The coral microbiome in sickness, in health and in a changing world.

Stony corals, the engines and engineers of reef ecosystems, face unprecedented threats from anthropogenic environmental change. Corals are holobionts that comprise the cnidarian animal host and a diverse community of bacteria, archaea, viruses and eukaryotic microorganisms. Recent research shows that the bacterial microbiome has a pivotal role in coral biology. A healthy bacterial assemblage contributes to nutrient cycling and stress resilience, but pollution, overfishing and climate change can break down these symbiotic relationships, which results in disease, bleaching and, ultimately, coral death. Although progress has been made in characterizing the spatial-temporal diversity of bacteria, we are only beginning to appreciate their functional contribution. In this Review, we summarize the ecological and metabolic interactions between bacteria and other holobiont members, highlight the biotic and abiotic factors influencing the structure of bacterial communities and discuss the impact of climate change on these communities and their coral hosts. We emphasize how microbiome-based interventions can help to decipher key mechanisms underpinning coral health and promote reef resilience. Finally, we explore how recent technological developments may be harnessed to address some of the most pressing challenges in coral microbiology, providing a road map for future research in this field.

Genome reduction occurred in early Prochlorococcus with an unusually low effective population size.

In the oligotrophic sunlit ocean, the most abundant free-living planktonic bacterial lineages evolve convergently through genome reduction. The cyanobacterium Prochlorococcus responsible for 10% global oxygen production is a prominent example. The dominant theory known as "genome streamlining" posits that they have extremely large effective population sizes (Ne) such that selection for metabolic efficiency acts to drive genome reduction. Because genome reduction largely took place anciently, this theory builds on the assumption that their ancestors' Ne was similarly large. Constraining Ne for ancient ancestors is challenging because experimental measurements of extinct organisms are impossible and alternatively reconstructing ancestral Ne with phylogenetic models gives large uncertainties. Here, we develop a new strategy that leverages agent-based modeling to simulate the changes in the genome-wide ratio of radical to conservative nonsynonymous nucleotide substitution rate (dR/dC) in a possible range of Ne in ancestral populations. This proxy shows expected increases with decreases of Ne only when Ne falls to about 10 k - 100 k or lower, magnitudes characteristic of Ne of obligate endosymbiont species where drift drives genome reduction. Our simulations therefore strongly support a scenario where the primary force of Prochlorococcus genome reduction is drift rather than selection.

Contaminant occurrence, water quality criteria and tiered ecological risk assessment in water: A case study of antifouling biocides in the Qiantang River and its estuary, Eastern China.

Antifouling biocides may cause adverse effects on non-target species. This study aims to determine the distribution, sources, and ecological risks of antifouling biocides in the surface waters of the Qiantang River and its estuary in eastern China. The concentrations of total antifouling biocides were ranged from 12.9 to 215 ng/L for all water samples. Atrazine, diuron and tributyltin were the major compounds in the water bodies of the study area. The acute and chronic toxicity criteria for tributyltin, diuron and atrazine were derived for freshwater and saltwater, respectively, based on the species sensitivity distribution approach. The freshwater and saltwater criteria were slightly different, and the toxicity to aquatic organisms could be summarized as tributyltin > diuron > atrazine. The graded ecological risk rating showed that the long-term risk of TBT was significant in coastal waters. The pollution of TBT in the Qiantang River deserves further attention.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Ecological divergence of syntopic marine bacterial species is shaped by gene content and expression.

Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species' divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species' pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.

Determination of Perfluorooctane Sulfonyl Fluoride and Perfluorohexane Sulfonyl Fluoride in Soil by Chemical Derivatization and Liquid Chromatography-Tandem Mass Spectrometry.

