 RPP accommodates LSFa of ~ 0.33 and LSFb of ~ 0.10 along - a and b directions, respectively. The n = 1 RPP has an average LSFb value of ~ 0.32 ± 0.01 within pressure of 2.51 GPa, while n = 2 RPP accommodates an average LSF-a value of ~ 0.32 ± 0.01 and LSFb value of ~ 0.14 ± 0.03 along - a and b directions within pressure of 2.31 GPa.The discontinuity in the n = 1 RPP results in a vertical strain of ~ 14.6 % within pressure of 2.51 GPa . To track the deviation from the initial angles at 0 GPa after compression, we monitor the difference in angles ∆θ between the final and initial state.To track the structural deformation and recoverability of the inorganic sublattice during compression-decompression cycles, we analyze several key structural parameters that represent the in-plane and out-of-plane components of the age Fig. , namely,n = 2 RPP undergoes lamellar contraction by the rotational isomerism of the softer BA cations, this allows the harder Pb-I lattice to remain in the elastic regime when the pressure is less than ~ 3 GPa. The apical angle α linking two octahedral cages serves as a pivot and bends by up to ~ 12.4° during vertical compression, and unbends a little bit more after decompression  were performed for both 27. The single crystal of n = 2 RPP was synthesized through a temperature-programmed crystallization method29, and the exfoliated flakes were loaded into the diamond anvil cell (DAC) for in situ high pressure Raman measurement. NaCl was used as the pressure transmitting medium (PTM) to obtain a clean Raman signal of the organic molecular vibrations.Raman spectroscopy is a powerful tool used to characterize the vibrations and rotations of organic molecules in organic-inorganic hybrid perovskites during the pressurization process+ gas molecule in the tt, tg-kind1 (tg-kind2) and g+g- conformations, as represented by the grey, dark (light) blue and red solid lines in Fig. n = 2 RPP structure, as shown in Table + gas molecules.As a reference, we have calculated the Raman spectra of BAn = 2 RPP under compression, as shown in Fig. + gas molecules. The measured Raman bands at 0 GPa in the frequency region of 450 − 890 cm−1 are the skeletal vibrational modes of BA molecules in the tt conformation, as represented by grey solid line in Fig. −1 belong to the scissoring modes of tt-BA, i.e., δ(CH2-CH2-CH3) (δ(CCC)) and δ(CH2-CH2-NH3+) (δ(CCN)), which are associated CH3 and HN3+ stretch in opposite directions  under compression, while the intensity of the Raman band at ~840 cm−1 is enhanced due to the formation of Raman-active tg and g+g- BA conformers , thus the BA conformers are strain-hardened and stabilized under high-pressure treatment. At ambient conditions, the high-energy isomers could partially transform back to the tt isomer according to the moderate barrier as shown by our DFT and AIMD simulations. This reversible isomerization renders the storage of the mechanical energy and hindrance of the deformation.Our measured in situ high-pressure Raman spectra of ons Fig. . The obsCH2 Fig. . Upon coely Fig. . Besidesers Fig. . From 0.n = 1 RPP, previous high-pressure Raman study by us showed that the BA chain remains linear under compression, and a totally reversible Raman spectrum is obtained after decompression21. This implies an interesting thickness (n)-selective response rooted in the n-dependent isomerization of the BA molecule.In contrast, in the case of 31. The exfoliated flakes are much thinner than bulk crystal and allow compression or relaxation effects to permeate much more quickly throughout the sample compared to the bulk  that are collected under compression, while Fig. 34. Upon compression to 2.5 GPa, the BS and SS emissions red shift continuously and merge into a single peak at 3.9 GPa  to organic slab (soft) increases in the RPP homologous series from n = 1 to 4, and varying bands of tensile and compressive stress develop respectively. When the proportion of BA organic fraction decreases, the ability to buffer compressive stress in the hybrid system decreases. As a result, there is increasing strain in the Pb-I inorganic lattice, leading to an increasingly lower pressure threshold from n = 1 to 4 RPPs for permanent deformation, as demonstrated by the in situ compression-decompression PL spectra shown in the Supplementary Fig. n = 1 to 4 RPPs, as summarized in Fig. n increases, the critical pressure point, above which irreversible deformation occurs, decreases, i.e., it requires