From ancestral diets to modern dysbiosis: evolutionary insights from ancient gut microbiomes

Abstract

The human gut microbiome has undergone profound ecological changes throughout human evolution, shaped by major dietary, environmental, and cultural transitions from hunter-gatherer societies to modern industrialized populations. Recent advances in paleomicrobiology, ancient DNA sequencing, and metagenomics have enabled the reconstruction of ancestral microbial communities preserved in coprolites, paleofeces, dental calculus, and mummified remains, providing unprecedented insights into the long-term coevolution between humans and their microbiota. This review synthesizes current evidence on the evolution of the human microbiome, highlighting how the adoption of agriculture, urbanization, industrialization, and widespread antibiotic use progressively reduced microbial diversity and altered key microbial functions. We discuss the principal archaeological sources used for ancient microbiome reconstruction, summarize major dietary and microbial transitions across human history, and examine their implications for the emergence of modern dysbiosis. Particular emphasis is placed on the evolutionary links between microbiome disruption and contemporary metabolic, inflammatory, and immune-mediated diseases through the concept of evolutionary mismatch. Finally, we explore the clinical implications of paleomicrobiological research, including microbiome-directed therapies, precision nutrition, next-generation probiotics, fecal microbiota transplantation, and emerging pharmacological approaches that modulate host–microbiome interactions. Rather than advocating restoration of ancestral microbial communities, evidence suggests that identifying and recovering evolutionarily conserved microbial functions may provide a more rational framework for preventing and treating chronic diseases in modern populations.

1 Introduction

The human microbiome represents one of the most complex biological components associated with the human body, comprising the entirety of microorganisms that colonize the gastrointestinal tract, oral cavity, skin, and other body surfaces (Bautista et al., 2025; Wardi et al., 2026). Although the terms microbiota and microbiome are frequently used interchangeably, they refer to distinct concepts. The microbiota denotes the community of living microorganisms inhabiting a specific ecological niche, whereas the microbiome encompasses not only these microorganisms but also their collective genomes, metabolic products, and interactions with the host and the surrounding environment (Grice and Segre, 2012; Hou et al., 2022; Hacioglu, 2026). The microbiome is now widely recognized as a functional “metabolic organ” that plays essential roles in nutrient digestion, vitamin synthesis, immune system development and regulation, and the maintenance of physiological homeostasis (Wardi et al., 2026).

Throughout human evolution, the gut microbiome developed under dietary and environmental conditions characterized by high consumption of diverse plant-derived foods, continuous exposure to environmental microorganisms, and the absence of many features of modern industrialized societies. Successive cultural transitions—including the adoption of agriculture, urbanization, and industrialization—progressively altered dietary composition, food processing, sanitation, antimicrobial exposure, and lifestyle, thereby reshaping host–microbiome interactions over time. Collectively, these cumulative dietary, environmental, and cultural transitions provide the evolutionary framework for understanding the emergence of modern dysbiosis and its association with chronic non-communicable diseases (Sonnenburg and Sonnenburg, 2019; Assaf et al., 2024; Alt et al., 2022). Importantly, these transitions cannot be explained by dietary change alone. Urbanization, sanitation, antimicrobial exposure, food processing, and reduced environmental microbial diversity acted together to reshape host–microbiome interactions, indicating that modern dysbiosis reflects cumulative ecological change rather than any single environmental factor (Sonnenburg and Sonnenburg, 2019; Assaf et al., 2024).

How the human microbiome has evolved over time has become possible through advances in paleopathology and, more recently, paleomicrobiology (Bos et al., 2019). A true paradigm shift, however, occurred with the introduction of molecular techniques, ranging from polymerase chain reaction (PCR) and ancient DNA (aDNA) analysis to next-generation metagenomic sequencing (Burbano et al., 2010). These approaches have enabled the direct identification of microorganisms and pathogens preserved in coprolites, paleofeces, dental calculus, and mummified tissues, making it possible to reconstruct ancestral microbial communities with unprecedented resolution (Nodari et al., 2021; Grasso et al., 2024). Consequently, paleomicrobiology now offers a unique perspective on how major transitions in diet, migration, urbanization, and industrialization have reshaped the human microbial ecosystem and contributed to the emergence of dysbiosis associated with numerous chronic diseases.

Over the past decade, the study of ancient microbiomes has emerged as a valuable tool for elucidating the long-term interactions between humans, their environment, and disease. The analysis of microorganisms preserved in coprolites, paleofeces, dental calculus, and mummified tissues enables not only the reconstruction of the gut microbiome composition of past populations but also the identification of microbial changes associated with major transitions in human history, including the adoption of agriculture, urbanization, large-scale migrations, and industrialization (Bos et al., 2019; Wibowo et al., 2021; Gancz et al., 2023). These transformations profoundly altered the structure and diversity of human microbial ecosystems, contributing to distinct patterns of biological adaptation, susceptibility to infectious diseases, and metabolic vulnerability (Rivera-Perez et al., 2016). Comparative studies of contemporary hunter-gatherer, rural agricultural, and industrialized populations, together with archaeological evidence derived from coprolites, dental calculus, and mummified remains, consistently demonstrate progressive reductions in microbial diversity, alterations in community composition, and changes in microbial metabolic function across major dietary and lifestyle transitions. These observations provide the evolutionary basis for the hypothesis that industrialization disrupted host–microbiome interactions that remained relatively stable throughout much of human evolution (Sonnenburg and Sonnenburg, 2019; Schnorr et al., 2016; Wibowo et al., 2021).

The aim of this review is not only to summarize current evidence regarding ancient human microbiomes but also to critically integrate archaeological, evolutionary, ecological, and clinical evidence into a unified framework explaining how long-term host–microbiome interactions have been reshaped during major dietary and lifestyle transitions. We discuss the principal discoveries derived from the analysis of coprolites and other sources of ancient DNA, the evolutionary changes in the gut microbiome across different historical periods and cultural contexts, and their implications for interpreting infectious, metabolic, and inflammatory diseases. By integrating evidence from paleopathology, ancient genomics, microbiology, and evolutionary medicine, this review seeks to illustrate how the microbial past of our species can provide novel insights into human health and disease in the present. Unlike previous reviews that have primarily focused on either ancient microbiomes, paleopathology, or evolutionary medicine as separate topics, the present review integrates archaeological evidence from coprolites, dental calculus, and mummified remains with contemporary microbiome ecology and clinical research to provide a unified evolutionary framework for modern dysbiosis. Particular emphasis is placed on critically evaluating methodological limitations, authentication challenges, ecological transitions associated with industrialization, and the translational relevance of evolutionarily conserved microbial functions. By combining paleomicrobiological evidence with current concepts in microbial ecology and evolutionary medicine, this review aims to provide a more comprehensive interpretation of how long-term host–microbiome interactions may contribute to modern metabolic and immune-mediated diseases. The overall conceptual framework of the present review, illustrating the integration of archaeological evidence, evolutionary transitions, methodological approaches, and their implications for modern dysbiosis and human disease, is summarized in Figure 1.

