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This study aimed to investigate the presence of antimicrobial resistance determinants (ARDs) in the Neanderthal microbiome through meticulous analysis of metagenomic data derived directly from dental calculus and fecal sediments across diverse Neanderthal sites in Europe. Employing a targeted locus mapping approach followed by a consensus strategy instead of an assembly-first approach, we aimed to identify and characterize ARDs within these ancient microbial communities. A comprehensive and redundant ARD database was constructed by amalgamating data from various antibiotic resistance gene repositories. Our results highlighted the efficacy of the KMA tool in providing a robust alignment of ancient metagenomic reads to the antibiotic resistance gene database. Notably, the KMA tool identified a limited number of ARDs, with only the 23S ribosomal gene from the dental calculus sample of Neanderthal remains at Goyet Troisieme Caverne exhibiting ancient DNA (aDNA) characteristics. Despite not identifying ARDs with typical ancient DNA damage patterns or negative distance proportions, our findings suggest a nuanced identification of putative antimicrobial resistance determinants in the Neanderthal microbiome's genetic repertoire based on the taxonomy-habitat correlation. Nevertheless, our findings are limited by factors such as environmental DNA contamination, DNA fragmentation, and cytosine deamination of aDNA. The study underscores the necessity for refined methodologies to unlock the genomic assets of prehistoric populations, fostering a comprehensive understanding of the intricate dynamics shaping the microbial landscape across history., Importance: The results of our analysis demonstrate the challenges in identifying determinants of antibiotic resistance within the endogenous microbiome of Neanderthals. Despite the comprehensive investigation of multiple studies and the utilization of advanced analytical techniques, the detection of antibiotic resistance determinants in the ancient microbial communities proved to be particularly difficult. However, our analysis did reveal the presence of some authentic ancient conservative genes, indicating the preservation of certain genetic elements over time. These findings raise intriguing questions about the factors influencing the presence or absence of antibiotic resistance in ancient microbial communities. It could be speculated that the spread of current antibiotic resistance, which has reached alarming levels in modern times, is primarily driven by anthropogenic factors such as the widespread use and misuse of antibiotics in medical and agricultural practices., Competing Interests: The authors declare no conflict of interest.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Elucidating the timescale of the evolution of Alphaproteobacteria, one of the most prevalent microbial lineages in marine and terrestrial ecosystems, is key to testing hypotheses on their co-evolution with eukaryotic hosts and Earth's systems, which, however, is largely limited by the scarcity of bacterial fossils. Here, we incorporate eukaryotic fossils to date the divergence times of Alphaproteobacteria, based on the mitochondrial endosymbiosis that mitochondria evolved from an alphaproteobacterial lineage. We estimate that Alphaproteobacteria arose ~1900 million years (Ma) ago, followed by rapid divergence of their major clades. We show that the origin of Rickettsiales, an order of obligate intracellular bacteria whose hosts are mostly animals, predates the emergence of animals for ~700 Ma but coincides with that of eukaryotes. This, together with reconstruction of ancestral hosts, strongly suggests that early Rickettsiales lineages had established previously underappreciated interactions with unicellular eukaryotes. Moreover, the mitochondria-based approach displays higher robustness to uncertainties in calibrations compared with the traditional strategy using cyanobacterial fossils. Further, our analyses imply the potential of dating the (bacterial) tree of life based on endosymbiosis events, and suggest that previous applications using divergence times of the modern hosts of symbiotic bacteria to date bacterial evolution might need to be revisited.

Ancient DNA studies provide genomic information about the origins, population structures, and physical characteristics of ancient humans that cannot be solely examined by archeological studies. The DNAs extracted from ancient human bones, teeth, or tissues are often contaminated with coexisting bacterial and viral genomes that contain DNA from ancient microbes infecting those of ancient humans. Information on ancient viral genomes is useful in making inferences about the viral evolution. Here, we have utilized metagenomic sequencing data from the dental pulp of five Jomon individuals, who lived on the Japanese archipelago more than 3000 years ago; this is to detect ancient viral genomes. We conducted de novo assembly of the non-human reads where we have obtained 277,387 contigs that were longer than 1000 bp. These contigs were subjected to homology searches against a collection of modern viral genome sequences. We were able to detect eleven putative ancient viral genomes. Among them, we reconstructed the complete sequence of the Siphovirus contig89 (CT89) viral genome. The Jomon CT89-like sequence was determined to contain 59 open reading frames, among which five genes known to encode phage proteins were under strong purifying selection. The host of CT89 was predicted to be Schaalia meyeri, a bacterium residing in the human oral cavity. Finally, the CT89 phylogenetic tree showed two clusters, from both of which the Jomon sequence was separated. Our results suggest that metagenomic information from the dental pulp of the Jomon people is essential in retrieving ancient viral genomes used to examine their evolution.

The key to evolution is reproduction. Pathogens can either kill the human host or can invade the host without causing death, thus ensuring their own survival, reproduction and spread. Tuberculosis, treponematoses and leprosy are widespread chronic infectious diseases whereby the host is not immediately killed. These diseases are examples of the co-evolution of host and pathogen. They can be well studied as the paleopathological record is extensive, spanning over 200 human generations. The paleopathology of each disease has been well documented in the form of published synthetic analyses recording each known case and case frequencies in the samples they were derived from. Here the data from these synthetic analyses were re-analysed to show changes in the prevalence of each disease over time. A total of 69,379 skeletons are included in this study. There was ultimately a decline in the prevalence of each disease over time, this decline was statistically significant (Chi-squared, p<0.001). A trend may start with the increase in the disease's prevalence before the prevalence declines, in tuberculosis the decline is monotonic. Increase in skeletal changes resulting from the respective diseases appears in the initial period of host-disease contact, followed by a decline resulting from co-adaptation that is mutually beneficial for the disease (spread and maintenance of pathogen) and host (less pathological reactions to the infection). Eventually either the host may become immune or tolerant, or the pathogen tends to be commensalic rather than parasitic., Competing Interests: The Authors have declared that no competing interests exist.

