Biology subjects

Hugenholtz, P.

Publications and source records attributed to Hugenholtz, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Nation-scale recovery of complete environmental plasmids pushes the boundaries of plasmid diversity through long-read sequencing

Plasmids are important agents of prokaryotic evolution and yet existing reference databases remain biased towards cultivable species, limiting our knowledge of their function and ecology in the environment. Here we present a major expansion of known plasmid diversity, through curation and annotation of 8,744 topologically complete plasmids from 9,972 short- and 154 long-read metagenomes as part of the Microflora Danica project. These were found to increase plasmid taxonomic unit diversity by 15% and 99% relative to all and environmental plasmids, respectively, from public sources. In particular, we identify a novel lineage of plasmids in the genus Methanosarcina, encoding genes associated with methanogenesis and cell surface properties, expanding our understanding of chromosome-plasmid complementarity and its potential contribution to host biology. Combining our plasmids with public sources, we curated a set of 17 replication-associated proteins that could be found in 82% of known plasmids and can serve as hallmarks for future detection of plasmids. We present this resource, and a tool for confirming topological completeness of plasmid assemblies, as a toolkit for future plasmid discovery.

microbiology↗

bioRxiv2026

Development of lyophilized faecal inoculation capsules for use in koala rehabilitation and conservation

As a specialist herbivore, koalas rely on their gut microbiomes to help digest their toxic and fibrous diet of Eucalyptus leaves. Without these critical microbes, koalas may not be able to obtain the nutrients and energy they need to survive. As such, a large proportion of koalas that undergo rehabilitation for chlamydiosis develop gut dysbiosis from the antibiotic treatment and are either euthanized or die. Here we aimed to modify previously developed fresh faecal inoculation capsules for an extended shelf-life such that they could be applied in a clinical setting to prevent or treat gut dysbiosis. Using 16S rRNA gene amplicon sequencing we demonstrated that air-drying faecal material leads to an overgrowth of facultative anaerobes, whereas lyophilised material retains a similar microbial composition to fresh material. Initial survival of koala faecal microbes as assessed by live/dead staining combined with microscopy was high after lyophilisation regardless of which excipient was used, except for 20% glycerol that resulted in ~15% lower survival than other treatments. Lyophilised fine particles extracted from koala faeces and packaged into acid-resistant capsules maintained their original microbial composition and had high microbial survival over a year, regardless of the excipient used. Dry-fill, single-layer capsules maintained integrity after 10 hrs in synthetic koala stomach acid. These capsules may be useful in adapting the gut microbiomes of koalas to novel diets e.g. during translocations. However, attempts to apply the capsules in the clinical setting were unsuccessful due to unanticipated difficulties in administrating the capsules to sick koalas.

microbiology↗

bioRxiv2026

A self-amplifying microbial-abiotic sulfur relay drives field feasible bauxite residue remediation

Bauxite residue (BR), the haloalkaline byproduct of alumina refining, represents the largest and most costly environmental challenge facing the global aluminium industry, yet sustainable remediation has remained elusive because no rapid and field-feasible technology can overcome its recalcitrant alkalinity. Here, we establish a self-amplifying microbial-abiotic sulfur relay that drives rapid in situ acid generation and sustained dealkalization of BR across laboratory and glasshouse experiments and a field trial, where dealkalized residue subsequently supported spontaneous pioneer-plant colonization. Mechanistic assays and multi-omics analyses show that the relay is initiated by microbial reduction of elemental sulfur (S8) to HS- under oxygen-limited conditions. The resulting HS- abiotically attacks and solubilizes solid S_8, generating a mobile pool of polysulfides (Sx2-). In anoxic microsites, polysulfide reduction regenerates HS^-, which mobilizes additional S8 and amplifies sulfur turnover by increasing sulfur mobilization and bioavailability. In oxic microsites, Sx(2-) are abiotically converted to thiosulfate and reactive S0, which are subsequently microbially oxidized to sulfate and acidity. By coupling biotic reductive initiation and regeneration with abiotic sulfur mobilization and oxidation, followed by biotic terminal oxidation, this relay overcomes the low bioavailability of S8 and the constraints of extreme haloalkaline conditions, providing a low-cost, field-feasible strategy for efficient and sustained BR remediation.