Figure 1

2 Methodology

This narrative review was conducted to provide a comprehensive overview of current knowledge regarding the evolution of the human gut microbiome from ancient populations to modern societies, with particular emphasis on evidence derived from paleomicrobiology and molecular paleopathology. Relevant literature was identified through searches performed in PubMed, Scopus, Web of Science, and Google Scholar up to June 2026. The final literature search was conducted in June 2026. Titles and abstracts retrieved through the database searches were initially screened for relevance to the objectives of the review, followed by full-text evaluation of potentially eligible articles. Reference lists of key review articles and landmark publications were manually screened to identify additional relevant studies not captured during the electronic database search. Search terms included combinations of the following keywords: “ancient microbiome”, “gut microbiome evolution”, “paleomicrobiology”, “molecular paleopathology”, “coprolites”, “paleofeces”, “ancient DNA”, “archaeogenomics”, “metagenomics”, “intestinal microbiota”, “dysbiosis”, and “human evolution”. Boolean operators (AND and OR) were used to combine search terms, and database-specific search syntax was applied where appropriate. Search terms were adapted to the indexing system of each database to maximize retrieval of relevant publications. Studies were selected according to their scientific relevance, methodological quality, and direct contribution to understanding the evolution of the human microbiome. Priority was given to original research articles reporting ancient DNA or metagenomic analyses of coprolites, paleofeces, dental calculus, mummified tissues, and archaeological human remains. Systematic reviews, meta-analyses, methodological studies, and landmark publications were also included when they provided important conceptual or methodological context. Studies focusing exclusively on modern microbiome composition without evolutionary relevance, conference abstracts, opinion articles lacking supporting evidence, and publications unavailable in full text were excluded. Because this work was designed as a narrative review rather than a formal systematic review, no quantitative meta-analysis or formal risk-of-bias assessment was performed. Nevertheless, methodological rigor was considered during study selection by prioritizing peer-reviewed publications with transparent study design, appropriate authentication of ancient DNA where applicable, and direct relevance to the objectives of the review. Additionally, relevant review articles, methodological papers, and landmark studies addressing the evolution of the human microbiome, dietary transitions, host–microbe interactions, and the relationship between ancient microbiomes and modern diseases were included. The included literature was synthesized narratively because of substantial heterogeneity in archaeological substrates, DNA preservation, sequencing technologies, authentication procedures, analytical pipelines, and study objectives. Findings were organized thematically according to archaeological evidence (coprolites, dental calculus, and mummified remains), evolutionary dietary and lifestyle transitions, and the clinical implications of microbiome evolution for contemporary human disease. The study selection process is summarized in the PRISMA-informed flow diagram (Figure 2).

Figure 2

3 Sources and methods for reconstructing ancient gut microbiomes

3.1 Coprolites and paleofeces

Coprolites and paleofeces constitute primary sources for reconstructing the intestinal microbiome of past human populations. These materials include desiccated, mineralized, or frozen fecal remains, preserved intestinal contents from mummified individuals, and archaeological fecal deposits recovered from contexts such as latrines and refuse pits. Their preservation enables the recovery of ancient biomolecules, including microbial DNA, dietary residues, and parasite remains, providing direct evidence of intestinal microbial communities and host-associated biological information (Appelt et al., 2016; Nodari et al., 2021). Because these materials preserve multiple categories of biological and archaeological evidence simultaneously, they have become the principal substrate for multidisciplinary paleomicrobiological investigations integrating microbiology, archaeology, parasitology, and dietary reconstruction.

The analytical potential of coprolites has increased substantially with the transition from microscopy, immunological assays, and targeted PCR to high-throughput 16S and 18S rRNA sequencing and shotgun metagenomics. Microscopy remains particularly informative for morphologically preserved parasite eggs, whereas immunological assays can detect selected antigens when nucleic acids are poorly preserved. Targeted PCR offers high analytical sensitivity but is susceptible to amplification bias, PCR inhibitors, and false-positive results while providing information only for predefined genetic targets. Amplicon sequencing enables broader community profiling but frequently lacks species-level and functional resolution. By contrast, shotgun metagenomics simultaneously recovers microbial, dietary, parasitic, and host-derived sequences and permits functional characterization of ancient gut ecosystems. However, this approach also requires higher endogenous DNA preservation, greater sequencing depth, and rigorous bioinformatic authentication to distinguish authentic ancient microbial DNA from environmental contaminants (Appelt et al., 2016; Nodari et al., 2021). Thus, although technological advances have substantially improved taxonomic resolution and functional interpretation, they have not eliminated the need for stringent contamination control and authentication procedures. Importantly, while Appelt et al. primarily emphasized methodological standardization and contamination control as prerequisites for reliable ancient DNA analyses, Nodari et al. highlighted the increasing integration of metagenomics with archaeobotanical, zooarchaeological, and paleoparasitological evidence, illustrating the transition from single-method investigations toward multidisciplinary reconstructions of ancient intestinal ecosystems. Together, these studies suggest that methodological integration, rather than the application of a single sequencing technology, provides the most robust framework for reconstructing ancient gut microbiomes.

A central limitation of coprolite research is that these specimens represent open biological systems whose molecular composition may be modified after deposition. Soil microorganisms and environmental DNA can infiltrate the specimen, whereas humidity, oxidation, microbial activity, temperature fluctuations, and differential DNA preservation may alter the original intestinal microbial profile. The study conducted by Cano et al. (2014) addressed this limitation experimentally by comparing paired cortical and core samples from 15 coprolites. Microbial communities differed significantly between the two compartments (Adonis R² = 0.191, p = 0.001), with cortical material containing substantially higher proportions of soil-associated microorganisms, thereby supporting the routine exclusion of external material during laboratory processing. Furthermore, significant differences between Huecoid and Saladoid microbiomes (Adonis R² = 0.287, p = 0.001; PERMANOVA pseudo-F = 1.98, p = 0.001) demonstrated that biologically meaningful variation could still be recovered despite long-term preservation. The paired core–cortex design represents one of the principal methodological strengths of this study because it experimentally validates contamination control rather than assuming its effectiveness. Nevertheless, only fifteen specimens were subjected to microbiome sequencing, all originated from a limited archaeological context, and the 16S/18S amplicon approach could not fully disentangle the relative contributions of diet, host ancestry, taphonomic processes, and environmental conditions to the observed microbial differences. Consequently, while the study provides robust evidence for microbiome differentiation between the sampled populations, it does not establish dietary variation as the sole determinant of community composition.

Overall, current evidence supports coprolites and paleofeces as among the most informative archives for reconstructing ancient intestinal ecosystems. However, methodological heterogeneity, including differences in sample preservation, excavation procedures, DNA extraction protocols, sequencing platforms, authentication criteria, and bioinformatic pipelines, continues to limit direct comparisons across studies (Appelt et al., 2016; Nodari et al., 2021). Moreover, dietary interpretations require careful consideration, as pollen, phytoliths, and plant residues may reflect environmental deposition, secondary consumption through prey gastrointestinal contents, or post-depositional contamination rather than direct human ingestion (Shipley and Kindscher, 2016). Consequently, integrating complementary lines of archaeological and molecular evidence within standardized analytical workflows will be essential for improving reproducibility and strengthening confidence in evolutionary reconstructions of the ancient human gut microbiome.

3.2 Dietary reconstruction from coprolite studies

Coprolites provide one of the most direct archives of ancient human diet and intestinal ecology, preserving dietary residues together with microorganisms, parasites and environmental DNA. Consequently, they have become an essential resource for reconstructing dietary adaptation, subsistence strategies and the long-term interactions between humans and their gut microbiota. Unlike isolated archaeobotanical or zooarchaeological remains, coprolites integrate dietary residues, microbial DNA, parasite remains, and environmental biomarkers within a single biological specimen, providing a more comprehensive reconstruction of ancient dietary practices and intestinal ecology.

Evidence from prehistoric populations consistently indicates diets characterized by substantial botanical diversity and high fiber intake. One of the earliest investigations, conducted in the Tehuacán Valley (Mexico), analyzed 116 coprolites spanning multiple phases of human occupation and identified dietary remains of cassava (Manihot spp.), pochote root (Ceiba parvifolia), and species belonging to the Opuntia and Agave genera (Project, 1967; Shillito et al., 2020a). These data are consistent with nutritional patterns rich in complex carbohydrates and microbiota-accessible plant polysaccharides before and during the early stages of agriculture.

The transition to farming did not immediately replace the exploitation of wild resources. At the Neolithic site of Tianluoshan (7000–6000 BP), pollen analysis demonstrated an overwhelming predominance of Typha (cattail), accounting for more than 93% of the pollen recovered from some coprolites (Zhang et al., 2020a; Qingjiang et al., 2024).

Together with archaeological evidence, these observations indicate that wetland plants remained an important dietary component despite the establishment of rice cultivation, illustrating the gradual rather than abrupt nature of the agricultural transition (Zhang et al., 2020b). Importantly, the integration of pollen analysis with archaeobotanical evidence provides greater confidence in dietary reconstruction than either approach alone, although pollen spectra should still be interpreted cautiously because they may also reflect environmental deposition rather than direct food consumption.

