Microbial Ecology

Stony coral tissue loss disease (SCTLD), a coral pathology with rapid tissue loss and high mortality rate has affected nearly 30 species with a variable degree of susceptibility across species. It has been suggested that SCTLD has a systemic effect within coral colonies, but little is known about within-colony changes of the microbial communities associated with this disease. Here we evaluate the microbial shifts within coral colonies: apparently healthy tissue and SCTLD tissue. The study was done in three species following a gradient of susceptibility to the disease: Dendrogyra cylindrus (Dcyl, n = 11) and Pseudodiploria strigosa (Pstr, n = 6) two highly susceptible species; and Orbicella faveolata (Ofav, n = 8), a moderately affected species. 16S rRNA Illumina sequences analysis showed differential microbial community structure within two species (Dcyl, p = 0.01, Pstr, p = 0.01) but not for Ofav (p = 0.28). Taxonomic profiles of bacterial groups were species-specific in SCTLD tissue, but healthy tissue shared similarities between species including Pirelullales, NB1-J and SAR324. Our results reveal that the microbial communities effects associated to the disease follow a similar pattern to the species susceptibility to SCTLD, providing new insights into the disease dynamics in the Mexican Caribbean.

Thermal stress can disturb microbial communities associated with host species. As microbes can support essential functions related to host metabolism, physiology, nutrition and immunity, changes in microbial communities can have severe fitness consequences for the host. Although the effects of thermal conditions on host-associated microbiomes have been demonstrated in controlled laboratory settings, how climate change might affect the structure and functionality of microbial communities in wild populations remain poorly understood. Here, we took advantage of the well-characterized long-term field survey of the Glanville fritillary butterfly (Melitaea cinxia) metapopulation on the [A]land islands, in the Baltic Sea, to fill this gap. We investigated whether bacterial communities associated with larvae show signs of gradual temporal change in response to slow environmental warming across a 28-year period, or whether these communities responded through abrupt change following a sudden drought event that triggered bottlenecks in their butterfly host population. Using a combination of 16S rRNA metabarcoding and metagenomic sequencing, we first showed that M. cinxia harbours a set of stable resident bacteria, including Pseudomonas, Telluria, and Enterobacteriaceae bacteria. But we also characterized a gradual shift in the M. cinxia associated bacterial community over three decades of increasing temperatures and decreasing precipitations. This shift was not unidirectional for all bacterial taxa, as the dominant Telluria and Pseudomonas showed opposing responses to environmental trends. Additionally, the 2018 extreme drought, which triggered acute host population bottlenecks, was associated with a severe disruption of M. cinxia microbiota, and the loss of key Enterobacteriaceae taxa. However, the M. cinxia bacterial community seemed to be able to recover towards pre-drought structure in subsequent years, suggesting a degree of resilience to acute climatic perturbations in this microbial system.

AO_SCPLOWBSTRACTC_SCPLOWUnderstudied bacterial genera present a dynamic phylogenetic landscape and opportunities for discovering new taxa as more strains are isolated and genomic data is added. Here, through phylogenomic analysis, we describe three novel species of the globally distributed cosmopolitan marine bacterial genus Microbulbifer. This genus is ubiquitous in saltwater microbiomes and is a validated source of biodegradation enzymes as well as high value small molecule natural products. Average nucleotide identity (ANI) to the closest known species, Microbulbifer variabilis ATCC 700307T, was less than 88.4% for all three novel species. Isolates of the three novel species, designated as PAAF003T (T = type strain), ZKSA006T, and SSSA003T were imaged to reveal their phormological characteristics. Based on phylogenetic data, strains PAAF003T, ZKSA006T, and SSSA003T represent three new species of the genus Microbulbifer, for which the names Microbulbifer maximicatervae sp. nov., Microbulbifer regidiadema sp. nov., and Microbulbifer mixtoriginis sp. nov. are proposed, respectively, under the SeqCode. We also reconstructed a robust phylogeny of available Microbulbifer genomes, which should faciliatate future isolation and strain description studies.