Perfluorooctane sulfonyl fluoride (PFOSF) and perfluorohexane sulfonyl fluoride (PFHxSF) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and 2022, respectively. To date, their concentrations in environmental samples have not been reported due to the lack of sensitive methods. Herein, a novel chemical derivatization was developed for quantitative analysis of trace PFOSF and PFHxSF in soil by derivatizing them to the corresponding perfluoroalkane sulfinic acids. The method showed good linearity in the range from 25 to 500 ng L-1 with correlation coefficients (R2) better than 0.99. The detection limit of PFOSF in soil was 0.066 ng g-1 with recoveries in the range of 96-111%. Meanwhile, the detection limit of PFHxSF was 0.072 ng g-1 with recoveries in the range of 72-89%. Simultaneously, perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) were also detected accurately without being affected by the derivative reaction. By applying this method in an abandoned fluorochemical manufacturing facility, PFOSF and PFHxSF were successfully detected at concentrations ranging from 2.7 to 357 ng g-1 and 0.23 to 26 ng g-1 dry weight, respectively. It is very interesting that 2 years after factory relocation, there still exists high concentrations of PFOSF and PFHxSF, which is of concern.

Water quality criteria derivation and tiered ecological risk evaluation of antifouling biocides in marine environment.

This study provides a comprehensive compilation of published toxicological and environmental data further used to assess the ecological risks of six antifouling biocides, including tributyltin (TBT), Irgarol 1051, Diuron, Chlorothalonil, 4,5-Dichloro-N-octyl-3(2H)-isothiazolone (DCOIT), and Dichlofluanid. The standard maximum concentration and standard continuous concentration of antifouling biocides were derived by the species susceptibility distribution method. Following that, the ecological risk assessment of antifouling biocides in the aquatic environment was conducted using the hazard quotient, margin of safety, joint probability curve, and Monte Carlo random sampling method. The following is a concise list of the antifouling biocide dangers associated with acute and chronic risks: Irgarol 1051 > TBT > Diuron > DCOIT > Chlorothalonil > Dichlofluanid. It is strongly advised that systematic and ongoing monitoring of these biocides in coastal areas take place, as well as the creation of acceptable and efficient environmental protection measures, to safeguard the coastal environment's services and functions.

A Computational Protocol for Dating the Evolution of Cyanobacteria.

Cyanobacteria are known to play important roles in driving biological and geochemical innovations in ancient Earth. The origin of Cyanobacteria is the key to understanding these evolutionary events and thus has gained much interest to biologists and geobiologists. Recent development of the molecular dating approaches provides us an opportunity to assess the timeline of Cyanobacteria evolution based on relaxed clock models. The implementation of Bayesian phylogenetic approaches accommodates the uncertainties from different sources, such as fossil calibrations and topological structure of the phylogenomic tree, and provides us converged estimates of posterior mean ages. In this chapter, by taking Cyanobacteria as an example, we introduce a refined strategy to perform molecular dating analysis, as well as a practical method to evaluate the precision of dating analysis.

Estimating the Divergence Times of Alphaproteobacteria Based on Mitochondrial Endosymbiosis and Eukaryotic Fossils.

Alphaproteobacteria is one of the most abundant bacterial lineages that successfully colonize diverse marine and terrestrial environments on Earth. In addition, many alphaproteobacterial lineages have established close association with eukaryotes. This makes Alphaproteobacteria a promising system to test the link between the emergence of ecologically important bacteria and related geological events and the co-evolution between symbiotic bacteria and their hosts. Understanding the timescale of evolution of Alphaproteobacteria is key to testing these hypotheses, which is limited by the scarcity of bacterial fossils, however. Based on the mitochondrial endosymbiosis which posits that the mitochondrion originated from an alphaproteobacterial lineage, we propose a new strategy to estimate the divergence times of lineages within the Alphaproteobacteria by leveraging the fossil records of eukaryotes. In this chapter, we describe the workflow of the mitochondria-based method to date Alphaproteobacteria evolution by detailing the software, methods, and commands used for each step. Visualization of data and results is also described. We also provide related notes with background information and alternative options. All codes used to build this protocol are made available to the public, and we strive to make this protocol user-friendly in particular to microbiologists with limited practical skills in bioinformatics.

Assessing a Role of Genetic Drift for Deep-Time Evolutionary Events.