microbiology↗

bioRxiv2026

No-take marine reserves promote oligotrophic reef bacterioplankton communities across the Great Barrier Reef

Australias Great Barrier Reef is a biodiversity hotspot critical to ocean health, yet it faces increasing threats from climate change and localised impacts requiring effective conservation and management action. Rezoning of the Great Barrier Reef Marine Park in 2004 expanded No-Take Marine Reserves (NTMRs) to restrict extractive activities like fishing and collecting, creating one of the largest networks of marine reserves globally. Benefits like increased biomass of fisheries-targeted species and improved coral community health metrics have been reported, though the effects of zoning on water chemistry and seawater microbiology remain unexplored. Using data from the Great Barrier Reef Microbial Genomics Database, we investigated the structure of seawater microbiomes on 48 offshore reefs within NTMRs and fished reefs. A supervised classification method (MINT sPLS-DA) identified 350 indicator species that predict zoning with [~]71% accuracy (range 58-85%). Microbial communities broadly reflected reef states, with NTMR zones enriched in streamlined microbial oligotrophs (Pelagibacter and SAR86) correlating with higher cover of hard coral, crustose coralline algae, and herbivore fish abundance under lower nutrient conditions. By contrast, fished reefs harbored opportunists (Flavobacteriales, especially UA16, and Pseudomonadales) associating with elevated nutrients and turf algae cover. Co-occurrence networks revealed stronger competitive interactions in fished reefs, where nutrient-responsive taxa may outcompete other microbes, underscoring the need to investigate how these shifts influence reef nutrient cycling and function. Our findings reveal ecosystem-wide effects of marine zoning beyond fish protection, with distinct seawater microbiomes between fished reefs and NTMRs, which will help build decision tools for more targeted reef health monitoring assessments.

microbiology↗

bioRxiv2026

The MiDAS global genome catalog: 53,501 long-read MAGs representing all core prokaryotic genera in the global activated sludge microbiome

Wastewater treatment relies on complex microbial communities, yet existing genome-resolved references for this essential engineered ecosystem remain dominated by short-read assemblies, limiting genome contiguity and linkage between taxonomic and metabolic function. We applied long-read sequencing to activated sludge from 83 globally distributed plants, reconstructing 53,501 metagenome-assembled genomes to establish the Microbial Database of Activated Sludge (MiDAS) global genome catalog. The catalog encompasses high-quality genomes for 12,047 prokaryotic species, 82% of which are not represented in GTDB release 226, and provides a median of 32 high-quality genomes for each of the 250 core prokaryotic genera previously defined in our MiDAS global 16S rRNA gene survey. This enables analyses of predicted functional traits and their ecological context, for example, we identified two sparsely represented Nitrospiraceae genera with conserved nitrite-oxidation genes that are abundant in higher-temperature wastewater treatment plants. In summary, the MiDAS genome catalog provides a framework for linking taxonomy, metabolism and ecological roles in wastewater treatment systems globally.

microbiology↗

bioRxiv2026

Models trained with noisy genomes extend bacterial phenotype prediction into deep time

Predicting phenotype from genotype in extant organisms is increasingly tractable through the accumulation of genome sequences and the development of machine-learning algorithms. Here we show that machine learning can be applied to reconstructed ancestral gene content, extending these predictions into the past. We trained models on a diverse set of bacterial phenotypes and found that introducing noise into gene content profiles allows predictions to generalize over larger evolutionary distances. For phenotypes with signal spread across many genes - such as metabolic oxygen use, cell envelope architecture and optimal growth temperature - noise augmentation extends resolution back to the root of the bacterial domain, while for other phenotypes - including GC content and sporulation - the range remains more limited. We therefore conclude that the last bacterial common ancestor (LBCA) was likely an anaerobic, double-membraned, and moderately thermophilic bacterium (46-75{degrees}C). Moreover, this work provides a general approach for learning about the genomic basis of phenotypes and drawing inferences about their early evolution.

evolutionary biology↗

bioRxiv2026

gTranslate: rapid and accurate translation table prediction for prokaryotic genomes