Bees and wasps are ecologically vital, but many species are declining due to anthropogenic stressors. Social bees harbour host-specific and dense gut microbiomes that affect their resilience to stress. However, there are tens of thousands of other bee and wasp species that vary in sociality and diet (including pollen-feeding and predatory guilds), traits known to influence host-microbe symbioses. The role of gut microbes in the biology of these species is largely unknown. Here, we measured the composition and absolute abundance of bacterial communities in adult abdomens across 61 genera and 14 families of field-collected bees, predatory wasps, and pollen wasps. We found that solitary bees and both wasp guilds harbor distinct bacterial taxa and lower bacterial abundances as compared with social bees. Bacterial abundances also varied extensively among and within genera of solitary bees, with little variation explained by body size, diet breadth, or nesting ecology. Further, microbiome composition was only weakly differentiated among solitary bees and the two wasp groups, even comparing herbivorous (pollen-feeding) and carnivorous taxa. We suggest that the sparse and somewhat stochastic microbiomes of solitary bees and wasps reflect weak host dependence on microbially mediated functions, a trait that may influence their responses to environmental change.

Heatwaves can affect species abundances by changing how species interact with one another in local communities. These effects can be complex and remain poorly understood, especially in cases where the outcome of species interactions is contingent on the history of species arrival. We studied how heatwaves affect interactions between the yeast Metschnikowia reukaufii and the bacterium Acinetobacter nectaris, both commonly found in the floral nectar of Diplacus aurantiacus, a hummingbird-pollinated shrub native to California. The microbes were introduced to artificial nectar in different orders of arrival in the presence or absence of simulated two-day heatwaves. We found that heatwaves made yeast-bacterium interactions more contingent on arrival history, thereby causing large variation in nectar acidity, a factor known to affect hummingbird preference and seed production. In the absence of heatwaves, Acinetobacter always became abundant regardless of arrival history, suppressing Metschnikowia and reducing nectar pH. In contrast, in the presence of heatwaves, Acinetobacter dominance depended on arrival history and heatwave timing. If Acinetobacter arrived after Metschnikowia during a heatwave, Metschnikowia suppressed Acinetobacter substantially enough to keep nectar pH at a high level. These results suggest that heatwaves shift species interactions from determinism to historical contingency, with both taxonomic and functional consequences.

Lyme disease is a tick-borne illness caused by the spirochaete bacterium Borrelia (Borreliella) burgdorferi. The black-legged tick Ixodes scapularis, which transmits B. burgdorferi and several other human pathogens, is endemic to the eastern United States and, due to climate change, is rapidly expanding into central and eastern Canada. Amplification and sequencing of bacterial DNA from I. scapularis is increasingly used to monitor the presence and abundance of B. burgdorferi and associated bacteria. However, variation in the nature of molecular data collected across studies presents challenges for analysis and interpretation. Here we use full-length Oxford Nanopore 16S ribosomal RNA gene amplicon sequencing to characterize the microbiome of I. scapularis, with an explicit focus on distinguishing between tick-adapted bacteria (endosymbionts and pathogens) and environmentally acquired bacteria (external sources, including soil, vegetation or vertebrate hosts). We show that environmental dominance strength differs between these two ecological classes of bacteria, and that environmental dominance does not appear to represent stochastic background alone; environmentally derived bacterial taxa detected in tick microbiomes are not mere contaminants. Paired soil microbiome profiling from tick collection sites will be required to test whether environmental dominance and associated co-occurrence structure track with seasonal changes in exposure and environmental microbial populations.

