Deep-branching Chloroflexota lineages illuminate the eco-evolutionary foundation of cross-ecosystem colonization

Domain: biology
Published: 2026-04-01
Editorial outcome: accept

Abstract
Bacteria span Earth’s ecosystems, coupling ecological versatility with genome-architectural reconfiguration across shifting physicochemical conditions. Yet the genomic routes by which free-living lineages cross ecosystem boundaries, and the consequences for genome architecture, remain poorly understood. Here, we use comparative and evolutionary genomics to investigate a soil-to-sediment-to-freshwater transition in Limnocylindria, an abundant clade within the Chloroflexota phylum. Two sister families show contrasting strategies. CSP1-4 expands genomes through niche-specific gene acquisition, whereas Limnocylindraceae undergoes genome reduction and metabolic simplification—revealing alternative evolutionary routes to similar ecological outcomes. In Limnocylindraceae, the loss of key DNA glycosylases coincides with degradation of base excision repair and is consistent with a hypermutator state that may have accelerated genomic erosion during freshwater specialization, potentially facilitating ecological expansion. This reductive genome trajectory is associated with a freshwater-adapted lineage with unexpectedly high GC content, challenging canonical links between base composition and genome size. While mutational processes appear to dominate genome restructuring, proteome-level patterns suggest selection favouring carbon- and nitrogen-efficient amino acid usage, implying that adaptive refinement can emerge alongside primarily non-adaptive dynamics. Overall, our findings are consistent with mutation-driven genome reduction and proteome optimization acting in concert to support cross-ecosystem boundary crossing and freshwater specialization in a free-living Chloroflexota lineage.

Introduction
Life on Earth likely originated during the early Archean Eon1,2—approximately 4.0 to 3.6 billion years ago—a period marked by the formation of Earth's first continental crust and its primordial oceans3. Although the exact nature and habitat of life’s origin2 remain subjects of debate, early life forms swiftly diversified, adapting to and colonizing the newly sprung environments4. Today, Bacteria and Archaea (the closest living descendants of these primordial organisms) numerically dominate Earth's ecosystems, with an estimated 1.2 × 1030 cells5. They are the most abundant and pervasive life forms, thriving in virtually every environment—from frozen polar caps and blazing hydrothermal vents to the abyssal depths of the ocean and the lofty heights of the atmosphere6–9.

Bacteria are notoriously adaptable, encompassing vast metabolic versatility10, high phylogenetic diversity11, and the ability to inhabit a wide array of ecosystems4. This adaptability stems from their capacity to reshape their genomic architecture through mechanisms such as horizontal gene transfer12, mutations13, and genetic rearrangements14. These processes enable bacteria to swiftly acquire new traits and adapt to changing environmental conditions, facilitating the exploration of novel ecological niches and fostering diversification15. Although bacteria can expand into related niches by exploiting new resources or undergoing metabolic rewiring, the ways and mechanisms through which they perform large-scale ecosystem transitions—that is, shifts into fundamentally different environments—remain poorly charted16.

Our primary understanding of how bacteria adapt to and colonize new environments stems from studies on pathogenic and parasitic lineages, which often originate from free-living environmental ancestors17–19. Such investigations have been instrumental in revealing how mechanisms like horizontal gene transfer, gene loss, duplication, and functional divergence enable bacteria to adapt to host, enhancing their virulence and survival20. However, transitions to host-associated habitats frequently involve mobile genetic element invasions21,22 or degenerative processes associated with small population sizes, including genome shrinkage, pseudogenization, and metabolic impairment20. In contrast, the evolutionary dynamics of free-living species—characterized by larger population sizes and likely different selective pressures—remain insufficiently explored23.

Ecosystem transitions in free-living bacteria represent rare yet pivotal evolutionary events that require intricate genomic innovation and ecological rewiring16. While relatively few lineages are known to breach ecosystem boundaries, several notable examples exist, including SAR1124 (marine to freshwater), Actinomarinales25 (freshwater to marine?), Salinispora26 (terrestrial to marine), and Methylophilaceae27 and Phycisphaerae28 (sediment to freshwater). These transitions are typically characterized by genomic rearrangements, reductions in metabolic capacity29, and the acquisition of habitat-specific gene sets16. Nevertheless, many aspects of the mechanisms driving these transformations and the eco-evolutionary processes influencing them remain unclear29. This information deficit is due not only to the limited number of documented cases but also to lineage extinctions over evolutionary timescales, which may have erased whole transitional clades30. Consequently, many crucial evolutionary events remain obscured, hindering our ability to fully comprehend the complexity of ecosystem-level transitions and their broader impact on bacterial ecology and evolution.