A comprehensive view of our evolutionary history cannot ignore the ancestral features of our gut microbiota. To provide some glimpse into the past, we searched for human gut microbiome components in ancient DNA from 14 archeological sediments spanning four stratigraphic units of El Salt Middle Paleolithic site (Spain), including layers of unit X, which has yielded well-preserved Neanderthal occupation deposits dating around 50 kya. According to our findings, bacterial genera belonging to families known to be part of the modern human gut microbiome are abundantly represented only across unit X samples, showing that well-known beneficial gut commensals, such as Blautia, Dorea, Roseburia, Ruminococcus, Faecalibacterium and Bifidobacterium already populated the intestinal microbiome of Homo since as far back as the last common ancestor between humans and Neanderthals.

During the two World Wars, Bartonella quintana was responsible for trench fever and is now recognised as an agent of re-emerging infection. Many reports have indicated widespread B. quintana exposure since the 1990s. In order to evaluate its prevalence in ancient populations, we used real-time PCR to detect B. quintana DNA in 400 teeth collected from 145 individuals dating from the 1st to 19th centuries in nine archaeological sites, with the presence of negative controls. Fisher's exact test was used to compare the prevalence of B. quintana in civil and military populations. B. quintana DNA was confirmed in a total of 28/145 (19.3%) individuals, comprising 78 citizens and 67 soldiers, 20.1% and 17.9% of which were positive for B. quintana bacteraemia, respectively. This study analysed previous studies on these ancient samples and showed that the presence of B. quintana infection followed the course of time in human history; a total of 14/15 sites from five European countries had a positive prevalence. The positive rate in soldiers was higher than those of civilians, with 20% and 18.8%, respectively, in the 18th and 19th centuries, but the difference in frequency was not significant. These results confirmed the role of dental pulp in diagnosing B. quintana bacteraemia in ancient populations and showed the incidence of B. quintana in both civilians and soldiers., Competing Interests: The authors have declared that no competing interests exist.

Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

Background: The identification of bona fide microbial taxa in microbiomes derived from ancient and historical samples is complicated by the unavoidable mixture between DNA from ante- and post-mortem microbial colonizers. One possibility to distinguish between these sources of microbial DNA is querying for the presence of age-associated degradation patterns typical of ancient DNA (aDNA). The presence of uracils, resulting from cytosine deamination, has been detected ubiquitously in aDNA retrieved from diverse sources, and used as an authentication criterion. Here, we employ a library preparation method that separates molecules that carry uracils from those that do not for a set of samples that includes Neandertal remains, herbarium specimens and archaeological plant remains., Results: We show that sequencing DNA libraries enriched in molecules carrying uracils effectively amplifies age associated degradation patterns in microbial mixtures of ancient and historical origin. This facilitates the discovery of authentic ancient microbial taxa in cases where degradation patterns are difficult to detect due to large sequence divergence in microbial mixtures. Additionally, the relative enrichment of taxa in the uracil enriched fraction can help to identify bona fide ancient microbial taxa that could be missed using a more targeted approach., Conclusions: Our experiments show, that in addition to its use in enriching authentic endogenous DNA of organisms of interest, the selective enrichment of damaged DNA molecules can be a valuable tool in the discovery of ancient microbial taxa.

Background: Recent advances in the next-generation sequencing (NGS) allowed the metagenomic analyses of DNA from many different environments and sources, including thousands of years old skeletal remains. It has been shown that most of the DNA extracted from ancient samples is microbial. There are several reports demonstrating that the considerable fraction of extracted DNA belonged to the bacteria accompanying the studied individuals before their death., Results: In this study we scanned 344 microbiomes from 1000- and 2000- year-old human teeth. The datasets originated from our previous studies on human ancient DNA (aDNA) and on microbial DNA accompanying human remains. We previously noticed that in many samples infection-related species have been identified, among them Tannerella forsythia, one of the most prevalent oral human pathogens. Samples containing sufficient amount of T. forsythia aDNA for a complete genome assembly were selected for thorough analyses. We confirmed that the T. forsythia-containing samples have higher amounts of the periodontitis-associated species than the control samples. Despites, other pathogens-derived aDNA was found in the tested samples it was too fragmented and damaged to allow any reasonable reconstruction of these bacteria genomes. The anthropological examination of ancient skulls from which the T. forsythia-containing samples were obtained revealed the pathogenic alveolar bone loss in tooth areas characteristic for advanced periodontitis. Finally, we analyzed the genetic material of ancient T. forsythia strains. As a result, we assembled four ancient T. forsythia genomes - one 2000- and three 1000- year-old. Their comparison with contemporary T. forsythia genomes revealed a lower genetic diversity within the four ancient strains than within contemporary strains. We also investigated the genes of T. forsythia virulence factors and found that several of them (KLIKK protease and bspA genes) differ significantly between ancient and modern bacteria., Conclusions: In summary, we showed that NGS screening of the ancient human microbiome is a valid approach for the identification of disease-associated microbes. Following this protocol, we provided a new set of information on the emergence, evolution and virulence factors of T. forsythia, the member of the oral dysbiotic microbiome.