Nitrogen-fixing symbioses, particularly those occurring in root nodules, are among the most consequential mutualisms in natural and agricultural systems and represent a globally important source of bioavailable nitrogen. Despite their importance, patterns of diversity and composition among diazotrophic symbionts--and the processes structuring those patterns in natural systems--remain poorly resolved, with competing hypotheses emphasizing ecological, or phylogenetic constraints on host-symbiont associations. Here, using a broad survey of nodulating plants from the southeastern United States, we examine how diazotrophic symbiont communities vary across host plant phylogeny, habitat context, and geographic origin. We find that host phylogeny is the primary determinant of symbiont composition, outweighing effects of fine-scale taxonomic identity. Symbiont associations are therefore structured mainly at deeper phylogenetic levels, consistent with phylogenetically constrained, or "fuzzy," host specificity. Likewise, nodule community diversity--potentially reflecting variation in host control over infection--differs primarily among higher-level clades rather than among closely related taxa. Habitat context also shapes nodule communities, but its influence is secondary and most evident in undisturbed environments. As well, nonnative legumes harbor distinct symbiont assemblages despite occupying similar habitats, whereas distantly related legume clades share symbionts across habitats, highlighting interactions among phylogeny, ecology, and geographic history. Overall, our results show that host phylogeny exerts the strongest influence on nodule microbial communities, likely reflecting evolutionary divergence in symbiotic function across major host lineages.

Invasive alien species (IAS) represent a major threat to biodiversity, human health, and economy. Yet the role of host-associated microbiomes in invasion processes remains poorly understood in natural systems. Here, we investigated the bacterial and fungal microbiomes of IAS and native Drosophila species collected along an anthropization gradient in French Guiana. Analyses of Drosophila communities revealed that IAS establishment is limited outside coastal habitats. Using bacterial 16S rRNA and fungal ITS metabarcoding of pooled individuals, we assessed microbial diversity, composition, and structure across host species, host category (invasive vs. native), and locality. Bacterial communities associated with IAS harboured reduced alpha diversity and distinct community structure compared to native Drosophila species, and host phylogenetic relatedness and locality also contributed to bacteriome variation. In contrast, fungal communities were mainly structured by locality and host species. We further identified core and exclusive taxa, compared bacteriome composition between native and IAS hosts using global reference datasets, and integrated information from the microbiome literature. This approach led to the identification of 45 bacterial and fungal candidate taxa potentially associated with host adaptation or competitiveness across coastal and rainforest environments. Overall, our results highlight microbiome variation as a potential component of invasion dynamics in Neotropical Drosophila.

Increasing global droughts exert large but poorly understood effects on the microbial communities and ecology of soil. Microbial communities generally show resilience and return to pre-drought conditions when short-term droughted soils are rewet; soils exposed to long-term drought, however, often show a lag upon rewetting, after which microbial communities may or may not return to their pre-stressed conditions. Though short-term droughts have been widely studied, long-term drought manipulation experiments remain rare, especially those that compare microbial response to short-term and long-term drought in tandem. We conducted a 1000-day drought simulation in controlled laboratory conditions with soil cores collected from a tidal freshwater ecosystem in Washington state, USA, and subsequently exposed them to rewetting for two weeks. We also included short-term (30-day and 90-day) drought and rewet treatments to directly compare microbial community and organic matter responses across drought durations. We found distinct microbial taxa belonging to Firmicutes and Actinobacteria enriched after the 1000-day drought, but not after the short-term droughts. While we hypothesized that the microbial community would recover from a short-term drought after rewetting to resemble pre-drought conditions, our results revealed community dissimilarities between rewet and pre-drought conditions across all drought durations. These findings suggest unique microbial life history strategies within certain microbial phyla that make them successful colonizers during an extended drought period, and the influence of environmental and physiological context on microbial responses to rewetting. ImportanceDroughts are increasing in frequency and intensity globally with severe implications for ecosystem services and soil functions. It is important to understand how long-term drought impacts soil microbial communities and organic matter chemistry to better predict future ecosystem responses to sustained moisture deficit conditions. We subjected soils to short-term (30 and 90 days) and long-term (1000 days) drought treatments and subsequently rewetted them to understand microbiome recovery to pre-drought conditions. Our results showed that prolonged drought drastically changes the microbial community and soil organic matter profile compared to short-term drought. While we expected the soil microbiome to recover upon rewetting after short-term drought, our results showed an altered microbiome composition, compared to pre-drought conditions, for both short-and long-term drought, suggesting microbial responses to soil rewetting was independent of drought duration imposed. These results provide important insights into soil biological and chemical functions that remain sensitive to change under fluctuating soil moisture conditions and future drought scenarios.