To address how microbial genomes evolve during ecosystem transitions, we analyse 236 metagenome-assembled genomes from Limnocylindria, an uncultivated, class-level lineage within the phylum Chloroflexota. This group spans soils, sediments, and freshwater environments. Within Limnocylindria, two sister families, CSP1-4 and Limnocylindraceae, exhibit contrasting adaptive strategies. CSP1-4 shows expansion into freshwater sediments and the water column, coupled to habitat-specialized gene acquisition, larger genomes, and signatures consistent with relaxed selection pressure. In contrast, Limnocylindraceae shows extensive genome reduction and metabolic simplification and is largely restricted to freshwater environments. This divergence provides a unique window into evolutionary alternatives for bacterial lineages crossing ecological boundaries, with one trajectory shaped by functional acquisition and the other by reductive specialization.

To dissect the reductive route in detail, we focus on Limnocylindraceae, a freshwater lineage whose unusually small yet GC-rich genome (median 1.307 Mbp; 63.15% GC) defies long-held assumptions about genome reduction and base composition in free-living bacteria31,32. By integrating phylogenomic reconstructions, gene-level selection analyses, and genome-informed metabolic reconstructions, we show that reductions in genome size and shifts in GC content are primarily driven by non-adaptive processes, likely exacerbated by the loss of DNA repair mechanisms. In parallel, adaptive signals emerge at the proteome level, where carbon and nitrogen economization suggests selective pressure to reduce biosynthetic costs. Together, these findings establish Limnocylindraceae as a model for how bacterial life can adapt and persist at the edge of genomic and metabolic minimalism while crossing ecosystem boundaries.

Results
Eco-evolutionary history
In this study, we expand upon our previously published pdCEL database15, designed to explore the eco-evolutionary forces driving adaptation and speciation in freshwater bacteria. pdCEL encompasses 5,500 metagenome-assembled genomes (MAGs) derived from time-series samples collected from lakes spanning a wide spectrum of trophic states, from oligotrophic to dystrophic.

To investigate the evolutionary history of the Limnocylindria class (Chloroflexota), we conduct a taxonomy-based retrieval, recovering 72 relevant MAGs from the pdCEL database (Supp. Fig. S2). We further augment this dataset with 164 publicly available MAGs from the GTDB R220 release (as of June 2024). This expanded dataset increases taxonomic breadth and resolution, enabling more accurate phylogenetic inferences and a clearer reconstruction of the clade's evolutionary history (Fig. 1a).

We built a rooted phylogenomic tree using maximum-likelihood methods based on 118 single-copy proteins, employing profile mixture models with the LG substitution matrix and 30 composition profiles to account for amino acid frequency variability and evolutionary rate heterogeneity across sites. This approach reveals four major phylogenetic clades, each with high support values (SH-aLRT = 100; UFBoot = 100), with the two evolutionary youngest forming a sister-group relationship (Fig. 1a).

We analyse the environmental distribution of the four major taxonomic clusters using the Sandpiper database, which employs SingleM to profile taxa from over 248,000 metagenomes, offering a comprehensive, high-resolution view of microbial diversity across diverse ecosystems33 (see Methods). By integrating distribution and abundance patterns within a phylogenetic framework, we reveal a clear evolutionary trajectory marked by ecosystem-level habitat transitions.

The ancestral P2-11E lineage (1,352 samples) is predominantly soil-associated (94.5%), with a peak environmental abundance of 9%. Its descendant, QHBO01 (2,441 samples), retains a strong preference for soil habitats (92.8%), where it reaches abundances of up to 30% (Fig. 1a). In contrast, the sister lineages CSP1-4 and Limnocylindraceae exhibit pronounced ecological shifts. CSP1-4 (9,454 samples), while primarily found in soil (71.4%), has significantly expanded into sediments (25%), achieving comparable peak abundances of 18% in both environments (Fig. 1a). Limnocylindraceae (1,352 samples), however, is almost exclusively freshwater-associated (98.7%)—presence reconfirmed through CARD-FISH (Supp. Fig. S1)— with peak abundances of 11%, particularly in shallow, runoff-affected lakes (high-abundance habitats shown in green; Fig. 1 upper left inset). These patterns highlight a cross-ecosystem transition by Limnocylindraceae, which maintains a tenuous link to soil habitats by thriving in freshwater systems heavily influenced by terrestrial inputs (Fig. 1a).

Evolution of genomic and metabolic architectures
We compiled a comprehensive genomic dataset for the Limnocylindria class, comprising genomes with a median completeness of 80% and contamination of 1.6% (n = 236). The pdCEL database significantly enhanced genomic representation, particularly for CSP1-4 (n = 5) and Limnocylindraceae (n = 67 genomes, representing five novel species), increasing the latter’s genomic coverage by 68% and expanding its phylogenetic diversity by 48.14%. A detailed examination of genome architectures across the four major phylogenetic lineages reveals significant evolutionary divergence. The families P2-11E, QHBO 01, and CSP1-4 exhibit comparable genomic features, including median genome sizes ranging from 2.4 to 3.2 Mbp and consistently high GC content between 68% and 68.6%. In stark contrast, Limnocylindraceae exhibits a dramatic genome reduction, with median genome sizes halving to 1.3 Mbp, accompanied by a slight decrease in GC content (median 63.15%) (Fig. 1b).