Ancient coprolites also reveal the remarkable dietary plasticity of prehistoric populations. Specimens recovered from Paisley Caves (Oregon, USA) contained seeds, insect remains, bone fragments and skeletal elements of small rodents, demonstrating the exploitation of a broad spectrum of plant and animal resources and, in some cases, the consumption of entire small vertebrates (Shillito et al., 2020a, b). Such findings contrast sharply with the dietary homogenization associated with contemporary industrialized societies. However, the studies conducted by Shillito et al., emphasized that botanical macroremains, phytoliths, and pollen should not be interpreted independently, as secondary ingestion, post-depositional processes, and environmental contamination may influence dietary reconstructions. Consequently, multiproxy analyses integrating botanical, zoological, and molecular evidence provide substantially more robust dietary interpretations than single-proxy approaches.

The progressive diversification of food systems accompanying urbanization is evident in later archaeological contexts. Metagenomic analyses of latrine deposits from Northern Europe and the Middle East (approximately 500 BC–1700 AD) identified DNA from numerous edible plant taxa, including Brassica, Fagopyrum, Hordeum, Pisum, Fragaria, Prunus, Pyrus, Rheum and Vaccinium. The additional detection of Vitis and Humulus DNA indicates the consumption of wine and beer, whereas the absence of potato and carrot DNA reflects their subsequent introduction into European diets following transcontinental exchange (Søe et al., 2018).

Further evidence from medieval and Renaissance Brussels (14th–17th centuries) illustrates how expanding trade networks and urban development reshaped dietary habits. Archaeological and palaeobiological analyses documented the consumption of cereals, fruits, vegetables, pork, beef, freshwater fish, beer and wine, reflecting increasing dietary complexity compared with prehistoric populations (Graff et al., 2020). Collectively, these studies demonstrate the progressive evolution of dietary reconstruction methods, from classical macrofossil and pollen analyses to integrated metagenomic approaches that simultaneously characterize dietary components and gut microbial communities. Although all investigations support a transition from diverse foraging diets toward increasingly specialized agricultural and urban food systems, methodological heterogeneity—including differences in sample preservation, analytical techniques, and taxonomic resolution—limits direct comparisons among archaeological populations. Consequently, dietary reconstructions should integrate archaeobotanical, zooarchaeological, molecular, and archaeological evidence rather than relying on a single proxy. Notably, these archaeological observations are consistent with a recent meta-analysis of 568 individuals spanning hunter-gatherer, agricultural, agropastoral, pastoral, and urban populations, which demonstrated a progressive decline in gut microbial diversity and gradual shifts in microbial community composition along the transition toward industrialized lifestyles (Rosas-Plaza et al., 2022). Together, these findings support the hypothesis that long-term dietary transitions contributed to shaping the human gut microbiome, although the relative contributions of diet and other environmental factors remain to be fully elucidated.

3.3 Evidence of infectious diseases and parasitic burden

Beyond dietary reconstruction, coprolites provide a valuable source of information for investigating health status and disease patterns in ancient populations. Advances in paleoparasitology, together with molecular and immunological techniques, have enabled the direct identification of intestinal parasites and enteric pathogens preserved in archaeological fecal material, offering insights into sanitation, dietary practices, host–pathogen interactions, and disease in past populations (Gonçalves et al., 2002; Mitchell, 2024).

Among the oldest evidence of ancient intestinal microbial communities are fecal deposits recovered from the Middle Paleolithic site of El Salt (Spain), attributed to Neanderthals. Molecular analyses identified bacterial taxa that are currently regarded as hallmarks of a healthy gut microbiome, including Faecalibacterium prausnitzii, Roseburia, Blautia, Coprococcus, Ruminococcus, Bifidobacterium, Prevotella, and Alistipes. Many of these microorganisms are involved in dietary fiber fermentation and short-chain fatty acid production, suggesting that key functional characteristics of the human gut microbiome were already established during the Paleolithic period (Sistiaga et al., 2014).

Evidence from later archaeological periods increasingly documents the burden of enteric infections. One of the earliest examples derives from two Iron Age latrines in Jerusalem (7th–6th centuries BCE), where Giardia duodenalis was identified using enzyme-linked immunosorbent assay (ELISA). This finding represents the earliest evidence of giardiasis in the Near East and indicates that protozoal enteric infections were already established in densely populated urban communities, most likely facilitated by inadequate sanitation and contaminated water sources (Mitchell et al., 2023).

Helminth infections became particularly well documented in medieval and post-medieval archaeological contexts. Metagenomic analyses of latrine sediments from Northern Europe and the Middle East identified Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, Diphyllobothrium latum, Taenia solium, and Fasciola hepatica. The detection of fish- and pork-borne helminths reflects the consumption of raw or insufficiently cooked food, whereas parasites associated with domesticated animals suggest frequent human–animal contact and suboptimal sanitary conditions (Søe et al., 2018; Côté and Le Bailly, 2018; Graff et al., 2020).

Comparable findings were reported from three latrines excavated in Brussels and dated between the 14th and 17th centuries, where analyses identified Ascaris spp., Trichuris spp., Taenia spp., Fasciola hepatica, Dicrocoelium dendriticum, Giardia duodenalis, and Entamoeba histolytica. Particularly high concentrations of Ascaris and Trichuris eggs indicate that fecal–oral transmission remained a major public health concern in medieval urban environments Notably, the overall parasitic profile changed little between the late Medieval and Renaissance periods, suggesting that improvements in sanitation were limited despite substantial demographic and urban development. Continued transmission was likely maintained by contaminated water sources, flooding, and the widespread agricultural use of human feces (Graff et al., 2020).

Additional evidence from naturally preserved intestinal contents of Joseon Dynasty mummies (14th–20th centuries CE) demonstrated a high prevalence of Ascaris lumbricoides and Trichuris trichiura, confirming that intestinal helminthiases remained common until relatively recent historical periods, before the implementation of modern sanitation systems (Hong et al., 2026).

Collectively, these studies demonstrate the long-standing association between humans and enteric microorganisms and illustrate how archaeological fecal material can be used to reconstruct both infectious disease burden and gut microbial ecology across different historical periods. When considered alongside evidence from ancient microbiome studies, these findings support the concept that progressive cultural transitions—including urbanization, changing dietary patterns, and improvements in hygiene—substantially altered the composition of the human gut microbiome, contributing to the gradual loss of ancestral microbial diversity and providing an evolutionary framework for understanding modern dysbiosis and microbiome-associated chronic diseases.

3.4 Dental calculus and oral microbiomes

Dental calculus is a mineralized oral biofilm formed through the progressive calcification of dental plaque by salivary minerals. Owing to its extensive mineralization, it represents one of the best-preserved biomolecular archives in the archaeological record, encapsulating microbial DNA, proteins, metabolites, food residues, starch granules, phytoliths, pollen and diverse pathogens over periods of months to years (Neves et al., 2026). Unlike coprolites, which primarily reflect short-term dietary intake and the intestinal microbiome, dental calculus preserves the long-term composition of the oral microbial ecosystem, providing unique insights into host–microbiome interactions across the lifespan (Warinner et al., 2015; Jacobson et al., 2020). Rather than representing an independent archive, dental calculus complements coprolites and mummified tissues by preserving a distinct microbial niche. Integrating these substrates enables reconstruction of oral and intestinal microbial ecosystems across different temporal and anatomical scales, providing substantially greater interpretative power than any single archaeological archive alone.

Metagenomic analyses have revealed that ancient dental calculus contains remarkably complex microbial communities composed of bacteria, archaea, fungi and viruses. Frequently identified oral commensals include Streptococcus, Actinomyces, Neisseria, Veillonella and Rothia, whereas pathogens associated with periodontal disease, including Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Streptococcus mutans, are consistently detected across diverse archaeological contexts. Viral DNA belonging to the Herpesviridae and Papillomaviridae families, together with numerous bacteriophages involved in regulating oral microbial communities, has also been identified, highlighting the exceptional capacity of dental calculus to preserve entire microbial ecosystems rather than isolated taxa (Huttenhower et al., 2012; Warinner et al., 2015). While early metagenomic investigations established dental calculus as an exceptional archive of ancient oral microorganisms (Warinner et al., 2015), subsequent studies progressively shifted toward ecological and functional interpretations of microbial communities. Building upon these observations, the study conducted by Jacobson et al. (2020) demonstrated that ancient dental calculus preserves ecologically structured microbial networks rather than merely taxonomic inventories, substantially improving our understanding of long-term host–microbiome interactions beyond simple species identification.