Animal microbiomes are shaped by both environmental exposure and host-associated filtering, but the relative importance of these processes remains poorly understood. Dung-associated insects provide an ideal model because they develop and feed in highly dynamic microbial environments. We investigated the gut microbiomes of six sympatric dung fly species of the genus Sepsis (Diptera: Sepsidae) and compared them with microbial communities in cow dung throughout a growing season in Switzerland. Using full-length 16S rRNA gene sequencing (PacBio), we characterized bacterial communities from 74 fly and 15 dung samples. Seasonal variation was the strongest predictor of microbiome composition, whereas host species exerted weaker effects that persisted after removing dung-associated taxa, indicating that gut communities are not merely passive reflections of environmental exposure. Only few gut microbiome reads were attributable to dung-associated taxa, and environmental overlap differed among fly species rather than season. A highly non-random core microbiome persisted across all six species: 36 bacterial genera (of 469) were shared by all hosts at [~]119-fold enrichment above random expectation and remained after removing dung-associated taxa. These findings support a two-layer model of microbiome assembly, in which seasonal environmental variation determines microbial availability while host-specific processes selectively retain a subset of taxa.

The critically endangered Caribbean staghorn coral Acropora cervicornis hosts microbiomes frequently dominated by the putatively parasitic intracellular bacterium Candidatus Aquirickettsia rohweri, which is associated with reduced coral growth and heightened disease susceptibility. Whether this dominance can be disrupted through antibiotic treatment and a sequential disturbance of thermal stress, remains unknown. In this study, we exposed disease-susceptible A. cervicornis fragments to broad-spectrum antibiotics, sub-bleaching thermal stress, or the combination of an antibiotic pre-treatment followed by thermal stress, and tracked changes in microbiome composition and diversity across all experimental phases using 16S rRNA amplicon sequencing and quantitative PCR (qPCR). We find that while the minor microbial fraction exhibits sustained compositional shifts in response to treatment, Ca. Aquirickettsia rohweri is resilient to antibiotic and thermal perturbation and may in fact increase in abundance following antibiotic exposure, suggesting that its dominance is actively maintained and not readily displaced by current disease mitigation strategies.These results indicate that antibiotic intervention is unlikely to be a viable strategy for disrupting Ca. A. rohweri dominance in disease-susceptible A. cervicornis, underscoring the urgency of understanding its transmission routes to inform microbiome rescue efforts.

Microbial succession can be used to estimate the postmortem interval (PMI); however, the impact of spatial variability within the cadaver decomposition island (CDI) is not well understood. This study examined spatial variation in necrobiome communities where soil samples were collected over time and across spatial locations from the CDIs of two human body donors. Microbial communities were characterized using 16S rRNA sequencing and statistical modelling of variation and PMI were conducted. Necrobiome community metrics showed no significant differences across anatomical sampling sites within the CDI at a single timepoint. Temporal modelling identified 11 taxa with significant relationships to PMI in one donor, with spatial sampling having a minimal impact on the PMI relationships. Non-linear approaches also identified taxa with likely PMI signals in the second donor. These findings demonstrate that opportunistic sampling can capture robust linear and non-linear PMI signals in later decomposition stages.