The CSP1-4 family comprises 16 genera and 51 strictly habitat-specific species—35 from soil, 4 from sediment, and 11 from freshwater. Genome-scale metabolic reconstructions, evaluated via a pairwise similarity matrix, reveal the greatest metabolic overlap between soil and sediment lineages (Fig. 2a,b). Soil genomes uniquely encoded enzymes for depolymerising plant polysaccharides and polyols (e.g., sucrose phosphorylase, 6-phospho-β-glucosidase, inosose dehydratase), along with nutrient-stress regulators (cstA, phoH/phoL, and phoB), reflecting possible adaptation to complex carbon inputs and episodic carbon–phosphate scarcity (Supp. Data S11). In contrast, sediment lineages harbour the catalytic α- and β-subunits of the nitrate reductase/nitrite oxidoreductase complex, a complete nitrate/nitrite ABC transporter, and the high-affinity cytochrome d terminal oxidase, consistent with respiration in oxygen-poor, nitrate-rich sediments. Freshwater representatives are distinguished by expanded flagellar and chemotaxis loci—traits rare in Chloroflexota34—suggesting capacity for motility-driven navigation of heterogeneous aquatic niches (Supp. Data S11). Genomic architecture differs consistently among habitats, reflecting distinct ecological constraints (Fig. 2c-f). Freshwater genomes have the largest estimated sizes and encode markedly fewer sigma factors (proxies for transcriptional regulatory complexity and ecological versatility) than those from soil or sediment, indicative of regulatory streamlining in planktonic environments (Fig. 2c). GC content and coding density were elevated in both sediment and freshwater lineages relative to soil representatives, suggesting habitat-specific selection for more compact and information-dense genomes outside terrestrial ecosystems. Together, these habitat-specific patterns in CSP1-4 gene content and genome structure support distinct ecological strategies shaped by carbon availability, nutrient dynamics, and redox conditions across terrestrial and aquatic environments (further details on Limnocylindria metabolism are provided in the Supplementary Note 1).

Striking genomic contrasts distinguish between the evolutionary younger sister families CSP1-4 and Limnocylindraceae (Fig. 1b). Limnocylindraceae exhibits significant genome size reduction, fewer sigma factors (key proteins essential for transcription initiation), and increased coding density resulting from reduced intergenic spacers (Fig. 1b, Supp. Fig. S3). While this genomic profile aligns with ‘classical’ models of genome reduction in free-living bacteria23,35, it also notably deviates by its high GC content (median = 63.15%). To elucidate the evolutionary processes that may have given rise to this particular genomic architecture, we first examine the evolutionary context of its emergence.

Phylogenetic analyses reveal that Limnocylindraceae shares a common ancestry with the CSP1-4 clade (Fig. 1a). The latter occupies an exceptionally short branch stemming from its most recent common ancestor—the shortest in our phylogeny—indicating reduced evolutionary change from the ancestral state (0.087 substitutions per site since divergence). To further understand the evolutionary landscape that may have favoured the emergence of Limnocylindraceae, we conduct a clade-specific selection force analysis on a set of approximately 800 genes conserved across the four clades. We observed that CSP1-4 exhibits a 60% increase in the number of genes under positive (diversifying) selection compared to its ancestral lineage (i.e., QHBO01), along with a 25% increase in the number of sites under positive selection (Supp. Table S1). The elevated levels of positive selection observed in CSP1-4, the highest among the four clades, suggest that this lineage is undergoing adaptive evolution, characterized by an accumulation of non-synonymous substitutions. Additionally, the relaxation of selective pressure in CSP1-4 is complemented by an increase in transposase density, a higher number of redundant proteins, and larger intergenic spacer regions—the longest among the four clades (Fig. 1b, Supp. Fig. S4). Together, these factors indicate decreased negative (purifying) selection and an enhanced potential for adaptive evolution15,36,37. These observations correlate with the habitat expansion of CSP1-4 in sediment and aquatic ecosystems.

GC dynamics
The extant genomic diversity of freshwater-adapted Limnocylindraceae is encompassed within a single genus-level clade, Limnocylindrus. The basal branch of this genus (blue arrow, Fig. 1) exhibits the defining features of the lineage: reduced genome size, high coding density, and low counts of sigma factors and signal transduction domains, consistent with other representatives (Fig. 3b, Supp. Fig. S5, Supp. Data S1). However, it retains a significantly higher GC content of 66.8% (n = 14; Fig. 1b) than the younger species of the genus (median = 63.1%; n = 85). This suggests that genome size reduction in Limnocylindraceae likely preceded the decrease in GC content. Therefore, the existence of such a genus-level clade with uniform genomic features—except for variations in GC content—offers a unique opportunity to study the evolution of GC content independently of genome size.