Beyond reconstructing oral ecology, dental calculus has emerged as a valuable source of information on ancient diet, mobility and infectious diseases. Preserved starch granules, phytoliths, pollen and plant-derived metabolites allow direct evidence of dietary practices and the use of medicinal plants, whereas microbial DNA can reveal broader aspects of population history (Warinner et al., 2015; Jacobson et al., 2020). For example, analyses of individuals from Oceania demonstrated that phylogenetic lineages of Tannerella forsythia exhibit clear geographic structure, suggesting that oral microorganisms may complement human genomic data in reconstructing past migration patterns (Velsko et al., 2024). In addition, the recovery of pathogen DNA, including Mycobacterium leprae, illustrates the potential of dental calculus to document long-term host–pathogen interactions and the evolutionary history of infectious diseases (Donoghue, 2013). Importantly, these applications extend the interpretative value of dental calculus beyond oral microbiology. Compared with coprolites, which predominantly reconstruct intestinal microbial ecology and recent dietary intake, dental calculus provides cumulative information on oral microbial succession, dietary exposure, mobility, and host–pathogen interactions, thereby offering complementary evidence for reconstructing ancient human biology.

Comparative metagenomic and pangenomic studies consistently indicate that the oral microbiome has undergone profound ecological restructuring during major cultural transitions. The adoption of agriculture and, subsequently, industrialization was accompanied by a progressive decline in microbial diversity and an increased prevalence of cariogenic and periodontopathogenic bacteria, supporting the hypothesis that dietary modernization reshaped not only the intestinal microbiome but also the oral microbial ecosystem (Jacobson et al., 2020). Recent pangenomic analyses have substantially expanded the evolutionary resolution previously achieved by metagenomic surveys, identifying previously undescribed Tannerella lineages preserved for up to 15,000 years. Compared with earlier taxonomic studies, these approaches enable reconstruction of microbial evolution, genome diversification, and the emergence of virulence-associated traits rather than simply documenting species presence (Galtier et al., 2026). Nevertheless, direct comparisons across archaeological cohorts remain challenging because differences in DNA preservation, sampling strategies, sequencing depth, and bioinformatic pipelines may influence taxonomic recovery and functional inference.

Evidence from ancient Egyptian mummies further supports the remarkable stability of key oral pathogens throughout human history. Metagenomic analyses identified microbial communities dominated by Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Fusobacteria and Spirochaetes, including the classical “Red Complex” bacteria—Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola—which remain strongly associated with periodontitis in modern populations. Their presence in individuals who lived more than two millennia ago suggests that oral dysbiosis and periodontal disease have deep evolutionary roots rather than representing exclusively modern conditions (Neukamm et al., 2020). Taken together, evidence derived from coprolites, dental calculus, and mummified tissues demonstrates that these archaeological archives provide complementary rather than redundant perspectives on ancient human microbiomes. Whereas coprolites primarily reconstruct intestinal microbial ecology and recent dietary intake, dental calculus records long-term oral microbial succession and cumulative dietary exposure, while mummified tissues preserve microorganisms within their anatomical context, enabling direct investigation of tissue-associated infections. The convergence of evidence across these distinct substrates strengthens confidence in evolutionary interpretations because similar ecological patterns emerge despite substantial differences in preservation, biomolecular degradation, DNA authentication, sequencing strategies, and analytical workflows. Consequently, integrating these complementary archives provides a considerably more robust reconstruction of ancient human microbiomes than reliance on any single substrate alone.

3.5 Mummified remains and intestinal contents

Mummified human remains, whether naturally or artificially preserved, provide a complementary source of evidence for reconstructing ancient human microbiomes. Unlike coprolites and dental calculus, mummified tissues enable the investigation of microorganisms associated with multiple anatomical sites, including the skin, respiratory tract, stomach and intestine, thereby offering a broader perspective on host–microbe interactions. Nevertheless, post-mortem degradation and taphonomic processes may alter DNA preservation and increase the risk of environmental contamination, requiring careful interpretation of molecular findings (Warinner et al., 2015).

One of the best-characterized examples is Ötzi, the approximately 5,300-year-old Tyrolean Iceman. Molecular analyses demonstrated that microbial DNA recovered from the skin and stomach largely reflected environmental contamination, whereas samples obtained from the colon preserved authentic signatures of the ancient intestinal microbiome based on 16S rRNA gene sequencing. Subsequent studies identified DNA belonging to Helicobacter pylori together with bacterial genera including Clostridium and Pseudomonas, demonstrating that both commensal microorganisms and opportunistic pathogens formed part of the human gut ecosystem during prehistory (Rollo et al., 2000; Lugli et al., 2017). Compared with earlier studies based primarily on targeted 16S rRNA sequencing, subsequent shotgun metagenomic analyses substantially increased the resolution of ancient microbiome reconstruction by enabling partial genome reconstruction, functional characterization, and assessment of virulence-associated genes. Nevertheless, the Ötzi investigations also demonstrated that data authenticity depends strongly on anatomical sampling location. Whereas intestinal samples preserved authentic endogenous microbial DNA, exposed tissues were considerably more susceptible to environmental contamination, emphasizing that tissue selection represents a critical determinant of data reliability in mummified remains (Lugli et al., 2017; Sarhan et al., 2026).

Comparable observations have been reported in pre-Columbian Andean mummies. Metagenomic analyses revealed microbial communities dominated by members of the Firmicutes, particularly the orders Clostridiales and Bacillales, together with representatives of the genus Bacteroides, one of the principal taxa of the modern human gut microbiome. The persistence of these microbial groups suggests that core components of the intestinal ecosystem have remained remarkably conserved throughout human history. In parallel, genetic sequences related to Trypanosoma cruzi and Leishmania donovani were identified, illustrating the capacity of mummified tissues to simultaneously document both commensal microbiota and infectious pathogens. Functional analyses further demonstrated the preservation of metabolic pathways involved in carbohydrate, amino acid and lipid metabolism, supporting the concept of long-term functional continuity in host–microbiome interactions despite substantial environmental and cultural change (Corthals et al., 2012; Santiago-Rodriguez et al., 2016).

The potential of mummified tissues extends beyond microbiome reconstruction to the evolutionary study of infectious diseases. Analyses of Egyptian mummies enabled the recovery of complete genomes of Mycobacterium leprae from bone tissue and hepatitis B virus (HBV) from soft tissues, providing direct molecular evidence of chronic infections in ancient populations while contributing to the reconstruction of pathogen evolution over millennia (Neukamm et al., 2020). Unlike earlier studies that mainly documented microbial community composition, Neukamm et al. demonstrated that rigorous ancient DNA authentication, damage-pattern analysis, negative controls, and pathogen genome reconstruction permit considerably greater confidence in distinguishing authentic ancient microorganisms from environmental contaminants (Neukamm et al., 2020). This methodological progress illustrates how advances in paleogenomic workflows have progressively improved the reliability and reproducibility of microbiome reconstruction from mummified tissues.

Collectively, evidence from coprolites, dental calculus, and mummified tissues demonstrates that these complementary archaeological archives provide a more comprehensive reconstruction of ancient human microbiomes than any single substrate alone. Although each archive is influenced by distinct preservation biases, contamination risks, and methodological limitations, the convergence of findings across multiple substrates strengthens confidence in current models of ancient human microbiome evolution. Future studies integrating standardized ancient DNA authentication and multi-omics approaches will further improve the reproducibility and interpretation of ancient microbiome reconstructions.

The major archaeological sources, representative microbial taxa, and their principal biological implications are summarized in Table 1.

Collectively, coprolites, dental calculus and mummified tissues provide complementary windows into the evolutionary history of the human microbiome. Although each archaeological archive captures distinct aspects of host–microbe interactions and presents unique preservation challenges, integrating evidence across these sources enables a more robust reconstruction of ancestral microbial ecosystems. To facilitate comparison across the heterogeneous paleomicrobiological literature, the principal studies included in this review are critically summarized in Table 2, highlighting their archaeological substrate, analytical methodology, principal findings, methodological limitations, and evolutionary contribution.