Animals harbor diverse gut microorganisms that influence host health and fitness. Synthetic microbial communities have been instrumental in enabling reductionist studies of host-microbiome interactions, but some questions require microbial communities with more natural-like complexity while preserving experimental tractability, in vivo monitoring, and quantitative analysis. Here, we describe a method optimized for longitudinal studies of host-microbiome-environment interactions in the nematode Caenorhabditis elegans. In this approach, complex microbial extracts (CMEs) are generated from environmental samples and applied to worm culture plates, providing a diverse yet experimentally convenient microbial environment. We show that CME composition remains stable during cold storage, enabling reproducible longitudinal experiments while minimizing confounding environmental drift over time. As a proof of principle, we apply this method to examine age-dependent changes in the worm gut microbiome, providing support for previous reports of age-dependent increase in the abundance of gut Enterobacteriaceae. CMEs provide a practical and reproducible framework that complements experiments using monocultures or synthetic communities, enabling longitudinal studies of host-microbiome interactions under conditions that better approximate natural microbial complexity.

Bacteriophages are abundant and diverse in soils, playing a major role in regulating bacterial communities and consequently affecting biogeochemical cycles. Such host-phage interactions may be influenced by fluctuations in soil moisture, as observed in wetlands soils which constitute a key feature of the ongoing climate change. Here, we investigated the spatial and temporal dynamics of both bacteria and T4-type bacteriophage community structures and diversities in soil of a freshwater wetland. Soil was sampled in three sites across a proximal soil transect presenting an increase moisture content at seven dates over an 18 months period with contrasted flooding periods. DNA was extracted and we applied amplicon sequencing of the bacterial 16S rRNA gene and viral g23 gene. Bacterial community composition varied across the proximal soil transect, with Methylomirabilia and Gammaproteobacteria being significantly enriched in the wettest site and comprising ASVs affiliated to methanotroph and denitrifying bacteria, respectively. We identified a large diversity of T4-type phages, among which a fraction was novel, while others were similar to phages previously sequenced from various biomes. These findings suggest that T4-type phages are capable of successfully colonizing diverse niches in the biosphere, contributing to their ubiquity and diversity. Viral community was however dominated by few vASVs, which were highly represented in one or two of the three studied sites supporting the Bank model. All together our results indicate that T4-type phages have broad host ranges and more likely follow bacterial population dynamics. The present study provides new insights into the role of phages in soil, highlighting their interactions with bacterial hosts involved in carbon and nitrogen cycles, interactions that are likely regulated by fluctuations in soil moisture, as observed in wetlands. HighlightsO_LIBoth bacterial and T4-type phages were structured across proximal sites C_LIO_LIBacterial 16S rRNA gene copy number was inversely correlated to the soil moisture C_LIO_LI26 viral ASVs did not cluster with reference sequences C_LIO_LIviral ASVs seem to be primarily controlled by host availability C_LIO_LISoil bacteria and phage diversities were significantly lower in the wettest site C_LI

Megasphaera elsdenii is best known as a prominent lactate consumer within the rumen microbial community in livestock, and its metabolic properties are relatively well studied. In humans, it can be isolated from healthy donors feces and, more often, from patients feces with diverse inflammatory conditions. It is currently unclear whether human and animal gut isolates are genetically related and perform the same metabolic function. In this study, we compared 86 M. elsdenii genomes from human feces (as a proxy for the human gut) to animal gut isolates and contrasted both with phylotypes colonizing the vaginal niche. Phylogenetic analysis showed that human gut samples form a genetically homogeneous branch with animal gut isolates, with samples intermingled and no evident host-specific clustering. Human gut samples shared most of their genes and biochemical pathways with isolates from swine and cattle, despite differences in their digestive tracts. When human and animal samples were labeled, supervised analysis detected only minor differences in pathway content. Genome-scale metabolic modeling suggests that human and animal gut samples likely share identical carbon and energy source requirements. Moreover, the requirements for lactate and acetate were conserved across all studied samples, regardless of the host or niche. Finally, we found no virulence genes, and lactate utilization remains a plausible explanation for M. elsdenii accumulation in different niches. IMPORTANCEMegasphaera elsdenii is considered a commensal in the human gut and animal rumen. However, M. elsdenii tends to be more abundant in patients feces with diverse inflammatory conditions. As we know little about strain diversity and biology in human isolates, comparison with better-studied isolates can be informative. In this study, we compared M. elsdenii genomes from the human gut to those in better-studied isolates from ruminal and non-ruminal animals. Human-derived samples from patients and healthy donors were genetically very similar to animal isolates and may have shared a common origin. We found that human and animal samples have a similar genomic makeup and metabolic potential, and that neither group harbors virulence genes. We hypothesize that M. elsdenii is a benign commensal that grows in response to lactate accumulation in the inflamed gut.