Selection-pressure analyses of 52 MAGs representing four freshwater Limnocylindrus species—defined by >95% average nucleotide identity and >70% conserved DNA—indicate that the basal lineage contains both fewer genes and sites per gene under negative selection than its evolutionary younger, lower-GC relatives (Fig. 3a,c). Thus, the approximately 3% decrease in GC content within Limnocylindrus is associated with an increase in both the number of genes and the number of sites under negative selection, along with a reduction in the number of fully conserved (invariable) genes—defined here as genes with no detectable nucleotide substitutions across the alignment, consistent with strong purifying selection. No substantial differences in GC content are observed between species-specific genes and the shared core genome (Suppl. Fig. S13). This indicates that negative selection affects a larger portion of the genome, while a higher mutation rate introduces more sequence variation, as evidenced by the predominance of synonymous substitutions—nucleotide changes that do not alter the amino acid sequence and are typically retained under negative selection— in most genes (Fig. 3c). Additionally, the decrease in invariable genes suggests that mutations occur more frequently in lower-GC genomes, resulting in fewer unchanged genes. Consequently, the combination of an increased mutation frequency and strong selection pressure leads to more genes under negative selection and fewer entirely invariable ones.

A further comparison of GC content of genes and intergenic spacers revealed that the P2-11E, QHBO01, and CSP1-4 main lineages exhibit higher GC content in their coding regions (i.e., genes) compared to their non-coding counterparts (Supp. Fig. S6a). These findings are consistent with previous research indicating that non-coding regions tend to accumulate more mutations and experience weaker selection pressure than protein-coding regions, often leading to a lower GC content38–40. Notably, the family Limnocylindraceae showed higher GC content in the intergenic regions (Supp. Fig. S6a). By examining four representative species, we found that in freshwater-adapted Limnocylindraceae, intergenic regions—despite their short median length (12 bp)—consistently exhibit higher GC content than coding regions (Fig. 3d). This pattern may reflect a reduced likelihood of mutation accumulation in these shorter sequences, as their limited length presents a smaller mutational target compared to longer coding regions. Overall, these findings suggest that the observed decrease in GC content within Limnocylindraceae is likely driven by the random accumulation of mutations. The selection pressure on the GC content of genes does not appear to be higher than that acting on intergenic spacers. Therefore, the differences in GC composition between the two are likely attributable to their overall size. Although GC content was not directly analysed in Bourguignon et al. (2020)41, their finding that genome reduction in prokaryotes is frequently associated with elevated mutation rates supports a broader mutational framework. Our results align with this model and suggest that mutation-driven genome erosion may also influence base composition—manifesting as GC content reduction—in genome-reduced, free-living lineages such as Limnocylindraceae.

The codon frequency analyses of 20 proteogenic amino acids reveal that synonymous substitutions are replacing GC-rich codons with AT-rich ones, mirroring the overall decrease in genomic GC content (Fig. 4). In the freshwater Limnocylindrus, this substitution pattern is particularly pronounced, with 75% of cases exhibiting a distinctive "funnel-shaped" dynamic where ancestral high-GC codons are progressively replaced by codons with higher AT content (Fig. 4a,b, Supp. Fig. S8). To determine whether this trend is consistent across the entire Limnocylindria class, we performed codon frequency analyses on all major families and observed a similar pattern (Fig. 3b,d; Supp. Fig. S8). The evolutionary decrease in GC content is accompanied by a reduction in GC-rich codons and an increase in AT-rich ones, resulting in a "wine bottle"-shaped dynamic in 70% of the cases. This pattern showcases how the initial spread in the distribution of codon GC content (low frequency for AT-rich and high frequency for GC-rich) is followed by a narrowing in Limnocylindraceae resembling the shape of a wine bottle. Overall, these findings indicate that the decrease in GC content in Limnocylindraceae is likely driven by a shift in synonymous codon usage from GC-rich to AT-rich codons. This suggests a mutation-driven mechanism rather than ecological adaptation, as the number of sites under positive selection is too limited to explain the observed GC content changes.

Emergence of reduced genomic architectures
Comparative genome-informed metabolic reconstructions reveal that genome shrinkage in Limnocylindraceae coincides with reductions in metabolic and biosynthetic capacities (Fig. 5, Supplementary Note 2). Remarkably, this lineage lacks most DNA glycosylases involved in the base excision repair (BER) pathway (Fig. 5a). BER is an essential DNA repair mechanism that corrects small, non-helix-distorting base lesions arising from alkylation, deamination, oxidative damage, or spontaneous base loss42. We propose that the absence of these DNA glycosylases may underlie the distinctive genomic architecture observed in freshwater-adapted Limnocylindraceae and explore the consequences of their loss on GC content evolution.