Effective population size (Ne) determines the amount of genetic diversity and the fate of genetic variants in a species and thus is an essential parameter in evolutionary genetics. There are standard approaches to determine the Ne of evolving species. For example, the long-term Ne of an extant species is calculated based on its unbiased global mutation rate and the neutral genetic diversity of the species. However, approaches for inferring Ne of ancestral lineages are less known. Here, we introduce an evolutionary genetic statistic and an analytical procedure to assess the efficiency of natural selection for deep nodes by calculating rates of nonsynonymous nucleotide substitutions leading to radical (dR) and conservative (dC) amino acid replacements, respectively. Given that radical variants are more likely to be deleterious than conservative ones, an elevated dR/dC ratio in gene families across the genome means an accelerated genome-wide accumulation of the more deleterious type of mutations (i.e., radical variants), which indicates that natural selection is less efficient and genetic drift becomes more powerful. Earlier approaches that calculate dR/dC do not consider the impact of nucleotide composition (G+C content) on the dR/dC result, which is partially accounted for in more recent methods. Here, we use these methods to demonstrate that genetic drift may have driven the early evolution of Prochlorococcus, the most abundant carbon-fixing photosynthetic bacteria in the ocean.

Phylogenomic Evidence for the Origin of Obligate Anaerobic Anammox Bacteria Around the Great Oxidation Event.

The anaerobic ammonium oxidation (anammox) bacteria can transform ammonium and nitrite to dinitrogen gas, and this obligate anaerobic process accounts for up to half of the global nitrogen loss in surface environments. Yet its origin and evolution, which may give important insights into the biogeochemistry of early Earth, remain enigmatic. Here, we performed a comprehensive phylogenomic and molecular clock analysis of anammox bacteria within the phylum Planctomycetes. After accommodating the uncertainties and factors influencing time estimates, which include implementing both a traditional cyanobacteria-based and a recently developed mitochondria-based molecular dating approach, we estimated a consistent origin of anammox bacteria at early Proterozoic and most likely around the so-called Great Oxidation Event (GOE; 2.32-2.5 Ga) which fundamentally changed global biogeochemical cycles. We further showed that during the origin of anammox bacteria, genes involved in oxidative stress adaptation, bioenergetics, and anammox granules formation were recruited, which might have contributed to their survival on an increasingly oxic Earth. Our findings suggest the rising levels of atmospheric oxygen, which made nitrite increasingly available, was a potential driving force for the emergence of anammox bacteria. This is one of the first studies that link the GOE to the evolution of obligate anaerobic bacteria.

Coastal Transient Niches Shape the Microdiversity Pattern of a Bacterioplankton Population with Reduced Genomes.

Globally dominant marine bacterioplankton lineages are often limited in metabolic versatility, owing to their extensive genome reductions, and thus cannot take advantage of transient nutrient patches. It is therefore perplexing how the nutrient-poor bulk seawater sustains the pelagic streamlined lineages, each containing numerous populations. Here, we sequenced the genomes of 33 isolates of the recently discovered CHUG lineage (~2.6 Mbp), which have some of the smallest genomes in the globally abundant Roseobacter group (commonly over 4 Mbp). These genome-reduced bacteria were isolated from a transient habitat: seawater surrounding the brown alga, Sargassum hemiphyllum. Population genomic analyses showed that: (i) these isolates, despite sharing identical 16S rRNA genes, were differentiated into several genetically isolated populations through successive speciation events; (ii) only the first speciation event led to the genetic separation of both core and accessory genomes; and (iii) populations resulting from this event are differentiated at many loci involved in carbon utilization and oxygen respiration, corroborated by BiOLOG phenotype microarray assays and oxygen uptake kinetics experiments, respectively. These differentiated traits match well with the dynamic nature of the macroalgal seawater, in which the quantity and quality of carbon sources and the concentration of oxygen likely vary spatially and temporally, though other habitats, like fresh organic aggregates, cannot be ruled out. Our study implies that transient habitats in the overall nutrient-poor ocean can shape the microdiversity and population structure of genome-reduced bacterioplankton lineages. IMPORTANCE Prokaryotic species, defined with operational thresholds, such as 95% of the whole-genome average nucleotide identity (ANI) or 98.7% similarity of the 16S rRNA gene sequences, commonly contain extensive fine-grained diversity in both the core genome and the accessory genome. However, the ways in which this genomic microdiversity and its associated phenotypic microdiversity are organized and structured is poorly understood, which disconnects microbial diversity and ecosystem functioning. Population genomic approaches that allow this question to be addressed are commonly applied to cultured species because linkages between different loci are necessary but are missing from metagenome-assembled genomes. In the past, these approaches were only applied to easily cultivable bacteria and archaea, which, nevertheless, are often not representative of natural communities. Here, we focus on the recently discovered cluster, CHUG, which are representative in marine bacterioplankton communities and possess some of the smallest genomes in the globally dominant marine Roseobacter group. Despite being over 95% ANI and identical in the 16S rRNA gene, the 33 CHUG genomes we analyzed have undergone multiple speciation events, with the first split event predominantly structuring the genomic diversity. The observed pattern of genomic microdiversity correlates with CHUG members' differential utilization of carbon sources and differential ability to explore low-oxygen niches. The available data are consistent with the idea that brown algae may be home to CHUG, though other habitats, such as fresh organic aggregates, are also possible.