BackgroundBioinformatic tools often require the prediction of protein-coding genes to make inferences about prokaryotic genomes. Typically, the genetic code used for translating genes to proteins must be specified by the user based on the taxonomic classification of a genome assembly or, for some widely used tools, established using a heuristic rule based on gene coding densities. Manual specification is at best inconvenient, but more challenging is that many bioinformatic tools are applied before taxonomic classifications have been established making specifying the translation table impractical. MethodsHere we provide a computationally efficient tool, gTranslate, that uses an ensemble of five machine learning methods to accurately predict translation tables for prokaryotic genomes. The feature vector used by gTranslate takes advantage of differences in gene coding densities when predicting genes under different translation tables along with features that consider the number and ratio of UGA stop codon reassignments to tryptophan or glycine. ResultsWe demonstrate that gTranslate correctly predicts the translation table of prokaryotic genomes >99.99% of the time (i.e. <1 error per 10,000 genomes) and outperforms a more computationally expensive prediction method and a coding density heuristic used by popular bioinformatic tools. Using gTranslate, we identify a basal lineage of Ca. Stammera capleta that uses the standard bacterial genetic code instead of the UGA stop codon to tryptophan reassignment common to other members of this species. We also identify the first instances of UGA-to-tryptophan reassignment in the Patescibacteriota making this the first bacterial phylum with members capable of using translation tables 4, 11, and 25.

bioinformatics↗

bioRxiv2025

First report of stop codon reassignment to tryptophan in members of the bacterial phylum Actinomycetota

Reassignment of stop codons is a significant evolutionary event with recoding of UGA to tryptophan being previously identified in only three bacterial phyla, the Bacillota, Pseudomonadota, and Verrucomicrobiota. Here, we present genomic evidence of this recoding in a fourth bacterial phylum, the Actinomycetota, specifically in the family Eggerthellaceae. We identify the UGA stop-to-tryptophan reassignment in 34 metagenome-assembled genomes recovered from the stool samples of diverse mammalian hosts, including equids and primates. Canonical markers for this reassignment are consistently observed including conserved UGA codons aligning to tryptophan, loss of release factor 2 (prfB), and presence of a tRNATrp(UCA) gene. We infer that this reassignment occurred at least twice as the lineages containing recoded genomes are paraphyletic, forming two distinct groups separated by a third lineage with strains that continue to use UGA as a stop codon. These lineages represent three new Eggerthellaceae genera for which we propose the type species Equivita altericodex, Gorillivita intestinalis, and Tapirivita inops reflecting isolation source and genomic properties. Genomes representing these genera, including the non-recoded Tapirivita lineage, have reduced genomes and complete or partial loss of biosynthetic pathways, suggesting a transition to obligate symbiosis. Increasing host dependency may have facilitated stop codon reassignment in Equivita and Gorillivita species. This work expands the known phylogenetic diversity of UGA stop-to-tryptophan reassignment in the bacterial domain and establishes the Eggerthellaceae as a new focal point for understanding the evolutionary drivers of genetic code plasticity.

evolutionary biology↗

bioRxiv2025

A molecular inventory of the faecal microbiomes of 23 marsupial species

Despite the recent expansion of culture-independent analyses of animal faecal microbiomes, many lineages remain understudied. Marsupials represent one such group, where despite their iconic status, direct sequencing-based analyses remain limited. Here we present a metagenomic and metabolomic exploration of the faecal microbiomes of 23 Diprotodontia marsupials, producing a reference set of 3,868 prokaryotic and 12,142 viral metagenome-assembled genomes, the majority (>80%) of which represent novel species. As with other animals, host phylogeny is the primary driver of microbiome composition, including distinct profiles for two eucalyptus folivore specialists (koalas and southern greater gliders), suggesting independent solutions to this challenging diet. Expansion of several bacterial and viral lineages were observed in these and other marsupial hosts that may provide adaptive benefits. Antimicrobial resistance genes were significantly more prevalent in captive than wild animals likely reflecting human interaction. This molecular dataset contributes to our ongoing understanding of animal faecal microbiomes. Impact statementDespite their ecological and evolutionary importance, marsupials remain underrepresented in microbiome research. Here, we present the most extensive faecal microbiome dataset to date for this group, encompassing metagenomic, metabolomic, and proximity ligation data from 23 marsupial species. As in other animals, we find the microbial community structure reflects the host species, and some marsupials carry expanded sets of certain microbial lineages indicative of within-host evolution. This work substantially expands the genomic landscape of host-associated microbes and viruses in a poorly studied mammalian clade. Data summaryRaw read data, prokaryotic MAGs [≥]50% complete with [≤]10% contamination are available via the European Nucleotide Archive under project PRJEB89408. The full set of viral genomes, clustered protein database and metabolite data (raw and processed) are available via https://doi.org/10.48610/14e37e9. Prokaryotic MAGs are also available via https://figshare.com/s/87443d80817f57aadc16.