Microorganisms, found in every environment, play an important role in recycling matter and providing energy to the ecosystem. Here, we used 18S and 16S metabarcoding to study bacteria, cyanobacteria and protist community diversity and functional ecology among different environments of typical lake, river, marine and soil from the Yangtze Delta of China. The results showed that the similarity of cyanobacteria and protozoa communities in soil and aquatic environments was both higher than that of bacteria and microalgae communities. While the diversity of cyanobacteria and bacteria in lake was higher than that in river, marine and soil, the diversity of microalgae and protozoa in river was higher than that in lake, marine and soil. The {beta} diversity of cyanobacteria and bacteria marked differences in lake from other environments. But The distribution of dominant families of cyanobacteria, bacteria, microalgae and protozoa is similar to diversity. While significant positive correlations were found among dominant species of cyanobacteria, protozoa, bacteria and microalgae in lake the dominant species among bacteria, microalgae and protozoa in river, marine and soil all showed more negative correlations. Our study provides the basis for understanding the functional ecology of microbes in the micro-food webs of different environments.

Soils are recognized as reservoirs of antibiotic resistance genes (ARGs) with the potential to transfer to clinical pathogens, creating antimicrobial resistance (AMR) that poses a threat to human health. While large-scale AMR surveys have profiled how diverse biomes shape soil resistomes, less is known about the influence of specific soil properties. Here, we combined metagenomics and 16S rRNA amplicon sequencing with isolate-based approaches to investigate drivers of soil AMR across a tropical watershed from beach to mountaintop in Waimea Valley, Oahu, Hawai{square}i. We leveraged functional- and taxonomic-classification of resistances to unravel how soil properties interact with bacterial taxa to structure resistomes. Metagenomic- and isolate-resistomes showed remarkable consistency, including a general gradient of increasing AMR from ridge to beach. Resistome functional composition was significantly correlated with total bacterial community structure. The relationship between resistances and soil properties was primarily dictated by taxonomic composition of each resistance. Rifampin- and Vancomycin-ARGs associated with Actinomycetes negatively correlated with soil physical properties, while resistant genes and isolates from Gammaproteobacteria positively correlated with enzymatic activity metrics. These findings indicate that soil properties structure the resistome indirectly through taxonomic filtering of microbial hosts and challenge the notion that AMR is decoupled from phylogenetic relatedness.

Resilient turbid coral reefs, found 1{degrees} north of the equator, experience fewer and less intense bleaching events despite being situated within the worlds busiest shipping port in highly urbanised Singapore. We hypothesised that bacteria within the coral holobiont play a role in maintaining coral diversity within this extreme environment by conferring traits that enhance host tolerance. Eleven Pseudovibrio isolates, whose genomes differ by only four SNPs, were isolated from the scleractinian coral Pachyseris speciosa. A [~]490 kbp megaplasmid (pCJH) was found in 7 of the 11 Pseudovibrio isolates. This study identified an opportunistic Pseudovibrio sp. pathogen of P. speciosa, accelerating bleaching disease. However, presence of the megaplasmid alters the ecological strategy of Pseudovibrio sp. toward mutualism, delaying coral bleaching. The megaplasmid enhances Pseudovibrios host colonisation and establishment of symbiosis through increased attachment and extends its bioactive genetic potential, but reduces fecundity. The Pseudovibrio genomes and megaplasmid encode several diffusible antibiotic biosynthetic gene clusters and contact-dependent inhibition mechanisms, with both types of inhibitory activity shown against local (i.e. P. speciosa) and type-strain Vibrio spp. Interaction analyses in experimentally heat-stressed corals revealed negative associations between Pseudovibrio and Vibrio ASVs corresponding to these cultured isolates. They also showed increased coral thermal tolerance by a full degree (1{degrees}C) when it is associated with the megaplasmid-bearing strain. Together, these findings support the Coral Probiotic Hypothesis that bacteria enhance coral resilience through chemical defense and identifies additional aspects to this symbiosis by a mobile genetic element which could play an important role in coral reef resilience.