The glycosylases Tag (K01246), AlkA (K01247), and MPG (K03652) play pivotal roles in excising alkylated bases such as 3-methyladenine (3-MeA) and 3-methylguanine (3-MeG), initiating BER. In bacteria lacking these enzymes, repair of alkylated bases is largely compromised. While 3-MeA lesions are highly cytotoxic due to their replication-blocking properties, they contribute minimally to mutagenesis as DNA polymerases rarely bypass them43. In contrast, 3-MeG lesions are less obstructive and can be misread during replication, leading to mispairing with thymine instead of cytosine. This mispairing will eventually lead to G→A transition mutations44. The pronounced impact of 3-MeG-induced mutations on GC content stems from their higher DNA polymerase bypass frequency and consistent mispairing behavior44,45. Uracil DNA glycosylases Family 4 (TIGR03914), Ung (K03648) and Mug (K03649) excise uracil from DNA, preventing mutations from cytosine deamination or uracil misincorporation during replication. In the absence of these enzymes, uracil-induced lesions will most probably persist. Uracil arises via spontaneous deamination of cytosine, forming U·G mismatches, or through misincorporation of dUMP opposite adenine, forming U·A pairs. Unrepaired uracil in U·G mismatches is read as thymine during replication, leading to the insertion of adenine opposite uracil and resulting in G→A transition mutations44,45. Accumulation of these mutations further decreases genomic GC content as G·C pairs are replaced by A·T ones.

The enzymes Nei (K05522) and MutT (K03574) are crucial for repairing oxidative DNA damage. Nei excises oxidatively damaged pyrimidines like thymine glycol, preventing mispairing during replication46. MutT sanitizes the nucleotide pool by hydrolyzing 8-oxo-dGTP, an oxidized and mutagenic form of dGTP. Without MutT, 8-oxo-dGTP accumulates and is incorporated opposite adenine, forming A·8-oxo-G mispairs. This mispairing leads to A·T→C·G transversions47. Conversely, the absence of Nei allows the accumulation of oxidized pyrimidines, causing mispairing during replication and resulting in G·C→T·A transversions, which will decrease GC content44.

Overall, we propose that the absence of key DNA glycosylases in Limnocylindraceae could lead to increased mutagenesis due to the accumulation of unrepaired DNA lesions, such as alkylated bases and deaminated cytosine residues. In the absence of repair, these lesions are likely to mispair during DNA replication, resulting in mutations. The elevated mutation frequency may promote the inactivation and subsequent deletion of non-essential or low-benefit gene sets, ultimately driving genome reduction through cumulative deletion events48. Moreover, the overall pattern of missing key enzymes in the BER pathway favours a decrease in GC content through G→A transitions. Thus, the absence of DNA glycosylases may effectively explain the observed increase in mutation frequency, genome reduction, and decline in GC content in Limnocylindraceae.

Proteome optimization
Limnocylindraceae genomes are missing the core enzymes for six canonical essential-amino-acid pathways (Fig. 4a), indicating a substantial, though not complete, contraction of their biosynthetic repertoire. This partial auxotrophy likely heightens dependence on the external amino acids or peptide‐rich organic matter. Freshwater lakes, which often contain dissolved amino acids and oligopeptides, provide a potential source of these essential substrates49. However, it remains unclear the extent to which reliance on external amino acids shaped the proteomic composition of Limnocylindraceae.

To investigate this, we calculated the C and N content of each protein and normalized these values by protein length (Fig. 6). Our analysis reveals that Limnocylindraceae possesses lower C and N contents per amino acid compared to its ancestors or the CSP1-4 sister lineage (Fig. 6a,b). When plotting the mean normalized values for C and N, we observe a direct relationship between the proteome's elemental requirements and a clear separation between Limnocylindraceae and the other clades, with the former exhibiting the lowest values (Fig. 6c). The loss of biosynthetic pathways for amino acids that are abundant in the proteome (e.g., alanine, arginine, histidine, etc.) likely made it reliant on external environmental supply and exerted selective pressure to minimize the cellular demand for essential elements such as C and N. Thus, by optimizing its proteome to favour amino acids with lower C and N content, Limnocylindraceae likely reduce the metabolic cost associated with protein synthesis and maintenance.

To determine whether the proteome streamlining strategy is unique to Limnocylindraceae, we conduct two case studies focusing on the Planctomycetota (class Phycisphaerae) and Gammaproteobacteria (family Methylophilaceae) clades—groups that include lineages believed to have transitioned to freshwater environments27,28. Our analyses reveal that in both instances, the freshwater lineages underwent C and N optimization, albeit in a niche-dependent manner (Fig. 6d). Specifically, freshwater Phycisphaerae exhibit overall lower C content per amino acid compared to their soil ancestors, reflecting a metabolism adapted for utilizing nitrogen-rich sinking aggregates28. Conversely, freshwater Methylophilaceae display reduced nitrogen requirements per amino acid, a trait that underlies their specialized metabolism for using reduced one-carbon compounds as energy and carbon sources27. These observations provide a novel perspective on previous studies suggesting that nutrient scarcity may foster proteome-wide nitrogen reduction, as demonstrated by comparative analyses of community-level protein sequences in open-ocean and coastal marine ecosystems50. Overall, our findings highlight the possible role of proteome streamlining as a broad evolutionary response to biosynthetic pathway loss and nutrient limitation following large habitat transitions.