Introduction: Dental pulp with special structure has become a good reference sample in paleomicrobiology-related blood-borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins., Objectives: This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe., Methods: The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: "ancient," "dental pulp," "teeth," "anatomy," "structure," "collection," "preservation," "selection," "photography," "radiography," "contamination," "decontamination," "DNA," "protein," "extraction," "bone," "paleomicrobiology," "bacteria," "virus," "pathogen," "molecular biology," "proteomics," "PCR," "MALDI-TOF," "LC/MS," "ELISA," "immunology," "immunochromatography," "genome," "microbiome," "metagenomics.", Results: The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty-seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species., Conclusions: Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Prevotella copri is a common human gut microbe that has been both positively and negatively associated with host health. In a cross-continent meta-analysis exploiting >6,500 metagenomes, we obtained >1,000 genomes and explored the genetic and population structure of P. copri. P. copri encompasses four distinct clades (>10% inter-clade genetic divergence) that we propose constitute the P. copri complex, and all clades were confirmed by isolate sequencing. These clades are nearly ubiquitous and co-present in non-Westernized populations. Genomic analysis showed substantial functional diversity in the complex with notable differences in carbohydrate metabolism, suggesting that multi-generational dietary modifications may be driving reduced prevalence in Westernized populations. Analysis of ancient metagenomes highlighted patterns of P. copri presence consistent with modern non-Westernized populations and a clade delineation time pre-dating human migratory waves out of Africa. These findings reveal that P. copri exhibits a high diversity that is underrepresented in Western-lifestyle populations., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Melanosomes (melanin-bearing organelles) are common in the fossil record occurring as dense packs of globular microbodies. The organic component comprising the melanosome, melanin, is often preserved in fossils, allowing identification of the chemical nature of the constituent pigment. In present-day vertebrates, melanosome morphology correlates with their pigment content in selected melanin-containing structures, and this interdependency is employed in the color reconstruction of extinct animals. The lack of analyses integrating the morphology of fossil melanosomes with the chemical identification of pigments, however, makes these inferences tentative. Here, we chemically characterize the melanin content of the soft tissue headcrest of the pterosaur Tupandactylus imperator by alkaline hydrogen peroxide oxidation followed by high-performance liquid chromatography. Our results demonstrate the unequivocal presence of eumelanin in T. imperator headcrest. Scanning electron microscopy followed by statistical analyses, however, reveal that preserved melanosomes containing eumelanin are undistinguishable to pheomelanin-bearing organelles of extant vertebrates. Based on these new findings, straightforward color inferences based on melanosome morphology may not be valid for all fossil vertebrates, and color reconstructions based on ultrastructure alone should be regarded with caution.

Lichens, symbiotic consortia of lichen-forming fungi and their photosynthetic partners have long had an extremely poor fossil record. However, recently over 150 new lichens were identified from European Paleogene amber and here we analyse crustose lichens from the new material. Three fossil lichens belong to the extant genus Ochrolechia (Ochrolechiaceae, Lecanoromycetes) and one fossil has conidiomata similar to those produced by modern fungi of the order Arthoniales (Arthoniomycetes). Intriguingly, two fossil Ochrolechia specimens host lichenicolous fungi of the genus Lichenostigma (Lichenostigmatales, Arthoniomycetes). This confirms that both Ochrolechia and Lichenostigma already diversified in the Paleogene and demonstrates that also the specific association between the fungi had evolved by then. The new fossils provide a minimum age constraint for both genera at 34 million years (uppermost Eocene).

Modern microorganisms growing in fossils provide major challenges for researchers trying to detect ancient molecules in the same fossils., Competing Interests: RE, AC No competing interests declared, (© 2019, Eisenhofer and Cooper.)

Ancient fermented food has been studied based on recipes, residue analysis, and ancient-DNA techniques and reconstructed using modern domesticated yeast. Here, we present a novel approach based on our hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. We developed a pipeline of yeast isolation from clay vessels and screened for yeast cells in beverage-related and non-beverage-related ancient vessels and sediments from several archaeological sites. We found that yeast cells could be successfully isolated specifically from clay containers of fermented beverages. The findings that genotypically the isolated yeasts are similar to those found in traditional African beverages and phenotypically they grow similar to modern beer-producing yeast strongly suggest that they are descendants of the original fermenting yeast. These results demonstrate that modern microorganisms can serve as a new tool in bio-archaeology research. IMPORTANCE So far, most of the study of ancient organisms has been based mainly on the analysis of ancient DNA. Here we show that it is possible to isolate and study microorganisms-yeast in this case-from ancient pottery vessels used for fermentation. We demonstrate that it is highly likely that these cells are descendants of the original yeast strains that participated in the fermentation process and were absorbed into the clay matrix of the pottery vessels. Moreover, we characterized the isolated yeast strains, their genomes, and the beer they produced. These results open new and exciting avenues in the study of domesticated microorganisms and contribute significantly to the fields of bio- and experimental archaeology that aim to reconstruct ancient artifacts and products., (Copyright © 2019 Aouizerat et al.)