3.6 Methodological challenges and authentication of ancient microbiomes

Ancient microbiome research presents methodological challenges that distinguish it fundamentally from studies of contemporary microbial communities. Unlike modern biological specimens, archaeological materials are affected by post-mortem DNA degradation, environmental contamination, variable preservation conditions, and low endogenous microbial biomass, all of which may influence taxonomic identification and biological interpretation (Warinner et al., 2015; Bos et al., 2019). Consequently, authentication of ancient microbial DNA has become a central methodological requirement in paleomicrobiology.

The authenticity of ancient DNA is commonly evaluated using multiple complementary criteria rather than a single analytical approach. Characteristic DNA damage patterns, including cytosine deamination at fragment termini, short fragment length, low levels of modern contamination, and reproducibility across independent laboratories represent the principal indicators supporting the ancient origin of recovered microbial DNA (Burbano et al., 2010; Warinner et al., 2015). The routine inclusion of extraction blanks, negative controls, dedicated clean-room facilities, and contamination monitoring has substantially improved confidence in ancient DNA analyses compared with early PCR-based investigations (Bos et al., 2019; Nodari et al., 2021).

Nevertheless, contamination remains one of the principal limitations of ancient microbiome research. Archaeological specimens may acquire exogenous microorganisms from surrounding soil, excavation procedures, museum storage, laboratory processing, or modern human handling. This challenge is particularly important for low-biomass samples, in which contaminant DNA may represent a considerable proportion of recovered sequencing reads. Consequently, interpretation of microbial taxa identified in ancient specimens requires careful evaluation of archaeological context, preservation conditions, and authentication criteria rather than relying solely on taxonomic composition (Warinner et al., 2015; Grasso et al., 2024).

Methodological heterogeneity further complicates direct comparisons among published studies. Differences in DNA extraction protocols, sequencing platforms, reference databases, bioinformatic pipelines, taxonomic classification algorithms, and authentication thresholds may influence both microbial composition and inferred functional profiles. As a result, discrepancies observed among archaeological populations may partly reflect methodological variation rather than true biological differences (Bos et al., 2019; Nodari et al., 2021).

Despite these limitations, confidence in current evolutionary interpretations is strengthened by the convergence of independent evidence obtained from multiple archaeological substrates, including coprolites, dental calculus, and mummified tissues, analyzed using different analytical approaches. Across geographically and temporally distinct populations, these complementary archives consistently support broad ecological patterns, including progressive changes in microbial diversity, dietary adaptation, and host–microbiome interactions during major cultural transitions. Nevertheless, continued standardization of ancient DNA authentication criteria, bioinformatic workflows, and reporting practices will be essential to improve reproducibility and facilitate quantitative comparisons across future paleomicrobiological studies.

4 Evolution of the human microbiome through dietary and lifestyle transitions

4.1 Dietary transitions and microbial adaptation

The evolution of the human microbiome has been driven not only by changes in food composition but also by major cultural and technological innovations that fundamentally altered human ecology. Among the earliest of these was the controlled use of fire and the thermal processing of food, which increased the digestibility of proteins and starch, enhanced energy extraction, and reduced exposure to foodborne pathogens (Moles and Otaegui, 2020). These innovations modified the quantity and composition of dietary substrates reaching the intestine, thereby reshaping the selective pressures acting on gut microbial communities (Zhang and Hamaker, 2009; Tannock, 2024). Although the evolutionary consequences of cooking are widely accepted, direct archaeological evidence linking thermal food processing to specific alterations in ancient gut microbiomes remains limited. Current interpretations therefore rely largely on comparative physiological, nutritional, and microbiome studies rather than direct paleomicrobiological observations, highlighting an important gap in the archaeological record.

An even more profound ecological transition accompanied the emergence of agriculture. The shift from mobile hunter-gatherer societies to sedentary communities based on cereal cultivation and animal domestication provided a more reliable food supply but substantially reduced dietary diversity while increasing starch consumption (Moles and Otaegui, 2020; Tannock, 2024). Comparative studies of traditional and industrialized populations suggest that these dietary changes were accompanied by marked alterations in gut microbial composition. Non-industrialized populations typically exhibit a greater abundance of Prevotella and retain taxa such as Treponema, microorganisms specialized in the degradation of complex plant polysaccharides and largely absent from industrialized populations (Angelakis et al., 2018; Prasoodanan et al., 2021).

Agricultural practices further transformed host–microbiome interactions by increasing the availability of digestible carbohydrates through grain milling and cooking, thereby improving caloric yield and supporting demographic expansion. These environmental changes were paralleled by host genetic adaptations, including increased copy number variation of the AMY1 gene, which encodes salivary amylase and has been associated with long-term consumption of starch-rich diets. Such adaptations likely influenced the metabolic interplay between the host and its gut microbiota, illustrating the coevolution of diet, human genetics and microbial communities (Perry et al., 2007; Tannock, 2024). This relationship is likely bidirectional, with host genetic adaptation influencing microbial metabolism while microbial fermentation capacity may also have contributed to the selective pressures favoring increased starch digestion. However, direct evidence demonstrating coevolution between AMY1 copy number expansion and specific microbial taxa remains limited.

The establishment of permanent settlements also transformed the epidemiological landscape. Increasing population density, close contact with domesticated animals and environmental modification facilitated the transmission of infectious diseases and zoonotic pathogens, creating novel ecological interactions among humans, microorganisms and their surroundings (Venkatakrishnan et al., 2021; Antush et al., 2024; Tannock, 2024), these transitions progressively reshaped the human microbiome and laid the biological foundations for the more profound microbial alterations associated with subsequent urbanization and industrialization.

4.2 Industrialization and the emergence of dysbiosis

Although the transition to agriculture substantially altered the composition of the human gut microbiome, the most profound ecological changes have occurred during industrialization. Comparative studies consistently demonstrate that individuals living in industrialized societies harbor markedly less diverse gut microbial communities than traditional populations and even closely related non-human primates. Indeed, humans from Western industrialized societies appear to possess the least diverse gut microbiome among all primate species studied to date, a phenomenon described as microbial depauperation or microbial extinction (Moeller, 2017). Importantly, a recent meta-analysis of 14 studies including 568 individuals representing hunter-gatherer, agricultural, agropastoral, pastoral, and urban populations confirmed that microbiome composition changes progressively along a continuum of lifestyle transitions rather than reflecting a simple dichotomy between industrialized and non-industrialized societies. This finding strengthens the evidence that industrialization is associated with gradual ecological restructuring of the gut microbiome across human populations while simultaneously highlighting substantial inter-population variability (Rosas-Plaza et al., 2022).

This progressive loss of microbial diversity reflects the combined effects of multiple lifestyle changes rather than a single environmental factor. Reduced consumption of dietary fiber and microbiota-accessible carbohydrates (MACs), increased intake of ultra-processed foods, widespread antibiotic use, cesarean delivery, formula feeding during early life and improved sanitation have collectively diminished the vertical transmission and ecological persistence of microbial taxa that coevolved with humans over hundreds of thousands of years (Sonnenburg and Sonnenburg, 2019; Zhou et al., 2023). Nevertheless, the relative contribution of each of these factors remains difficult to determine because dietary habits, sanitation, antibiotic exposure, environmental microbial diversity, and medical interventions changed simultaneously during industrialization. Consequently, current evidence supports a multifactorial ecological model of dysbiosis rather than a direct causal relationship between any individual lifestyle factor and microbiome disruption (Rosas-Plaza et al., 2022; Sreenikitha et al., 2025).

Industrialization also fundamentally transformed human nutrition. From the seventeenth to the nineteenth centuries, expanding global trade and the Industrial Revolution promoted increasing consumption of refined sugar, coffee, tea and alcoholic beverages while progressively replacing traditional fiber-rich and fermented foods (Lewandowska-Pietruszka et al., 2022; Bobu et al., 2025a). These dietary changes altered not only the taxonomic composition of the gut microbiome but also its metabolic capacity, accelerating ecological shifts at a pace unprecedented in human evolutionary history (Lewandowska-Pietruszka et al., 2022).