An eco-evolutionary synthesis
Bacterial ecosystem transitions have fundamentally shaped Earth's natural history by instigating transformative changes in the biosphere. For instance, the colonization of land by bacteria51 is thought to have initiated biogeochemical rock weathering and soil formation, creating environments that later supported the emergence and diversification of early plants—a key milestone in life’s evolution52. Yet, despite the recognized importance of such events, the selective pressures that drove them, as well as the evolutionary tempo (rates of change) and mode (patterns of adaptation), remain poorly charted.

The Chloroflexota phylum designates a major branch in the tree of life, thought to have emerged as a direct descendant of the earliest bacterial lineages that colonized land51. Among its clades, Limnocylindria is notable as one of the oldest and most prominent divisions53. This uncultivated lineage, identified through metagenomic surveys of freshwater lakes54, derives its name from the aquatic habitats where its first representatives were recognized.

Our results indicate that within terrestrial Limnocylindria, the capacity for cross-ecosystem colonization likely emerges during a period of reduced selective pressure, as suggested by clade-specific selection force analyses (see Evolution of genomic architectures). In CSP1-4, cross-ecosystem colonization appears to have been driven by genome expansion and the acquisition of habitat-specific functions, whereas in Limnocylindraceae, the same ecological shift coincided with pronounced genome reduction—revealing two distinct evolutionary strategies within a shared evolutionary ancestry.

Ecological transitions in Limnocylindraceae and CSP1-4 exemplify niche-directed genome evolution28, yet the sister clades appear to sit at opposite ends of that continuum. CSP1-4 presents genomic signatures consistent with diversifying selection accompanied by net gene acquisition. Soil representatives retain a broad plant-polymer catabolic arsenal; sediment-dwelling taxa carry additional nitrate-respiratory modules and microaerobic terminal oxidases; and freshwater representatives uniquely encode flagellar and chemotaxis loci—features uncommon in other Chloroflexota34 (Supp. Data S11). By contrast, Limnocylindraceae shows only modest habitat-linked gene gains, most notably the acquisition of proton-pumping rhodopsins (Supplementary Note 1).

This asymmetry may reflect divergent responses to relaxed selective constraint. In Limnocylindraceae, such relaxation appears to have permitted the accumulation of loss-of-function mutations in DNA repair genes—including Tag/AlkA/MPG, UDG-family, Nei, and MutT (Fig. 4a)—leading to the degradation of base excision repair (BER) pathways that defend against alkylated, deaminated, and oxidized bases. The resulting hypermutator state likely accelerated GC content erosion and contributed to the loss of multiple biosynthetic pathways (Fig. 5b). Thus, while CSP1-4 seems to have navigated habitat transitions through adaptive gene gain and genome expansion, Limnocylindraceae appears to have undergone reductive genome evolution and metabolic simplification under sustained mutation pressure following BER collapse.

In Limnocylindraceae, the concerted loss of Tag-/AlkA-/MPG-mediated alkyl repair, UDG-driven uracil repair, Nei-directed oxidation repair, and MutT nucleotide sanitising strips the lineage of its chief defences against the three most pervasive small-base lesions42. Because nucleotide-excision repair, mismatch repair, homologous recombination and AlkB-type dioxygenases cannot excise 3-methyl-guanine, uracil, thymine glycol or 8-oxo-dGTP with comparable efficiency, these lesions will likely persist into replication42,45,55. Laboratory strains deprived of the same activities manifest 10- to 10⁴-fold mutator phenotypes and strongly GC-eroding spectra—G→A (tag alkA), G·C→A·T (ung) and GC→TA (nei) 56–59. Such biases furnish a direct mechanistic path to the lineage’s AT-enrichment and gene loss, echoing broad phylogenomic evidence that links elevated mutation pressure to bacterial genome reduction 41 and the rapid GC erosion produced in BER-deficient Salmonella typhimurium evolution experiments 60. Thus, in Limnocylindraceae, the capacity to cross ecological boundaries was likely accelerated by DNA glycosylase losses (Fig. 5a) that gave rise to hypermutator descendants (as inferred from branch length elongation, Fig. 1a) capable of rapid adaptation. Elevated mutation rates and ecological plasticity appear tightly linked; hypermutation likely enabled dramatic shifts in genome content and metabolic function, generating the genetic variation required to colonize novel environments. However, the very processes that facilitated this swift adaptation also sowed the seeds of constraint. The freshwater-adapted lineage evolved and diverged rapidly—as evidenced by its elongated phylogenetic branch, indicative of numerous non-synonymous substitutions—but eventually reached an evolutionary slowdown. Having shed a large part of its metabolic repertoire (Fig. 5bc) and accumulated extensive sequence changes (Fig. 3), Limnocylindraceae ended up with a highly reduced genomic architecture. While this reduction likely facilitates niche specialization, the combination of genome shrinkage (Fig. 1b) and elevated mutation pressure (Fig. 3a) may also limit the lineage’s capacity to explore new adaptive landscapes. Similar trade-offs have been documented in other microbial systems: in Prochlorococcus, genome reduction restricts physiological plasticity and niche range 61,62, while hypermutator strains of Pseudomonas aeruginosa lose fitness when introduced to new environments 63. Thus, although Limnocylindraceae remains evolutionarily viable, its current genomic configuration may reduce future evolutionary flexibility—a possible example of the tension between hyperadaptability and hyperspecialization63.