Over the last few years, genomic studies on Yersinia pestis , the causative agent of all known plague epidemics, have considerably increased in numbers, spanning a period of about 5,000 y. Nonetheless, questions concerning historical reservoirs and routes of transmission remain open. Here, we present and describe five genomes from the second half of the 14th century and reconstruct the evolutionary history of Y. pestis by reanalyzing previously published genomes and by building a comprehensive phylogeny focused on strains attributed to the Second Plague Pandemic (14th to 18th century). Corroborated by historical and ecological evidence, the presented phylogeny, which includes our Y. pestis genomes, could support the hypothesis of an entry of plague into Western European ports through distinct waves of introduction during the Medieval Period, possibly by means of fur trade routes, as well as the recirculation of plague within the human population via trade routes and human movement., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Chemical mobility of crystalline and amorphous SiO 2 plays a fundamental role in several geochemical and biological processes, with silicate minerals being the most abundant components of the Earth's crust. Although the oldest evidences of life on Earth are fossilized in microcrystalline silica deposits, little is known about the functional role that bacteria can exert on silica mobility at non-thermal and neutral pH conditions. Here, a microbial influence on silica mobilization event occurring in the Earth's largest orthoquartzite cave is described. Transition from the pristine orthoquartzite to amorphous silica opaline precipitates in the form of stromatolite-like structures is documented through mineralogical, microscopic and geochemical analyses showing an increase of metals and other bioessential elements accompanied by permineralized bacterial cells and ultrastructures. Illumina sequencing of the 16S rRNA gene describes the bacterial diversity characterizing the consecutive amorphization steps to provide clues on the biogeochemical factors playing a role in the silica solubilization and precipitation processes. These results show that both quartz weathering and silica mobility are affected by chemotrophic bacterial communities, providing insights for the understanding of the silica cycle in the subsurface.

Tuberculosis (TB) has been described in Native American populations prior to the arrival of European explorers, and in Brazilian populations dating from the Colonial Period. There are no studies demonstrating TB infection in native Brazilians, and the history and epidemiological scenario of TB in Brazil is still unknown. The aim of this study was to verify the presence of TB infection among the native Tenetehara-Guajajara population from Maranhão State, Brazil, 210 ± 40 years ago. A Tenetehara-Guajajara skeleton collection was submitted to paleopathological analysis, and rib bone samples (n = 17) were used for paleogenetic analysis based on Mycobacterium tuberculosis complex (MTC) targets. Porotic hyperostosis and cribra orbitalia were found in 10 and 13 individuals, respectively. Maternal ancestry analysis revealed Native American mtDNA haplogroups A and C1 in three individuals. Three samples showed osteological evidence suggestive of TB. katG and mtp40 sequences were detected in three individuals, indicating probable TB infection by two MTC lineages. Tuberculosis infection in the Tenetehara-Guajajara population since the 18th century points to a panorama of the disease resulting, most probably, from European contact. However, the important contribution of African slaves in the population of Maranhão State, could be also considered as a source of the disease. This study provides new data on TB during the Brazilian Colonial Period. This is the first report integrating paleopathological and paleogenetic data for the study of TB in Brazil., Competing Interests: The authors have declared that no competing interests exist.

Early Triassic microbialites are distributed widely in the shallow marine facies of the Tethys Region, especially in the carbonate platform where they were deposited immediately after the latest Permian mass extinction (LPME). Ten Griesbachian domed microbial mounds were found in an outcrop of the uppermost first member of the Feixianguan (FXG) Formation at Baimiaozi, which is located in Beibei in the Upper Yangtze Region of southwest China. Field investigations and thin-sections analyses indicated that oolitic limestone, bioclastic limestone, microbialite, marl, and mudstone deposits are present in the first and second members of the FXG Formation, among which the thickness of the microbial mound above the massive oolitic limestone at the carbonate platform was approximately 3-4 m. Three facies were identified at the microbial mounds, namely, a mound base, mound body, and mound cap. Irregular laminae were found in the brown-colored microbialite of the mound base. The main mound body, which is composed of gray microbialite, is 1.0-1.5 m high and 2.0-3.0 m in diameter at the base. Dark gray grainstone found in the mound cap is covered by a thin layer of shelly limestone containing intact fossils of bivalves and gastropods, which are indicative of a simple ecosystem consisting of microbes and primary consumers. Brown-colored mudstone and marl layers of the second member of the FXG Formation overlie the microbialite, and this indicates that growth of the microbial mounds was halted by a sudden increase of terrestrial inputs and rapid transgression. Early Griesbachian conodonts of Hindeodus parvus? were identified from the mound limestone and the overlying strata of the second member of the FXG Formation, which is suggestive of the presence of a microbialite-dominated ocean in the Upper Yangtze Region during a certain interval after the LPME., Competing Interests: The authors have declared that no competing interests exist.

The Iberian Mediterranean Basin is home to one of the largest groups of prehistoric rock art sites in Europe. Despite the cultural relevance of prehistoric Spanish Levantine rock art, pigment composition remains partially unknown, and the nature of the binders used for painting has yet to be disclosed. In this work, we present the first omic analysis applied to one of the flagship Levantine rock art sites: the Valltorta ravine (Castellón, Spain). We used high-throughput sequencing to provide the first description of the bacterial communities colonizing the rock art patina, which proved to be dominated by Firmicutes species and might have a protective effect on the paintings. Proteomic analysis was also performed on rock art microsamples in order to determine the organic binders present in Levantine prehistoric rock art pigments. This information could shed light on the controversial dating of this UNESCO Cultural Heritage, and contribute to defining the chrono-cultural framework of the societies responsible for these paintings.