Comparative analyses between humans and other primates further suggest that industrialization has resulted in the disappearance of microbial lineages maintained throughout hominin evolution. Members of the Bacteroidaceae, Bifidobacteriaceae and Lachnospiraceae families have become substantially less prevalent in industrialized populations, while studies of contemporary traditional societies have identified the so-called VANISH (Volatile and/or Associated Negatively with Industrialized Societies of Humans) taxa, microorganisms that remain common in non-industrialized communities but are rare or absent in Western populations (Moeller, 2017). Because many of these bacteria participate in the degradation of complex plant polysaccharides and the production of beneficial microbial metabolites, their disappearance likely represents not only a reduction in taxonomic diversity but also the erosion of ancestral microbial functions. These observations have given rise to the hypothesis that components of the ancestral human microbiome may already have been irreversibly lost, contributing to an evolutionary mismatch between the modern microbiome and the host physiology shaped during most of human evolution (Sonnenburg and Sonnenburg, 2019). However, the evolutionary significance of VANISH taxa remains an area of active investigation. Although their disappearance has frequently been interpreted as evidence of microbial extinction associated with industrialization, alternative hypotheses suggest that some ancestral functions may be retained through functionally redundant microorganisms or low-abundance taxa that are not consistently detected across populations. Therefore, functional loss should not be inferred solely from taxonomic depletion.

The widespread introduction of antibiotics during the twentieth century imposed an additional selective pressure on the human microbiome. Although these agents transformed the management of infectious diseases and substantially increased life expectancy, they frequently disrupt intestinal microbial ecosystems by reducing both taxonomic and functional diversity. Antibiotic exposure has been associated with rapid declines in alpha diversity and persistent alterations in microbial community structure that may remain detectable months after treatment cessation. Particularly affected are beneficial taxa such as Faecalibacterium prausnitzii, Roseburia spp., Eubacterium spp., Bifidobacterium spp., Ruminococcus spp. and Anaerostipes spp., organisms responsible for fermenting dietary fiber and producing short-chain fatty acids (SCFAs), which are essential for intestinal homeostasis (Fishbein et al., 2023; Habtemariam, 2026). Their depletion facilitates the expansion of opportunistic microorganisms, including members of the Enterobacteriaceae, Enterococcus spp. and Clostridioides difficile, thereby promoting dysbiosis and increasing susceptibility to inflammatory and metabolic disorders (Lathakumari et al., 2024; Ottria et al., 2026). Although antibiotic-induced reductions in microbial diversity are consistently reported, the magnitude and persistence of these alterations vary considerably among populations. Recent evidence indicates that baseline microbiome composition, geographical differences, dietary habits, strain-level diversity, antibiotic-resistance gene carriage, and the specific antibiotic class all influence microbiome resilience and recovery following treatment. Furthermore, most longitudinal studies have been conducted in high-income countries, whereas populations from low- and middle-income regions remain markedly underrepresented, limiting the generalizability of current evidence (Jaya and Brito, 2025). Consequently, although antibiotics are recognized as major ecological perturbators of the gut microbiome, their long-term contribution to dysbiosis should be interpreted within the broader context of industrialization-associated environmental and lifestyle changes rather than as an isolated causal factor.

Taken together, these findings indicate that industrialization has reshaped the human microbiome through the convergence of dietary, environmental, medical and societal changes rather than through any single selective pressure. Although declining microbial diversity represents one of the most consistent observations across comparative studies, the relative contribution of individual drivers remains difficult to disentangle because these ecological transitions occurred simultaneously. Future research should prioritize geographically diverse longitudinal cohorts integrating ancient microbiome data with metagenomics, metabolomics, and standardized antibiotic exposure data to improve causal inference and better characterize microbiome resilience across populations (Jaya and Brito, 2025). A schematic overview of these transitions is presented in Figure 3.

Figure 3

5 Ancient microbiomes and modern disease

Comparative analyses indicate that ancient populations and contemporary traditional communities harbored more diverse gut microbial ecosystems, including fiber-degrading taxa such as Prevotella and Treponema, which are now rare or absent in many Western industrialized populations (Levy, 2013). These observations suggest that ancestral microbiomes provide an evolutionary reference for understanding modern dysbiosis and its contribution to metabolic, inflammatory and immune-mediated diseases.

5.1 Evolutionary basis of modern dysbiosis

Evidence derived from archaeological microbiomes, comparative primate studies, and contemporary microbial ecology provides an evolutionary framework for understanding the increasing burden of chronic non-communicable diseases. Rather than demonstrating direct causal relationships, these complementary lines of evidence suggest that the human gut microbiome evolved under dietary and environmental conditions that differed profoundly from those characterizing modern industrialized societies. Consequently, the concept of host–microbiome mismatch has emerged to explain how rapid cultural and ecological transitions may have disrupted host–microbiome interactions that remained relatively stable throughout most of human evolution (Sonnenburg and Sonnenburg, 2019; Franck et al., 2022). Comparative analyses further indicate that the transition toward industrialized lifestyles was accompanied not only by changes in microbial composition but also by the progressive loss of evolutionarily conserved microbial diversity. Studies comparing humans with African apes demonstrated that modern industrialized populations harbor the least diverse gut microbiomes among extant hominids, suggesting that ecological changes associated with recent human evolution, subsequently amplified by industrialization, exceeded those expected from normal evolutionary divergence (Moeller et al., 2014; Moeller, 2017). However, these observations should be interpreted cautiously. Most available evidence is derived from cross-sectional comparative studies involving geographically distinct populations that differ simultaneously in diet, sanitation, environmental microbial exposure, antibiotic consumption, and socioeconomic conditions. Consequently, although reduced microbial diversity is one of the most reproducible findings across studies, the independent contribution of each ecological factor remains difficult to determine.

One of the principal biological mechanisms linking these evolutionary transitions to modern disease involves disruption of microbial metabolic function. Throughout most of human evolution, the gut microbiome coexisted with fiber-rich diets that supported microbial communities specialized in fermenting complex polysaccharides into SCFAs, particularly acetate, propionate, and butyrate. These metabolites play essential roles in maintaining intestinal barrier integrity, regulating immune responses, modulating epithelial energy metabolism, and preserving systemic metabolic homeostasis (Schnorr et al., 2016; Xie et al., 2025). Industrialization profoundly altered this ecological equilibrium through reduced consumption of dietary fiber and microbiota-accessible carbohydrates (MACs), increased intake of ultra-processed foods, widespread antibiotic exposure, and diminished environmental microbial diversity. These changes have consistently been associated with depletion of major SCFA-producing and immunomodulatory taxa, including Faecalibacterium prausnitzii, Roseburia spp., Eubacterium rectale, Ruminococcus bromii, Bifidobacterium spp., and Akkermansia muciniphila, taxa frequently regarded as hallmarks of the ongoing process of microbial extinction in industrialized populations (Xie et al., 2025; Mahanta et al., 2026). Nevertheless, SCFA depletion represents only one aspect of industrialization-associated dysbiosis. Multiple microbial functions and host–microbe interactions are disrupted simultaneously, suggesting that ecosystem dysfunction rather than depletion of individual taxa underlies metabolic disease.

Recent eco-evolutionary research has further expanded this conceptual framework by demonstrating that the gut microbiome should not be regarded as a static community but as a dynamic ecosystem undergoing continuous genetic adaptation within individual hosts. Experimental and computational studies indicate that microbial evolution and ecological interactions occur simultaneously, with bacterial adaptation influencing community composition while ecological changes create new selective pressures for microbial evolution (Dapa et al., 2023; Good and Rosenfeld, 2023). These findings suggest that dysbiosis represents a dynamic eco-evolutionary process rather than a fixed taxonomic state and may partly explain the considerable inter-individual variability observed in microbiome composition and therapeutic responses. Importantly, these findings also provide a biological rationale for personalized microbiome-based therapies while emphasizing that microbial communities continue to evolve following therapeutic intervention.