Bacteria inhabiting leaf surfaces - the phyllosphere - are crucial to plant health and ecosystem functioning. Methylobacterium is a taxonomically diverse, growth-promoting genus, ubiquitous on leaves. Different plant species host distinct Methylobacterium communities, but the genomic and functional basis of Methylobacterium symbiotic associations with particular host species remains poorly understood. Here, we used a metapangenomic approach to quantify the influence of host species on Methylobacterium assemblages, to identify genes potentially involved in Methylobacterium adaptations to host species, and to evaluate the contribution of Methylobacteriums accessory pangenome to these adaptations. We sequenced the metagenomes of 25 phyllosphere communities spanning five host species in a temperate forest in Quebec, Canada, and mapped these metagenomes onto Methylobacteriums pangenome to obtain nucleotide-level coverage and composition for each population on each individual host. We revealed strong divergences in the species- and gene-level community structure of Methylobacterium, driven by host phylogeny and plant form. Conifer communities were notably enriched in genes involved in amino acid, lipid, and carbohydrate metabolism; broadleaves, in genes involved in cell membrane, signalling, defense, and chemotaxis; and trees, in genes related to photosynthesis, oxidative phosphorylation, and translation. The shrub Corylus cornuta was a reservoir of Methylobacterium taxonomic diversity, and harboured numerous accessory genes under positive selection. Methylobacteriums accessory pangenome, evolving under weaker purifying selection, contributed importantly to gene-host associations, supporting its adaptive role. By linking genes to phyllosphere niches, our study shed light on the genetic basis of host adaptation and highlighted the crucial role of forest biodiversity in shaping microbial ecology and evolution.

Host-associated microbiomes play vital roles in organismal health, ecological interactions, and evolution, yet little is known about how microbial communities shift during the transition from aquatic to increasingly terrestrial habitats. Here, we characterize the skin microbiomes of three combtooth blenny species (Blenniella paula, Praealticus labrovittatus, and Alticus arnoldorum) that occupy distinct positions along the intertidal gradient in Guam--from fully subtidal (and exclusively aquatic) to intertidal (amphibious) and supratidal environments (exclusively terrestrial). Using 16S rRNA amplicon sequencing, we compared skin-associated bacterial communities with those in surrounding seawater and substrate biofilms to assess habitat influences on microbiome structure. Skin microbiomes were distinct from environmental microbial communities, indicating strong ecological filtering by the host. The divergence between skin and substrate microbiomes in the three species parallels their distribution along progressively higher zones of the intertidal gradient. The most divergent skin microbiome was that of the supratidal fish A. arnoldorum, characterized by higher Gammaproteobacteria abundance and enrichment of epiphytic and mucus-associated taxa. Across all species, we identified 32 microbial orders significantly enriched on the skin relative to environmental samples, including taxa commonly associated with fish mucosa (e.g., Vibrio, Alteromonas, Cetobacterium) and others rarely reported in aquatic marine fish (e.g., Rubritalea, Granulosicoccus). Several rare taxa with potential pathogenicity were also detected at low abundances. Together, these findings suggest that habitat-specific selective pressures strongly shape fish skin microbiomes along subtidal (aquatic) to supratidal (terrestrial) habitats and suggest that microbial symbionts may contribute to the ecological and physiological adaptations enabling amphibious lifestyles. This study provides the first comparative assessment of skin microbiome divergence across amphibious fish species along an intertidal gradient and offers a framework for predicting microbiome responses to environmental change.