a lower compressive stress to deform the inorganic lattice, as judged by the appearance of the broadband PL. This clearly corroborates the fact that the organic cation helps to buffer the stress during compression. Therefore, when the proportion of organic cations is increasingly diluted by thicker inorganic slab, the deformation resistance decreases.The role of the organic cations in buffering stress becomes clear when we examine the deformation resistance of the inorganic lattice for n = 1 RPP, the compressive stress is first accommodated by the tilting of the organic cation, and this acts in concert with the in-plane slip of the inorganic slab, which results in phase change of the crystal upon compression. The absence of methyl ammonium ions occupying the cubo-octahedral site in n = 1 RPP imposes less steric hindrance, and this may allow the organic chain to tilt and un-tilt in a reversible way. By contrast, the compressive stress in n = 2 RPPs is accommodated firstly by the rotational isomerism of the organic cations. The rotation to high energy conformer is accompanied by the in-plane tensile strain along a and b axis. Following decompression, elastic recovery of the inorganic octahedral cages in n > 1 can be achieved, but the pressure threshold for irreversible deformation is much lower than the n = 1 case. Our results provide the essential basis for understanding how multilayer organic-inorganic system act synergistically under compressive stress, which may be useful for understanding the isothermal barocaloric effects in perovskites, and for engineering new types of deformation-resistant multilayer coatings.In 4H9NH3I (BAI) and CH3NH3I (MAI) via a temperature-programmed crystallization method37. For (BA)2MAPb2I7 (n = 2), PbO, BAI, and MAI  were dissolved in a concentrated mixture of HI and H3PO2 . The solution was then heated at 110 °C while stirring for 40 min to give clear solutions. Subsequently, the solutions were quickly transferred to an oven set at 110 °C and allowed to cool slowly down to room temperature at a rate of 3 °C h−1, in which the 2D RPP crystals crystallized. The crystals were isolated via vacuum filtration and subsequently dried under vacuum at room temperature29. Thin flakes of RPPs were exfoliated by following the method developed for graphene exfoliation and transferred onto a culet of 500 µm of the diamond anvil cell in the glovebox21.The RPP single crystals were synthesized using three precursors: PbO, Cn = 2 RPP. The pressure was calibrated using the pressure-dependent ruby fluorescent technique31.Two identical diamond anvils with a culet of 500 µm were employed to generate pressure. A stainless-steel gasket was pre-indented to 50 µm with a drilled hole 300 µm in diameter serving as the sample chamber. A small ruby ball was loaded together with 38, and perovskite flakes with different thicknesses were then selected and transferred onto the pressing surface (500 µm) of the DAC using a transfer stage. The exfoliation and transfer processes were carried out in an argon-filled glove box. The RPP microsheets were covered with a silicone oil before taking out of the glove box for in situ PL measurements under compression and decompression. PL spectra were excited by 532 nm laser using WITec alpha 300RAS microscope.Raman spectra were conducted by using Renishaw Invia Raman microscope. A solid-state 785 nm laser was used to excite Raman scattering to avoid strong PL background as well as any sample degradation. For the high-pressure PL measurements, the bulk RPP crystals were first exfoliated on polydimethylsiloxane (PDMS) following the method developed for graphene exfoliation39. An additional vacuum layer of thickness greater than 15 Å was adopted. The Perdew-Burke-Ernzerhof functional for the exchange-correlation potential was used together with a kinetic energy cutoff of 500 eV and a 3×3×1 Monkhorst-Pack grid. All the structures are fully relaxed until the force on each atom is less than 0.005 eV Å−1. The van der Waal’s interactions were treated by using DFT-D3 method. The hydrostatic model is based on the calculations and experimental results of low-dimensional materials under pressure42. More details about model construction, molecular dynamics calculations and Raman calculation can be found in the Supplementary Discussion.The structural evolutions under compression and decompression were simulated by using Vienna ab initio simulation package (VASP)Supplementary Information