Despite growing evidence supporting this evolutionary framework, several limitations remain. Archaeological studies are constrained by preservation bias, limited sample size, and methodological heterogeneity, whereas contemporary microbiome research is characterized by substantial variation in population characteristics, sequencing methodologies, and analytical pipelines. Consequently, current evidence supports robust evolutionary associations but remains insufficient to establish definitive causal relationships between industrialization-associated microbiome alteration and chronic disease. Collectively, current evidence suggests that modern dysbiosis reflects disruption of an evolutionarily dynamic host–microbiome ecosystem rather than the simple loss of individual bacterial taxa. Integrating archaeological microbiomes with comparative evolutionary biology provides a biologically grounded framework for identifying conserved microbial functions that may become future targets for metabolic and immune-directed therapies. Although the evolutionary mismatch hypothesis provides a useful conceptual framework, it does not represent the only explanation for contemporary microbiome variation. Alternative perspectives propose that the human microbiome remains highly adaptable and continues to evolve in response to changing environmental conditions, suggesting that at least some microbiome alterations observed in industrialized populations may reflect ongoing ecological adaptation rather than exclusively maladaptive processes (Good and Rosenfeld, 2023; Dapa et al., 2023).

5.2 Metabolic diseases: obesity, type 2 diabetes and cardiovascular risk

Building upon the evolutionary framework outlined above, obesity, type 2 diabetes (T2D), and cardiovascular disease represent the clinical conditions most consistently associated with industrialization-related alterations of the gut microbiome.

Obesity provides one of the clearest examples of the interaction between evolutionary pressures and modern environmental exposures. Rather than resulting from a single evolutionary mechanism, contemporary obesity appears to arise from the convergence of dietary change, reduced physical activity, host genetics, immune adaptation, and microbiome evolution. Recent evolutionary models further suggest that the gut microbiome should be considered an integral component of obesity susceptibility, contributing to host metabolic flexibility through continuous interactions with immune and energy-regulatory pathways rather than acting as an isolated environmental factor (Saad and Santos, 2025). Consistent with this evolutionary framework, obesity is generally associated with reduced microbial diversity and depletion of SCFA-producing bacteria. However, no universal microbial signature has been reproduced across populations, reflecting substantial heterogeneity in dietary habits, ethnicity, sequencing methodologies, and environmental exposures (Xie et al., 2025; Mahanta et al., 2026). Similarly, considerable inter-population variability has been reported across human microbiome studies. Geographic location, ethnicity, habitual diet, medication use, socioeconomic factors, and environmental microbial exposure all contribute substantially to microbial composition, indicating that no single “healthy” ancestral microbiome can be universally defined (Al Bataineh et al., 2023; Xie et al., 2025).Similar observations have been reported in T2D. Mechanistic studies consistently implicate impaired SCFA production, altered bile acid metabolism, microbial endotoxins, and other microbiota-derived metabolites in insulin resistance and glucose dysregulation. However, these pathways interact with host metabolism through complex and interconnected mechanisms, making it unlikely that any single metabolite explains disease development (Mendis et al., 2025; Xie et al., 2025). This complexity is reflected in human studies, where differences in microbial composition are highly reproducible at the community level but considerably less consistent at the level of individual taxa.

Human studies further support an association between gut dysbiosis and T2D but remain heterogeneous. While case–control investigations have identified significant alterations in microbial composition (Yao et al., 2022; Razavi et al., 2024), multi-omics approaches integrating bacterial, fungal, metabolomic, and dietary data demonstrate that disease-associated microbial variation is strongly influenced by dietary habits and other environmental factors (Al Bataineh et al., 2023). Although these integrative approaches provide greater functional insight than taxonomic profiling alone, most studies remain limited by relatively small cohorts and cross-sectional designs, restricting causal inference (Al Bataineh et al., 2023; Razavi et al., 2024). Cardiovascular disease represents one of the most intensively investigated areas of microbiome research. Beyond traditional cardiovascular risk factors, gut dysbiosis has been associated with endothelial dysfunction, chronic vascular inflammation, accelerated atherosclerosis, and altered production of microbial metabolites, particularly trimethylamine N-oxide (TMAO), bile acids, and SCFAs (Cao et al., 2023). Nevertheless, the relative contribution of these pathways remains uncertain because microbial metabolite production is influenced by diet, renal function, medication use, host genetics, and considerable inter-individual variation in microbiome composition. Consequently, microbial metabolites should currently be regarded as promising biomarkers and therapeutic targets rather than established causal mediators of cardiovascular disease (Cao et al., 2023). These uncertainties are reflected in current therapeutic research, which increasingly focuses on microbiome-directed interventions aimed at restoring microbial function rather than individual bacterial taxa.

Likewise, early clinical studies suggest that dietary modulation may improve metabolic and cardiovascular parameters, but these findings remain preliminary because intervention trials generally involve small sample sizes, short follow-up periods, and heterogeneous treatment protocols (Pinel and Marette, 2025; Valencia et al., 2025).

Collectively, current evidence supports microbiome modulation as a promising adjunct to established therapies for obesity, T2D, and cardiovascular disease. Nevertheless, substantial heterogeneity in study populations, dietary assessment, sequencing methodologies, and intervention protocols continues to limit causal inference and clinical implementation. Future longitudinal multi-omics studies and adequately powered randomized clinical trials will be essential to identify reproducible microbial biomarkers and determine whether microbiome-directed therapies provide sustained cardiometabolic benefit beyond conventional treatment (Pinel and Marette, 2025; Valencia et al., 2025).

5.3 Inflammatory and immune-mediated diseases

Unlike metabolic disorders, which primarily arise from disrupted metabolic homeostasis, inflammatory and immune-mediated diseases may reflect the breakdown of long-standing evolutionary relationships between the human immune system and the microbiome. Throughout human evolution, immune maturation occurred in the presence of diverse microbial communities that promoted immune tolerance, maintained mucosal barrier integrity and balanced pro- and anti-inflammatory responses (Quan et al., 2023).

Industrialization has profoundly altered this ecological context. Reduced microbial diversity and the loss of evolutionarily conserved taxa may represent a form of evolutionary mismatch, whereby an immune system adapted to continuous microbial exposure functions within an increasingly microbe-depleted environment (Quan et al., 2023; Adawi, 2025). Consistent with this concept, the microbiome regulates immune homeostasis through the production of immunomodulatory metabolites and the induction of regulatory T cells, whereas disruption of these interactions promotes chronic inflammation and loss of immune tolerance (Fyhrquist, 2019; Zhao et al., 2023). Experimental and animal studies provide strong evidence that microbial metabolites and host–microbiome interactions contribute to immune regulation. However, translation of these findings to humans remains more challenging because most available clinical studies are observational and involve substantial heterogeneity in population characteristics, environmental exposures, microbiome composition, and analytical methodologies. Consequently, although impaired host–microbiome interactions are consistently associated with immune-mediated diseases, direct causal relationships remain difficult to establish (Quan et al., 2023; Zhao et al., 2023).

This evolutionary framework is further supported by the Old Friends Hypothesis, which proposes that the disappearance of microorganisms that coevolved with humans contributes to the rising incidence of Crohn’s disease, ulcerative colitis, asthma, atopic dermatitis and rheumatoid arthritis (Rook, 2013; Rook et al., 2014; Sonnenburg and Sonnenburg, 2019). Although the Old Friends Hypothesis represents one of the most influential evolutionary models of immune dysregulation, it should not be regarded as the sole explanation for the increasing incidence of immune-mediated diseases. Current evidence indicates that dietary modernization, antibiotic exposure, urbanization, reduced environmental biodiversity, and host genetic susceptibility probably interact with microbiome alterations to influence immune homeostasis. This multifactorial perspective is supported by the considerable inter-individual variability observed across immune-mediated disorders and explains why no single microbial signature has been consistently identified (Adawi, 2025; Quan et al., 2023). Nevertheless, competing hypotheses emphasize microbial resilience and ecological adaptation rather than irreversible microbial loss. According to these perspectives, microbial communities may retain considerable functional redundancy despite taxonomic turnover, allowing maintenance of essential metabolic and immunological functions even when community composition differs substantially among populations (Good and Rosenfeld, 2023; Dapa et al., 2023).Collectively, current evidence supports an association between industrialization-associated microbiome alteration and immune-mediated diseases, although direct causal mechanisms remain incompletely understood. Consequently, ancestral microbiomes should be regarded primarily as functional reference ecosystems that help identify conserved microbial pathways relevant for future preventive and therapeutic strategies rather than templates to be directly restored (Rook, 2013; Rook et al., 2014; Sonnenburg and Sonnenburg, 2019).