We observe that genome reduction precedes the decline in GC content (Fig. 1), indicating that deletion events outpace mutation accumulation during the transition to freshwater environments. Once Limnocylindraceae adapted to these ecosystems, negative (purifying) selection likely emerged as a dominant force (Fig. 3), preserving the primary structures of essential proteins. Concurrently, the loss of DNA glycosylases (Fig. 5a) most probably facilitated the accumulation of GC-lowering mutations60, predominantly at third codon positions where they are often synonymous and minimally impact protein function. The pervasive negative selection across numerous genes (Fig. 3a) underscores that while mutations arose randomly, only those occurring at neutral sites persisted. We observe that the substitution of third codon positions with AT-rich nucleotides was a principal driver behind the decrease of GC content in freshwater-adapted Limnocylindraceae (Fig. 3, Supp. Fig. S9). Thus, it is probable that the intricate interplay between mutation-induced GC reduction and selective pressures preserving protein functionality orchestrates the retention of a high GC content in the genome-reduced freshwater lineage (Supp. Fig. S10).

Genome size and GC content are hallmarks that define the architectural blueprint of bacterial genomes. Understanding the forces that shape these features has been a central pursuit of evolutionary microbiology23,32,64,65. Traditionally, two paradigms—the adaptive35 and the non-adaptive66—have competed to explain the remarkable variation observed across bacterial taxa. The adaptive paradigm portrays genomes as finely tuned instruments, honed by natural selection to maximize efficiency. Deterministic mechanisms like genome streamlining35 act as evolutionary chisels, chipping away non-essential DNA to reduce genome size and minimize resources required for replication in response to nutrient limitations. The ‘black queen hypothesis’ enriches this narrative, suggesting that selective pressures eliminate costly communal functions, offloading them onto other community members and thereby lightening the genomic load67. Ancient adaptations are further integrated into this framework, with remnants of past selective pressures continuing to shape contemporary genomic traits such as GC content 68. In stark contrast, the non-adaptive paradigm69 posits that the evolution of genome size and GC content is more a product of chance than design. Here, neutral processes such as elevated mutation rates and random genetic drift—operating independently of selective pressures—introduce genomic variability23.

Our findings in freshwater Limnocylindraceae support the non-adaptive paradigm, suggesting that its genome reduction and GC content are likely driven by mutational biases rather than selective optimization. Unlike other free-living marine bacteria that adaptively streamline their genomes and lower GC content in response to nutrient-poor environments35, Limnocylindraceae genomic architecture is likely shaped by inherent processes linked to elevated mutation rates and the environmental niche it occupies. This rationale resonates with experimental evolution studies where GC content decrease in microbial isolates was attributed to mutational biases in the absence of DNA repair systems60 and align with recent observations regarding genome reduction in Prochlorococcus70.

Considering that DNA constitutes approximately 3% of a bacterial cell’s dry weight while proteins account for about 53%71, we hypothesized that any adaptive mechanisms in Limnocylindraceae would likely operate at the proteomic level. Our analysis of the carbon and nitrogen content of amino acids reveals that Limnocylindraceae preferentially utilize amino acids with lower C and N content (Fig. 6). This selective utilization could be particularly significant given that proteins represent the largest fraction of bacterial biomass. Thus, the transition to freshwater environments appears to have driven a strategic optimization of carbon and nitrogen usage within the proteome. This proteomic optimization likely emerged during the initial phases of adaptation to freshwater habitats, characterized by a substantial increase in non-synonymous substitutions (long branch in Fig. 1a). Furthermore, we show that similar proteome-level C or N optimizations occur in other microbial lineages believed to have transitioned into freshwater habitats (Fig. 6d), suggesting that proteome streamlining may represent a more widespread adaptive strategy among freshwater taxa.

Together, these findings reveal how divergent genomic strategies—one expansive and acquisitive, the other reductive and minimalist—can both drive microbial transitions across ecosystem boundaries. Arising from a shared terrestrial ancestor, CSP1-4 and Limnocylindraceae exemplify contrasting routes to ecological success: the former through gene acquisition and metabolic diversification, the latter through mutation-driven genome erosion and functional contraction. In Limnocylindraceae, genome reduction was highly likely catalysed by the collapse of base excision repair, fostering a hypermutator state that promoted rapid genetic change and facilitated ecological flexibility. This mutational pressure reshaped genome architecture, eroded biosynthetic capacity, and constrained functional breadth. Yet even within this reductive trajectory, selective pressures at the proteomic level favoured elemental efficiency, sustaining survival in nutrient-variable freshwater environments. These results demonstrate how cross-ecosystem transitions can reconfigure bacterial genomes through the intertwined forces of adaptive refinement and non-adaptive mutation dynamics, offering an expanded mechanistic framework for genome reduction and ecological specialization in free-living bacteria.