microbiology↗

bioRxiv2025

The planktonic microbiome of the Great Barrier Reef

Large genome databases have markedly improved our understanding of marine microorganisms. Although these resources have focused on prokaryotes, genomes from many dominant marine lineages, such as Pelagibacter and Prochlorococcus, are conspicuously underrepresented. Here, we present the Great Barrier Reef Microbial Genomes Database (GBR-MGD) comprising 5,283 prokaryotic genomes obtained from GBR seawater samples using Nanopore sequencing, including a collection of high quality genomes of underrepresented groups. We show that standard short read assemblies miss these populations due to a combination of strain heterogeneity and low GC% sequencing bias. The GBR-MGD also comprises 20 chromosome-level picoeukaryote and 808,585 viral genomes, including a newly described clade of marine Crassvirales. We demonstrate the use of the GBR-MGD to identify indicator taxa that can reliably predict the effects of reef management practices, such as the establishment of marine protected zones.

genomics↗

bioRxiv2024

Recovery of highly contiguous genomes from complex terrestrial habitats reveals over 15,000 novel prokaryotic species and expands characterization of soil and sediment microbial communities

Genomes are fundamental to understanding microbial ecology and evolution. The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the microbial genome catalogue of soils and sediments has been challenging due to the enormous complexity of these environments. Here, we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected across Denmark and through an optimised bioinformatics pipeline, we recovered genomes of 15,314 novel microbial species, including 4,757 high-quality genomes. The recovered microbial genomes span 1,086 novel genera and provide the first high-quality reference genomes for 612 previously known genera, expanding the phylogenetic diversity of the prokaryotic tree of life by 8 %. The long-read assemblies also enabled the recovery of thousands of complete rRNA operons, biosynthetic gene clusters and CRISPR-Cas systems, all of which were underrepresented and highly fragmented in previous terrestrial genome catalogues. Furthermore, the incorporation of the recovered MAGs into public genome databases significantly improved species-level classification rates for soil and sediment metagenomic datasets, thereby enhancing terrestrial microbiome characterization. With this study, we demonstrate that long-read sequencing and optimised bioinformatics, allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain the largest untapped source of biodiversity for expanding genome databases and filling in the gaps of the tree of life.

bioinformatics↗

bioRxiv2024

Metagenomic analysis of marsupial gut microbiomes provides a genetic basis for the low methane economy

The potent greenhouse gas methane is an end-product of plant biomass digestion by gut microbiota, though the amount produced and/or released varies among herbivorous animals. On a per unit of feed basis, macropodid marsupials (e.g. kangaroos) are widely thought to be low methane-emitting herbivores compared to high methane-producing ruminant livestock. How the gut microbiome contributes to the low methane status of marsupials is not well understood but of high potential value for a low methane economy. Here, we analyse the faecal metagenomes of 14 different marsupial species and 1,394 derived metagenome-assembled genomes (MAGs), focusing on the functional distinction of the bacterial and archaeal communities compared to ruminant faecal microbiomes. Though composition and function of the marsupial gut microbiome considerably varied across and within animal species, there was a clear host-associated bacterial signature for the community that differed significantly between marsupial hosts and compared to ruminants. Of particular note was a range of Bacteroidota, Campylobacterota, Desulfobacterota, Pseudomonadota and Verrucomicrobiota species that were enriched in marsupials and encode H2-uptake hydrogenases that mediate hydrogenotrophic respiration. Additionally, in support of an enrichment of electron sinks, enzymes for butyrate, propionate, and glutamate production, as well as nitrate, nitrite, and fumarate respiration were enriched in marsupials. Collectively, these data suggest that, by favoring an enrichment of alternate hydrogen sinks of bacterial origin, the low methane phenotype reported for marsupials is feasible and offers a genetic basis to pursue reductions of livestock methane emissions.

microbiology↗

bioRxiv2024

Proposal of Patescibacterium danicum gen. nov., sp. nov. in the ubiquitous ultrasmall bacterial phylum Patescibacteriota phyl. nov.