The ability to encrust in order to secure and maintain growth on a substrate is a key competitive innovation in benthic metazoans. Here we describe the substrate growth dynamics, mode of biomineralization and possible affinity of Namapoikia rietoogensis , a large (up to 1 m), robustly skeletal, and modular Ediacaran metazoan which encrusted the walls of synsedimentary fissures within microbial-metazoan reefs. Namapoikia formed laminar or domal morphologies with an internal structure of open tubules and transverse elements, and had a very plastic, non-deterministic growth form which could encrust both fully lithified surfaces as well as living microbial substrates, the latter via modified skeletal holdfasts. Namapoikia shows complex growth interactions and substrate competition with contemporary living microbialites and thrombolites, including the production of plate-like dissepiments in response to microbial overgrowth which served to elevate soft tissue above the microbial surface. Namapoikia could also recover from partial mortality due to microbial fouling. We infer initial skeletal growth to have propagated via the rapid formation of an organic scaffold via a basal pinacoderm prior to calcification. This is likely an ancient mode of biomineralization with similarities to the living calcified demosponge Vaceletia. Namapoikia also shows inferred skeletal growth banding which, combined with its large size, implies notable individual longevity. In sum, Namapoikia was a large, relatively long-lived Ediacaran clonal skeletal metazoan that propagated via an organic scaffold prior to calcification, enabling rapid, effective and dynamic substrate occupation and competition in cryptic reef settings. The open tubular internal structure, highly flexible, non-deterministic skeletal organization, and inferred style of biomineralization of Namapoikia places probable affinity within total-group poriferans., (© 2018 The Author(s).)

The deep biosphere is one of the least understood ecosystems on Earth. Although most microbiological studies in this system have focused on prokaryotes and neglected microeukaryotes, recent discoveries have revealed existence of fossil and active fungi in marine sediments and sub-seafloor basalts, with proposed importance for the subsurface energy cycle. However, studies of fungi in deep continental crystalline rocks are surprisingly few. Consequently, the characteristics and processes of fungi and fungus-prokaryote interactions in this vast environment remain enigmatic. Here we report the first findings of partly organically preserved and partly mineralized fungi at great depth in fractured crystalline rock (-740 m). Based on environmental parameters and mineralogy the fungi are interpreted as anaerobic. Synchrotron-based techniques and stable isotope microanalysis confirm a coupling between the fungi and sulfate reducing bacteria. The cryptoendolithic fungi have significantly weathered neighboring zeolite crystals and thus have implications for storage of toxic wastes using zeolite barriers.Deep subsurface microorganisms play an important role in nutrient cycling, yet little is known about deep continental fungal communities. Here, the authors show organically preserved and partly mineralized fungi at 740 m depth, and find evidence of an anaerobic fungi and sulfate reducing bacteria consortium.

Metagenome analysis has become a common source of information about microbial communities that occupy a wide range of niches, including archaeological specimens. It has been shown that the vast majority of DNA extracted from ancient samples come from bacteria (presumably modern contaminants). However, characterization of microbial DNA accompanying human remains has never been done systematically for a wide range of different samples. We used metagenomic approaches to perform comparative analyses of microorganism communities present in 161 archaeological human remains. DNA samples were isolated from the teeth of human skeletons dated from 100 AD to 1200 AD. The skeletons were collected from 7 archaeological sites in Central Europe and stored under different conditions. The majority of identified microbes were ubiquitous environmental bacteria that most likely contaminated the host remains not long ago. We observed that the composition of microbial communities was sample-specific and not correlated with its temporal or geographical origin. Additionally, traces of bacteria and archaea typical for human oral/gut flora, as well as potential pathogens, were identified in two-thirds of the samples. The genetic material of human-related species, in contrast to the environmental species that accounted for the majority of identified bacteria, displayed DNA damage patterns comparable with endogenous human ancient DNA, which suggested that these microbes might have accompanied the individual before death. Our study showed that the microbiome observed in an individual sample is not reliant on the method or duration of sample storage. Moreover, shallow sequencing of DNA extracted from ancient specimens and subsequent bioinformatics analysis allowed both the identification of ancient microbial species, including potential pathogens, and their differentiation from contemporary species that colonized human remains more recently., (© The Authors 2017. Published by Oxford University Press.)

The pattern and sequence of the decomposition of the Pipidae African dwarf frog (Hymenochirus boettgeri) is tracked in an experiment with microbial mats in order to explore soft tissue preservation over three years. Frog decay in microbial mats is preceded by rapid entombment (25-30 days) and mediated by the formation of a sarcophagus, which is built by a complex microbial community. The frog carcasses maintained a variety of soft tissues for years. Labile organic structures show greater durability within the mat, cells maintain their general shape (bone marrow cells and adipocytes), and muscles and connective tissues (adipose and fibrous tendons) exhibit their original organic structures. In addition, other soft tissues are promptly mineralized (day 540) in a Ca-rich carbonate phase (encephalic tectum) or enriched in sulphur residues (integumentary system). The result is coherent with a bias in soft-tissue preservation, as some tissues are more likely to be conserved than others. The outcomes support observations of exceptionally preserved fossil anurans (adults and tadpoles). Decomposition in mats shows singular conditions of pH and dissolved oxygen. Mineralization processes could be more diverse than in simple heterotrophic biofilms, opening new taphonomic processes that have yet to be explored.

Trilobites were one of the most successful groups of marine arthropods during the Palaeozoic era, yet their soft-part anatomy is only known from a few exceptionally-preserved specimens found in a handful of localities from the Cambrian to the Devonian. This is because, even if the sclerotized appendages were not destroyed during early taphonomic stages, they are often overprinted by the three-dimensional, mineralised exoskeleton. Inferences about the ventral anatomy and behavioural activities of trilobites can also be derived from the ichnological record, which suggests that most Cruziana and Rusophycus trace fossils were possibly produced by the actions of trilobites. Three specimens of the asaphid trilobite Megistaspis (Ekeraspis) hammondi, have been discovered in the Lower Ordovician Fezouata Konservat-Lagerstätte of southern Morocco, preserving appendages and digestive tract. The digestive structures include a crop with digestive caeca, while the appendages display exopodal setae and slight heteropody (cephalic endopods larger and more spinose than thoracic and pygidial ones). The combination of these digestive structures and the heteropody has never been described together among trilobites, and the latter could assist in the understanding of the production of certain comb-like traces of the Cruziana rugosa group, which are extraordinarily abundant on the shallow marine shelves around Gondwana.