5.4 Evolutionary medicine perspective

From an evolutionary medicine perspective, the gut microbiome should be regarded as a dynamic biological ecosystem that has coevolved with humans over millions of years under relatively stable environmental and dietary conditions. In contrast, major cultural transitions—including agriculture, urbanization and, most recently, food industrialization—have occurred on a timescale far too short for equivalent biological adaptation, creating an evolutionary mismatch between the human host, its microbiota and the modern environment (Barone et al., 2019). Alt et al. estimate that hunter-gatherer lifestyles characterized approximately 99% of human evolutionary history, whereas agriculture emerged only around 12,000 years ago and industrialized diets represent an even more recent phenomenon. These transitions were accompanied by reduced dietary diversity, increased reliance on cereal-based foods and the emergence of novel disease patterns, including dental caries, malnutrition, infectious diseases and, ultimately, chronic non-communicable disorders (Alt et al., 2022; Assaf et al., 2024). Importantly, this evolutionary framework is supported not only by comparisons between contemporary industrialized and non-industrialized populations but also by independent archaeological evidence presented throughout this review. Findings from coprolites, dental calculus, and mummified remains consistently indicate that ancestral human microbiomes developed under conditions of greater dietary diversity, broader microbial functional capacity, and continuous environmental microbial exposure, thereby strengthening the evolutionary interpretation of modern dysbiosis (Appelt et al., 2016; Warinner et al., 2015; Corthals et al., 2012).

Experimental evidence further suggests that dietary changes may exert consequences extending beyond a single generation. In murine models colonized with human microbiota, long-term consumption of diets deficient in microbiota-accessible carbohydrates (MACs) progressively reduced microbial diversity and, after several generations, resulted in irreversible loss of bacterial taxa despite subsequent fiber reintroduction. These findings suggest that persistent nutritional changes may impair the vertical transmission of microbial functions and reduce ecological resilience. However, because this evidence derives primarily from experimental animal models, its direct translation to human populations remains uncertain and requires confirmation in long-term longitudinal studies (Li et al., 2025; Nguyen et al., 2026).

The concept of the evolutionary metabolome extends this framework by emphasizing that ancestral diets supplied a far broader repertoire of bioactive compounds than contemporary ultra-processed foods. Because diet represents the principal exogenous contributor to the human metabolome, the progressive reduction in phytochemicals, dietary fiber and structurally complex nutrients may have generated a metabolically impoverished environment for both the host and its microbiota. This reduction in metabolic diversity has been proposed as one mechanism contributing to obesity, type 2 diabetes, cardiovascular disease and age-related alterations in body composition (Zamboni et al., 2015; Assaf et al., 2024).

Importantly, evolutionary medicine also provides a framework for understanding why microbiome-based therapies may exhibit variable efficacy. The intestinal microbiome is not a static community but an evolving ecosystem capable of rapid adaptation through mutation, horizontal gene transfer and ecological competition. Consequently, probiotics, live biotherapeutic products and fecal microbiota transplantation should be considered dynamic ecological interventions rather than simple microbial replacement strategies. As highlighted by Leeflang et al., evolutionary processes occurring after administration may influence both therapeutic efficacy and safety, including microbial adaptation, gene exchange and long-term restructuring of the intestinal ecosystem (Leeflang et al., 2025).

Collectively, evolutionary medicine provides a valuable framework for interpreting the relationship between ancient microbiomes and modern disease, but it should not be viewed as evidence that restoration of ancestral microbial communities is either feasible or clinically desirable. Instead, paleomicrobiology helps identify conserved microbial functions and host–microbe interactions that may inform future preventive and therapeutic strategies. Although microbiome-directed interventions are increasingly investigated, robust evidence from adequately powered randomized clinical trials remains necessary before these concepts can be translated into routine clinical practice (Blaser, 2018; Leeflang et al., 2025).

6 Future perspectives and clinical implications

The translation of paleomicrobiological and evolutionary insights into clinical practice represents one of the most promising directions in microbiome research. Current evidence consistently supports the importance of diet in maintaining gut microbial function, and increasing consumption of diverse plant-derived fibers while limiting ultra-processed foods remains the microbiome-directed strategy best supported by clinical evidence (Gulliver et al., 2022). By contrast, the routine use of microbiome profiling, metagenomics, and metabolomics for disease prediction, prognosis, or treatment selection remains investigational despite considerable technological progress (Alt et al., 2022; Caesar, 2025; Tannock, 2024).

Growing interest has focused on microbiome-directed interventions, including personalized nutrition, prebiotics, postbiotics, next-generation probiotics, live biotherapeutic products, and fecal microbiota transplantation. Although encouraging results have been reported, substantial heterogeneity in patient populations, intervention protocols, outcome measures, and follow-up duration currently prevents these approaches from being considered standard clinical care. Consequently, most microbiome-directed interventions should presently be regarded as promising investigational strategies rather than established therapeutic options (Gulliver et al., 2022).

Pharmacological therapies are also increasingly recognized as modulators of the gut microbiome. Among these, glucagon-like peptide-1 receptor agonists (GLP-1 RAs), originally developed for obesity and type 2 diabetes, appear to exert effects extending beyond glycemic control. Experimental and clinical evidence indicates that liraglutide, semaglutide and tirzepatide may modify gut microbial composition, strengthen intestinal barrier integrity and promote microbial metabolites associated with improved metabolic health. Although these observations suggest that modulation of host–microbiome interactions may contribute to the metabolic benefits of GLP-1 receptor agonists, current evidence remains insufficient to establish this mechanism as a clinically validated therapeutic pathway because most available studies are experimental or observational (Bobu et al., 2025b; Brockmann et al., 2025; Alali and Shori, 2026; Dinkov and Pendicheva-Duhlenska, 2026; Singh et al., n.d).

Future research should prioritize integrating functional microbiome profiling with adequately powered randomized clinical trials to identify reproducible microbial biomarkers and validate microbiome-directed interventions across diverse populations. Until such evidence becomes available, evolutionary medicine should primarily be regarded as a framework for generating biologically informed therapeutic hypotheses rather than a source of clinically validated treatment strategies (Levy, 2013; Abavisani et al., 2025; Alali and Shori, 2026).

7 Conclusions

This review integrates archaeological microbiology, paleogenomics, microbial ecology, and evolutionary medicine to provide a comprehensive synthesis of how long-term host–microbiome interactions have changed across major dietary and cultural transitions. Rather than simply summarizing differences between ancestral and modern microbiomes, the review critically evaluates methodological challenges, competing evolutionary hypotheses, and the clinical relevance of conserved microbial functions, thereby providing an updated framework for interpreting modern dysbiosis.

Compared with ancestral and traditional populations, modern industrialized societies are characterized by reduced microbial diversity, the loss of evolutionarily conserved microbial taxa, and alterations in key metabolic functions involved in immune regulation, intestinal barrier maintenance, and energy homeostasis. These findings support the concept that part of the growing burden of metabolic, inflammatory, and immune-mediated diseases may reflect an evolutionary mismatch between a host shaped by long-term coevolution with diverse microbial communities and a rapidly changing modern environment.

Importantly, paleomicrobiological research suggests that the relevance of ancestral microbiomes lies not in reconstructing a prehistoric microbial state, but in identifying microbial functions that have been progressively lost during modernization and that may be important for human health. Integrating paleomicrobiology, microbiome science, evolutionary medicine, and systems biology offers a unique framework for understanding the origins of contemporary diseases and may guide the development of more effective preventive and therapeutic strategies.

As the field continues to evolve, ancient microbiomes are increasingly recognized not merely as historical records of past human populations, but as valuable biological archives capable of revealing fundamental principles governing host–microbe coevolution, resilience, and health across the human lifespan.

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