Candidatus Patescibacteria is a diverse bacterial phylum that is notable for members with ultrasmall cell size, reduced genomes, limited metabolic capabilities and dependence on other prokaryotic hosts. Despite the prevalence of the name Ca. Patescibacteria in the scientific literature, it is not officially recognized under the International Code of Nomenclature of Prokaryotes (ICNP) and lacks a nomenclatural type. Here, we rectify this situation by describing two closely related circular metagenome-assembled genomes (MAGs) and by proposing one of them (ABY1TS) to serve as the nomenclatural type for the species Patescibacterium danicumTS gen. nov., sp. nov. according to the rules of the SeqCode. Rank-normalized phylogenomic inference confirmed the stable placement of P. danicumTS in the class ABY1 within Ca. Patescibacteria. Based on these results, we propose Patescibacterium gen. nov. to serve as the type genus for associated higher taxa, including the phylum Patescibacteriota phyl. nov. We complement our proposal with a genomic characterization, metabolic reconstruction, and biogeographical analysis of Patescibacterium. Our results confirm small genome sizes (< 1Mbp), low GC content (>36%), and the occurrence of long gene coding insertions in the 23S rRNA sequences, along with reduced metabolic potential, inferred symbiotic lifestyle, and a global distribution. In summary, this effort, focused on the fourth-largest phylum in the bacterial domain, aims to provide nomenclatural stability in the scientific literature.

microbiology↗

bioRxiv2024

A rational approach for the targeted discovery and characterisation of microbiome-derived therapeutics

The human gut microbiome is intrinsically involved in health and disease, representing a wealth of untapped therapeutic potential. Here, we demonstrate the utility and potential of a metagenome guided, large cohort-based approach for the rational selection of live biotherapeutics from the human gut. We applied this approach to Inflammatory Bowel Disease (IBD), identifying several lead candidates that were significantly depleted in individuals with IBD compared to healthy controls. Their therapeutic potential was assessed in preclinical models of IBD where they improved markers of disease pathology by reducing inflammation and promoting mucosal healing and wound repair. All leads had excellent safety profiles in silico and in vitro, and several additionally presented favourable manufacturing properties, supporting their progression into clinical trials. We believe that this rational approach will be generalisable to any disease state with underlying microbiome aetiology and will expedite the development of novel microbiome-derived therapeutics to improve human health.

microbiology↗

bioRxiv2023

Fecal microbial transfer and complex carbohydrates mediate protection against COPD

ObjectiveChronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DesignUsing an in vivo mouse model of cigarette smoke-induced COPD and fecal microbial transfer (FMT), we characterized the fecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology, and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in sixteen COPD patients using a randomized, double-blind, placebo-controlled pilot study of inulin supplementation. ResultsFMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in cigarette smoke-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. ConclusionThe gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically. What is already known on this topicO_LIChanges in gut microbiota are associated with COPD but the underlying host and microbial mechanisms are unclear, limiting the therapeutic applications. C_LI What this study addsO_LIMicrobiome composition and metabolism is reproducibly correlated with lung and gastrointestinal pathology in experimental COPD. C_LIO_LIMicrobiome modifying interventions effectively alleviate disease, including protective effects supplementing smoking cessation. C_LIO_LINutritional interventions targeting the microbiome in COPD patients demonstrate efficacy in a small pilot study. C_LI How this study might affect research, practice or policyO_LIMicrobiome-targeting therapeutics and nutritional interventions may be developed for COPD, including as supplements to smoking cessation. C_LI

physiology↗

bioRxiv2023

An evolutionary timescale for Bacteria calibrated using the Great Oxidation Event