The development of paleomicrobiology with new molecular techniques such as metagenomics is revolutionizing our knowledge of microbial evolution in human history. The study of microbial agents that are concomitantly active in the same biological environment makes it possible to obtain a picture of the complex interrelations among the different pathogens and gives us the perspective to understand the microecosystem of ancient times. This research acts as a bridge between disciplines such as archaeology, biology, and medicine, and the development of paleomicrobiology forces archaeology to broaden and update its methods. This chapter addresses the archaeological issues related to the identification of cemeteries from epidemic catastrophes (typology of burials, stratigraphy, topography, paleodemography) and the issues related to the sampling of human remains for biomolecular analysis. Developments in the field of paleomicrobiology are described with the example of the plague. Because of its powerful interdisciplinary features, the paleomicrobiological study of Yersinia pestis is an extremely interesting field, in which paleomicrobiology, historical research, and archeology are closely related, and it has important implications for the current dynamics of epidemiology.

The 407-million-year-old Rhynie Chert (Scotland) contains the most intact fossilised remains of an early land-based ecosystem including plants, arthropods, fungi and other microorganisms. Although most studies have focused on the terrestrial component, fossilised freshwater environments provide critical insights into fungal-algal interactions and the earliest continental branchiopod crustaceans. Here we report interactions between an enigmatic organism and an exquisitely preserved fungus. The fungal reproductive structures are intermixed with exceptionally well-preserved globular spiny structures interpreted as branchiopod resting eggs. Confocal laser scanning microscopy enabled us to reconstruct the fungus and its possible mode of nutrition, the affinity of the resting eggs, and their spatial associations. The new fungus (Cultoraquaticus trewini gen. et sp. nov) is attributed to Chytridiomycota based on its size, consistent formation of papillae, and the presence of an internal rhizoidal system. It is the most pristine fossil Chytridiomycota known, especially in terms of rhizoidal development and closely resembles living species in the Rhizophydiales. The spiny resting eggs are attributed to the crustacean Lepidocaris rhyniensis, dating branchiopod adaptation to life in ephemeral pools to the Early Devonian. The new fungal interaction suggests that, as in modern freshwater environments, chytrids were important to the mobilisation of nutrients in early aquatic foodwebs., Competing Interests: The authors have declared that no competing interests exist.

The paleomicrobiology of coprolites, which are fossilized fecal materials, has already yielded data about various organisms, including micro-eukaryotes, bacteria, and archaea, thus expanding our comprehension of ancient human dietary habits, gut microbiota, and intestinal and systemic infections. This mini-review briefly describes previous works and summarizes the main techniques used in handling coprolites and the findings obtained about ancient gut microbiota. Past intestinal and systemic infections are outlined.

The use of paleomicrobiological techniques in leprosy has the potential to assist paleopathologists in many important aspects of their studies on the bones of victims, solving at times diagnostic problems. With Mycobacterium leprae, because of the unique nature of the organism, these techniques can help solve problems of differential diagnosis. In cases of co-infection with Mycobacterium tuberculosis, they can also suggest a cause of death and possibly even trace the migratory patterns of people in antiquity, as well as explain changes in the rates and level of infection within populations in antiquity.

Epidemic typhus caused by Rickettsia prowazekii is one of the oldest pestilential diseases of humankind. The disease is transmitted to human beings by the body louse Pediculus humanus corporis and is still considered a major threat by public health authorities, despite the efficacy of antibiotics, because poor sanitary conditions are conducive to louse proliferation. Epidemic typhus has accompanied disasters that impact humanity and has arguably determined the outcome of more wars than have soldiers and generals. The detection, identification, and characterization of microorganisms in ancient remains by paleomicrobiology has permitted the diagnosis of past epidemic typhus outbreaks through the detection of R. prowazekii. Various techniques, including microscopy and immunodetection, can be used in paleomicrobiology, but most of the data have been obtained by using PCR-based molecular techniques on dental pulp samples. Paleomicrobiology enabled the identification of the first outbreak of epidemic typhus in the 18th century in the context of a pan-European great war in the city of Douai, France, and supported the hypothesis that typhus was imported into Europe by Spanish soldiers returning from America. R. prowazekii was also detected in the remains of soldiers of Napoleon's Grand Army in Vilnius, Lithuania, which indicates that Napoleon's soldiers had epidemic typhus. The purpose of this article is to underscore the modern comprehension of clinical epidemic typhus, focus on the historical relationships of the disease, and examine the use of paleomicrobiology in the detection of past epidemic typhus outbreaks.

There are several scenarios regarding how burial sites in archaeological contexts are discovered. We will focus on two scenarios according to the degree of historical knowledge regarding the studied sector. The excavation may be performed in a known funeral place or a highly suspected place (e.g., the interior or immediate exterior space in a religious monument or a parish cemetery). Also, the excavation of unexpected graves or graves discovered by chance may occur in places that had unknown or forgotten funeral purposes.