Prochlorococcus have low global mutation rate and small effective population size.

Prochlorococcus are the most abundant free-living photosynthetic carbon-fixing organisms in the ocean. Prochlorococcus show small genome sizes, low genomic G+C content, reduced DNA repair gene pool and fast evolutionary rates, which are typical features of endosymbiotic bacteria. Nevertheless, their evolutionary mechanisms are believed to be different. Evolution of endosymbiotic bacteria is dominated by genetic drift owing to repeated population bottlenecks, whereas Prochlorococcus are postulated to have extremely large effective population sizes (Ne) and thus drift has rarely been considered. However, accurately extrapolating Ne requires measuring an unbiased global mutation rate through mutation accumulation, which is challenging for Prochlorococcus. Here, we managed this experiment over 1,065 days using Prochlorococcus marinus AS9601, sequenced genomes of 141 mutant lines and determined its mutation rate to be 3.50 × 10-10 per site per generation. Extrapolating Ne additionally requires identifying population boundaries, which we defined using PopCOGenT and over 400 genomes related to AS9601. Accordingly, we calculated its Ne to be 1.68 × 107, which is only reasonably greater than that of endosymbiotic bacteria but surprisingly smaller than that of many free-living bacteria extrapolated using the same approach. Our results therefore suggest that genetic drift is a key driver of Prochlorococcus evolution.

Cryptic niche differentiation of novel sediment ecotypes of Ruegeria pomeroyi correlates with nitrate respiration.

Marine intertidal sediments fluctuate in redox conditions and nutrient availability, and they are also known as an important sink of nitrogen mainly through denitrification, yet how denitrifying bacteria adapt to this dynamic habitat remains largely untapped. Here, we investigated novel intertidal benthic ecotypes of the model pelagic marine bacterium Ruegeria pomeroyi DSS-3 with a population genomic approach. While differing by only 1.3% at the 16S rRNA gene level, members of the intertidal benthic ecotypes are complete denitrifiers whereas the pelagic ecotype representative (DSS-3) is a partial denitrifier lacking a nitrate reductase. The intertidal benthic ecotypes are further differentiated by using non-homologous nitrate reductases and a different set of genes that allow alleviating oxidative stress and acquiring organic substrates. In the presence of nitrate, the two ecotypes showed contrasting growth patterns under initial oxygen concentrations at 1 vol% versus 7 vol% and supplemented with different carbon sources abundant in intertidal sediments. Collectively, this combination of evidence indicates that there are cryptic niches in coastal intertidal sediments that support divergent evolution of denitrifying bacteria. This knowledge will in turn help understand how these benthic environments operate to effectively remove nitrogen.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes.

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Snowball Earth, population bottleneck and Prochlorococcus evolution.

Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean. A massive DNA loss event occurred in their early evolutionary history, leading to highly reduced genomes in nearly all lineages, as well as enhanced efficiency in both nutrient uptake and light absorption. The environmental landscape that shaped this ancient genome reduction, however, remained unknown. Through careful molecular clock analyses, we established that this Prochlorococcus genome reduction occurred during the Neoproterozoic Snowball Earth climate catastrophe. The lethally low temperature and exceedingly dim light during the Snowball Earth event would have inhibited Prochlorococcus growth and proliferation, and caused severe population bottlenecks. These bottlenecks are recorded as an excess of deleterious mutations accumulated across genomic regions and inherited by descendant lineages. Prochlorococcus adaptation to extreme environmental conditions during Snowball Earth intervals can be inferred by tracing the evolutionary paths of genes that encode key metabolic potential. Key metabolic innovation includes modified lipopolysaccharide structure, strengthened peptidoglycan biosynthesis, the replacement of a sophisticated circadian clock with an hourglass-like mechanism that resets daily for dim light adaption and the adoption of ammonia diffusion as an efficient membrane transporter-independent mode of nitrogen acquisition. In this way, the Neoproterozoic Snowball Earth event may have altered the physiological characters of Prochlorococcus, shaping their ecologically vital role as the most abundant primary producers in the modern oceans.

Mechanisms driving genome reduction of a novel Roseobacter lineage.

Members of the marine Roseobacter group are key players in the global carbon and sulfur cycles. While over 300 species have been described, only 2% possess reduced genomes (mostly 3-3.5 Mbp) compared to an average roseobacter (>4 Mbp). These taxonomic minorities are phylogenetically diverse but form a Pelagic Roseobacter Cluster (PRC) at the genome content level. Here, we cultivated eight isolates constituting a novel Roseobacter lineage which we named 'CHUG'. Metagenomic and metatranscriptomic read recruitment analyses showed that CHUG members are globally distributed and active in marine pelagic environments. CHUG members possess some of the smallest genomes (~2.6 Mb) among all known roseobacters, but they do not exhibit canonical features of typical bacterioplankton lineages theorized to have undergone genome streamlining processes, like higher coding density, fewer paralogues and rarer pseudogenes. While CHUG members form a genome content cluster with traditional PRC members, they show important differences. Unlike other PRC members, neither the relative abundances of CHUG members nor their relative gene expression levels are correlated with chlorophyll a concentration across the global samples. CHUG members cannot utilize most phytoplankton-derived metabolites or synthesize vitamin B12, a key metabolite mediating the roseobacter-phytoplankton interactions. This combination of features is evidence for the hypothesis that CHUG members may have evolved a free-living lifestyle decoupled from phytoplankton. This ecological transition was accompanied by the loss of signature genes involved in roseobacter-phytoplankton symbiosis, suggesting that relaxation of purifying selection owing to lifestyle shift is likely an important driver of genome reduction in CHUG.

Dating Alphaproteobacteria evolution with eukaryotic fossils.

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

Evolutionary origin and ecological implication of a unique nif island in free-living Bradyrhizobium lineages.

The alphaproteobacterial genus Bradyrhizobium has been best known as N2-fixing members that nodulate legumes, supported by the nif and nod gene clusters. Recent environmental surveys show that Bradyrhizobium represents one of the most abundant free-living bacterial lineages in the world's soils. However, our understanding of Bradyrhizobium comes largely from symbiotic members, biasing the current knowledge of their ecology and evolution. Here, we report the genomes of 88 Bradyrhizobium strains derived from diverse soil samples, including both nif-carrying and non-nif-carrying free-living (nod free) members. Phylogenomic analyses of these and 252 publicly available Bradyrhizobium genomes indicate that nif-carrying free-living members independently evolved from symbiotic ancestors (carrying both nif and nod) multiple times. Intriguingly, the nif phylogeny shows that the vast majority of nif-carrying free-living members comprise an independent cluster, indicating that horizontal gene transfer promotes nif expansion among the free-living Bradyrhizobium. Comparative genomics analysis identifies that the nif genes found in free-living Bradyrhizobium are located on a unique genomic island of ~50 kb equipped with genes potentially involved in coping with oxygen tension. We further analyze amplicon sequencing data to show that Bradyrhizobium members presumably carrying this nif island are widespread in a variety of environments. Given the dominance of Bradyrhizobium in world's soils, our findings have implications for global nitrogen cycles and agricultural research.

Population differentiation of Rhodobacteraceae along with coral compartments.

Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.