Most of lifes diversity and history is microbial but it has left a meagre fossil record, greatly hindering understanding of evolution in deep time. However, the co-evolution of life and the Earth system has left signatures of bacterial metabolism in the geochemical record, most conspicuously the Great Oxidation Event (GOE) [~]2.33 billion years ago (Ga, (Poulton et al. 2021)), in which oxygenic photosynthesis and tectonism (Eguchi, Seales, and Dasgupta 2019) transformed Earths biosphere from dominantly anaerobic to aerobic. Here, we combine machine learning and phylogenetic reconciliation to infer ancestral transitions to aerobic lifestyles during bacterial evolution. Linking these transitions to the GOE provides new constraints to infer the timetree of Bacteria. We find that extant bacterial phyla are truly ancient, having radiated in the Archaean and the Proterozoic: the oldest include Bacillota (Firmicutes), which radiated 3.1-3.7 Ga, Cyanobacteria (3.3-3.5 Ga) and Patescibacteria (3-3.5 Ga). We show that most bacterial phyla were ancestrally anaerobic and that most transitions to an aerobic lifestyle post-dated the GOE. Our analyses trace oxygen production and consumption back to Cyanobacteria. From that starting point, horizontal transfer seeded aerobic lifestyles across bacterial diversity over hundreds of millions of years. Our analyses demonstrate that the diversification of aerobes proceeded in two waves corresponding to the GOE and to a second sustained rise in atmospheric O2 at the dawn of the Palezoic (Krause et al. 2022).

evolutionary biology↗

bioRxiv2023

Anaerobic gut fungal communities in marsupial hosts

The anaerobic gut fungi (AGF) inhabit the alimentary tracts of herbivores. In contrast to placental mammals, information regarding the identity, diversity, and community structure of AGF in marsupials is extremely sparse. Here, we characterized AGF communities in sixty one fecal samples from ten marsupial species belonging to four families in the order Diprotodontia: Vombatidae (wombats), Phascolarctidae (koalas), Phalangeridae (possums), and Macropodidae (kangaroos, wallabies, and pademelons). Amplicon-based diversity survey using the D2 region in the large ribosomal subunit (D2 LSU) as a phylogenetic marker indicated that marsupial AGF communities were dominated by eight genera commonly encountered in placental herbivores (Neocallimastix, Caecomyces, Cyllamyces, Anaeromyces, Orpinomyces, Piromyces, Pecoramyces, and Khoyollomyces). Community structure analysis revealed a high level of stochasticity, and ordination approaches did not reveal a significant role for animal host, gut type, dietary preferences, or lifestyle in structuring marsupial AGF communities. Marsupial foregut and hindgut communities displayed diversity and community structure patterns comparable to AGF communities typically encountered in placental foregut hosts, while exhibiting a higher level of diversity and a distinct community structure compared to placental hindgut communities. Quantification of AGF load using quantitative PCR indicated a significantly smaller load in marsupial hosts compared to their placental counterparts. Isolation efforts were only successful from a single red kangaroo fecal sample and yielded a Khoyollomyces ramosus isolate closely related to strains previously isolated from placental hosts. Our results suggest that AGF communities in marsupials are in low abundance, and show little signs of selection based on ecological and evolutionary factors. The observed lack of host-fungal coevolutionary signal suggests a potential recent acquisition and/or a transient nature of AGF communities in marsupial herbivores.

microbiology↗

bioRxiv2023

The power and limitations of species tree-aware phylogenetics

Species tree-aware phylogenetic methods model how gene trees are generated along the species tree by a series of evolutionary events, including the duplication, transfer and loss of genes. Over the past ten years these methods have emerged as a powerful tool for inferring and rooting gene and species trees, inferring ancestral gene repertoires, and studying the processes of gene and genome evolution. However, these methods are complex and can be more difficult to use than traditional phylogenetic approaches. Method development is rapid, and it can be difficult to decide between approaches and interpret results. Here, we review ALE and GeneRax, two popular packages for reconciling gene and species trees, explaining how they work, how results can be interpreted, and providing a tutorial for practical analysis. It was recently suggested that reconciliation-based estimates of duplication and transfer frequencies are unreliable. We evaluate this criticism and find that, provided parameters are estimated from the data rather than being fixed based on prior assumptions, reconciliation-based inferences are in good agreement with the literature, recovering variation in gene duplication and transfer frequencies across lineages consistent with the known biology of studied clades. For example, published datasets support the view that transfers greatly outnumber duplications in most prokaryotic lineages. We conclude by discussing some limitations of current models and prospects for future progress. Significance statementEvolutionary trees provide a framework for understanding the history of life and organising biodiversity. In this review, we discuss some recent progress on statistical methods that allow us to combine information from many different genes within the framework of an overarching phylogenetic species tree. We review the advantages and uses of these methods and discuss case studies where they have been used to resolve deep branches within the tree of life. We conclude with the limitations of current methods and suggest how they might be overcome in the future.

evolutionary biology↗