Tuberculosis is a significant global disease today, so understanding its origins and history is important. It is primarily a lung infection and is transmitted by infectious aerosols from person to person, so a high population density encourages its spread. The causative organism is Mycobacterium tuberculosis, an obligate pathogen in the M. tuberculosis complex that also contains closely related species, such as Mycobacterium bovis, that primarily infect animals. Typical bone lesions occur in about 5% of untreated infections. These can be recognized in historical and archaeological material, along with nonspecific paleopathology such as new bone formation (periostitis), especially on ribs. Based on such lesions, tuberculosis has been found in ancient Egypt, pre-Columbian America, and Neolithic Europe. The detection of M. tuberculosis ancient DNA (aDNA) by using PCR led to the development of the new field of paleomicrobiology. As a result, a large number of tuberculosis cases were recognized in mummified tissue and bones with nonspecific or no lesions. In parallel with these developments, M. tuberculosis cell wall lipid biomarkers have detected tuberculosis suggested by paleopathology and confirmed aDNA findings. In well-preserved cases, molecular typing has identified M. tuberculosis lineages and genotypes. The current interest in targeted enrichment, shotgun sequencing, and metagenomic analysis reveals ancient mixed infections with different M. tuberculosis strains and other pathogens. Identification of M. tuberculosis lineages from samples of known age enables the date of the emergence of strains and lineages to be calculated directly rather than by making assumptions on the rate of evolutionary change.

Paleomicrobiology, or the study of ancient microorganisms, has raised both fascination and skepticism for many years. While paleomicrobiology is not a recent field, the application of emerging techniques, such as DNA sequencing, is proving essential and has provided novel information regarding the evolution of viruses, antibiotic resistance, saprophytes, and pathogens, as well as ancient health and disease status, cultural customs, ethnic diets, and historical events. In this review, we highlight the importance of studying ancient microbial DNA, its contributions to current knowledge, and the role that forensic paleomicrobiology has played in deciphering historical enigmas. We also discuss the emerging techniques used to study the microbial composition of ancient samples as well as major concerns that accompany ancient DNA analyses.

Paleopathology studies the traces of disease on human and animal remains from ancient times. Infectious diseases have been, for over a century, one of its main fields of interest. The applications of paleogenetics methods to microbial aDNA, that started in the 90s combined to the recent development of new sequencing techniques allowing 'paleogenomics' approaches, have completely renewed the issue of the infections in the past. These advances open up new challenges in the understanding of the evolution of human-pathogen relationships, integrated in "One Health" concept.In this perspective, an integrative multidisciplinary approach combining data from ancient texts and old bones to those of old molecules is of great interest for reconstructing the past of human infections. Despite some too optimistic prediction of their eradication in the late 20th century, some of these ancient human diseases, such as plague, leprosy or tuberculosis, are still present and continue their evolution at the beginning of this 21rst century. Better know the past to predict a part of the future of human diseases remains, more than ever, the motto of the paleopathological science.

Antibiotic resistance is an ancient biological mechanism in bacteria, although its proliferation in our contemporary world has been amplified through antimicrobial therapy. Recent studies conducted on ancient environmental and human samples have uncovered numerous antibiotic-resistant bacteria and resistance genes. The resistance genes that have been reported from the analysis of ancient bacterial DNA include genes coding for several classes of antibiotics, such as glycopeptides, β-lactams, tetracyclines, and macrolides. The investigation of the resistome of ancient bacteria is a recent and emerging field of research, and technological advancements such as next-generation sequencing will further contribute to its growth. It is hoped that the knowledge gained from this research will help us to better understand the evolution of antibiotic resistance genes and will also be used in drug design as a proactive measure against antibiotic resistance.

The origins of human infectious diseases have long fascinated scientists worldwide. Paleomicrobiology offers a unique access to the history of these infections and sheds light on ancient and historical epidemics. In this chapter, we review the paleomicrobiological evidence for Bartonella infections.

Microbial mats have been hypothesized to improve the persistence and the preservation of organic remains during fossilization processes. We test this hypothesis with long-term experiments (up to 5.5 years) using invertebrate and vertebrate corpses. Once placed on mats, the microbial community coats the corpses and forms a three-dimensional sarcophagus composed of microbial cells and exopolymeric substances (EPS). This coverage provides a template for i) moulding superficial features, resulting in negative impressions, and ii) generating replicas. The impressions of fly setulae, fish scales and frog skin verrucae are shaped mainly by small cells in an EPS matrix. Microbes also replicate delicate structures such as the three successive layers that compose a fish eye. The sarcophagus protects the body integrity, allowing the persistence of inner organs such as the ovaries and digestive apparatus in flies, the swim bladder and muscles in fish, and the bone marrow in frog legs. This study brings strong experimental evidence to the idea that mats favour metazoan fossilization by moulding, replicating and delaying decay. Rapid burial has classically been invoked as a mechanism to explain exceptional preservation. However, mats may play a similar role during early fossilization as they can preserve complex features for a long time.

Welwitschia mirabilis is an ancient and rare plant distributed along the western coast of Namibia and Angola. Several aspects of Welwitschia biology and ecology have been investigated, but very little is known about the microbial communities associated with this plant. This study reports on the bacterial and fungal communities inhabiting the rhizosphere of W. mirabilis and the surrounding bulk soil. Rhizosphere communities were dominated by sequences of Alphaproteobacteria and Euromycetes, while Actinobacteria, Alphaproteobacteria, and fungi of the class Dothideomycetes jointly dominated bulk soil communities. Although microbial communities within the rhizosphere and soil samples were highly variable, very few "species" (OTUs defined at a 97% identity cut-off) were shared between these two environments. There was a small 'core' rhizosphere bacterial community (formed by Nitratireductor, Steroidobacter, Pseudonocardia and three Phylobacteriaceae) that together with Rhizophagus, an arbuscular mycorrhizal fungus, and other putative plant growth-promoting microbes may interact synergistically to promote Welwitschia growth.

The aim of this study was to investigate the bioweathering of copper minerals present in the alkaline, copper-bearing and organic-rich Kupferschiefer black shale through the action of a consortium of indigenous lithobiontic, heterotrophic, neutrophilic bacteria isolated from this sedimentary rock. The involvement of microorganisms in the direct/enzymatic bioweathering of fossil organic matter of the rock was confirmed. As a result of bacterial activity, a spectrum of various organic compounds such as urea and phosphoric acid tributyl ester were released from the rock. These compounds indirectly act on the copper minerals occurring in the rock and cause them to weather. This process was reflected in the mobilization of copper, iron and sulfur and in changes in the appearance of copper minerals observed under reflected light. The potential role of identified enzymes in biodegradation of fossil organic matter and role of organic compounds released from black shale as a result of this process in copper minerals weathering was discussed. The presented results provide a new insight into the role of chemical compounds released by bacteria during fossil organic matter bioweathering potentially important in the cycling of copper and iron deposited in the sedimentary rock. The originality of the described phenomenon lies in the fact that the bioweathering of fossil organic matter and, consequently, of copper minerals occur simultaneously in the same environment, without any additional sources of energy, electrons and carbon., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Patterns of geographic distribution and composition of fungal communities are still poorly understood. Widespread occurrence in terrestrial ecosystems and the unique richness of interactions of Sebacinales with plants make them a target group to study evolutionary events in the light of nutritional lifestyle. We inferred diversity patterns, phylogenetic structures and divergence times of Sebacinales with respect to their nutritional lifestyles by integrating data from fossil-calibrated phylogenetic analyses. Relaxed molecular clock analyses indicated that Sebacinales originated late Permian within Basidiomycota, and their split into Sebacinaceae and Serendipitaceae nom. prov. likely occurred during the late Jurassic and the early Cretaceous, coinciding with major diversifications of land plants. In Sebacinaceae, diversification of species with ectomycorrhizal lifestyle presumably started during the Paleocene. Lineage radiations of the core group of ericoid and cavendishioid mycorrhizal Sebacinales started probably in the Eocene, coinciding with diversification events of their hosts. The diversification of Sebacinales with jungermannioid interactions started during the Oligocene, and occurred much later than the diversification of their hosts. Sebacinales communities associated either with ectomycorrhizal plants, achlorophyllous orchids, ericoid and cavendishioid Ericaceae or liverworts were phylogenetically clustered and globally distributed. Major Sebacinales lineage diversifications started after the continents had drifted apart. We also briefly discuss dispersal patterns of extant Sebacinales.

Well-preserved mycelia of fungal- or saprolegnia-like biota mineralised by ferromanganese oxides were found for the first time in long bones of Late Cretaceous dinosaurs from the Gobi Desert (Nemegt Valley, Mongolia). The mycelia formed a biofilm on the wall of the bone marrow cavity and penetrated the osteon channels of the nearby bone tissue. Optical microscopy, Raman, SEM/EDS, SEM/BSE, electron microprobe and cathodoluminescence analyses revealed that the mineralisation of the mycelia proceeded in two stages. The first stage was early post-mortem mineralisation of the hyphae by Fe/Mn-oxide coatings and microconcretions. Probably this proceeded in a mildly acidic to circumneutral environment, predominantly due to heterotrophic bacteria degrading the mycelial necromass and liberating Fe and Mn sorbed by the mycelia during its lifetime. The second stage of mineralisation, which proceeded much later following the final burial of the bones in an alkaline environment, resulted from the massive precipitation of calcite and occasionally barite on the iron/manganese-oxide-coated mycelia. The mineral phases produced by fungal biofilms colonising the interiors of decaying dinosaur bones not only enhance the preservation (fossilisation) of fungal remains but can also be used as indicators of the geochemistry of the dinosaur burial sites.

Studying ancient infectious diseases is a challenge, as written contemporary descriptions, when available, are often imprecise and do not allow for accurate discrimination among the pathogens endemic at that time. Paleomicrobiology offers a unique access to the history of these infections by identifying precisely the causative agents. Body louse-transmitted infections are amongst the most epidemic diseases in history, especially in war and famine periods. Of these, Bartonella quintana was detected by suicide PCR in 4000-year-old human remains, thus representing the oldest evidence to date of an arthropod-transmitted infection to human beings. This species has also been detected in human specimens from the 11th to 15th, 18th and 19th centuries. In addition, Bartonella henselae, a cat- and flea-associated pathogen, was detected in cat specimens from the 13th to 18th centuries, therefore demonstrating an association of the bacterium and its reservoir for over 800 years. Therefore, pathogenic Bartonella species have been involved in several outbreaks in the past millennia and should systematically be investigated in human remains from suspected epidemics., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Microbial corrosion textures in volcanic glass from Cenozoic seafloor basalts and the corresponding titanite replacement microtextures in metamorphosed Paleoarchean pillow lavas have been interpreted as evidence for a deep biosphere dating back in time through the earliest periods of preserved life on earth. This interpretation has been recently challenged for Paleoarchean titanite replacement textures based on textural and geochronological data from pillow lavas in the Hooggenoeg Complex of the Barberton Greenstone Belt in South Africa. We use this controversy to explore the strengths and weaknesses of arguments made in support or rejection of the biogenicity interpretation of bioalteration trace fossils in Cenozoic basalt glasses and their putative equivalents in Paleoarchean greenstones. Our analysis suggests that biogenicity cannot be taken for granted for all titanite-based textures in metamorphosed basalt glass, but a cautious and critical evaluation of evidence suggests that biogenicity remains the most likely interpretation for previously described titanite microtextures in Paleoarchean pillow lavas.