Methods
Limnocylindria Genomic Database
To build a comprehensive genomic database for the bacterial class-level lineage Limnocylindria, we began by reclassifying metagenome-assembled genomes (MAGs) from the recently published pdCEL v1.0 database15 using GTDB-Tk72 version 2.4.0 (database release R09-RS220). The pdCEL database contains approximately 5,500 high-quality MAGs sourced from Central European freshwater lakes with varying trophic statuses. These MAGs were subjected to meticulous curation, including taxonomic validation, checks for GC content consistency, and the application of stringent thresholds for genome completeness and contamination to ensure data integrity15. From this reclassified dataset, we identified 72 Limnocylindria MAGs based on the updated taxonomy. To expand our dataset further, we incorporated an additional 164 MAGs indexed in the Genome Taxonomy Database (GTDB)73, resulting in a curated collection of 236 high-quality MAGs. This extensive repository provides a robust foundation for in-depth genomic and evolutionary analyses, enabling us to investigate Limnocylindria adaptations across diverse environments such as soil, sediment, and freshwater.

To estimate genome sizes accurately, we corrected the assembled lengths of the MAGs for both completeness and contamination. This was achieved by dividing each MAG length by its estimated completeness and then multiplying the result by the difference between 100 and the MAG contamination value. This approach accounted for missing genomic content and excluded contamination15.

Because short-read MAGs can under-recover parts of the flexible (accessory) genome, we applied conservative criteria for interpreting gene presence/absence. Specifically, we restricted downstream analyses to quality-filtered, curated MAGs and inferred lineage-level gene depletion only when a gene was consistently undetected across multiple MAGs, rather than from single-genome non-detections. Accordingly, “absence” is interpreted as loss from the conserved (core) gene repertoire at the lineage level, while rare strain-specific occurrences (e.g., via horizontal transfer) cannot be formally excluded.

Evolutionary history reconstructions
Phylogenetic analyses were based on a set of 118 single-copy proteins previously identified as phylogenetically informative and suitable for deep evolutionary inference74. To maintain balanced representation and uphold the genomic diversity of Chloroflexota lineages, the initial collection of 236 genomes was subsampled to 50. This subsampling retained all 10 QHBO01 family representatives and chose additional genomes from other lineages to ensure their genomic diversity was adequately represented. Protein-coding genes were predicted using PRODIGAL75 v2.6.3 with default settings for each selected MAG (n = 50). The resulting proteomes were scanned using HMMER hmmscan76 v3.1b2 against the TIGRFAMs77 v15.0 database (parameters: E-value ≤ 1E-10; -prcov 70; -hmcov 70). Based on TIGRFAM accession numbers, 118 ubiquitous single-copy protein sequences were extracted from the genomes. For each phylogenomic marker, phylogeny-aware multiple sequence alignments (MSAs) were constructed using PRANK78 v.170427 with default settings. The alignments were trimmed with BMGE79,80 v1.12 using the -g 0.5 setting to retain regions suitable for phylogenetic inference. The individual MSAs were concatenated into a supermatrix comprising 39,854 aligned sites prior to phylogenetic analysis. A maximum-likelihood phylogeny was constructed using IQ-TREE80 v2.0.6. To account for amino acid frequency variability and evolutionary rate heterogeneity across sites, we employed mixture models with the LG substitution matrix and 30 composition profiles. Node support was assessed using ultrafast bootstrapping with 1,000 replicates and the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) with 1,000 replicates. Outgroup rooting was performed by using members of the Eremiobacterota phylum, chosen due to their phylogenetic position related to the Chloroflexota (1). This approach was performed to confer directionality to the phylogenomic tree. A jack‐knifed family-centric genome-based phylogeny of the Limnocylindria class is further presented in Supp. Fig. S11.

Phylogenetic gain
To evaluate the increase in phylogenetic diversity from incorporating MAGs from the pdCEL database, we conducted phylogenetic analyses using both the newly added MAGs (n = 72) as well as the existing database representatives (n = 113) based on 118 single-copy proteins known to be phylogenetically informative and appropriate for deep evolutionary inference.

Protein-coding genes were predicted using PRODIGAL v2.6.3 with default settings for each selected genome (n = 185). The resulting proteomes were then scanned HMMER hmmscan v3.1b2 against the TIGRFAMs77 v15.0 database (parameters: E-value ≤ 1E-7; -prcov 70; -hmcov 70). Based on TIGRFAM accession numbers, 118 ubiquitous single-copy protein sequences were identified and extracted from the genomes. For each phylogenomic marker, phylogeny-aware multiple sequence alignments (MSAs) were constructed using FAMSA v1.281 with default settings. All individual MSAs were concatenated into a supermatrix comprising 53,775 aligned sites prior to phylogenetic analysis. A maximum-likelihood phylogeny was constructed with IQ-TREE75,80 v2.1.3, employing mixture models with the LG substitution matrix and 60 composition profiles. Node support was assessed using ultrafast bootstrapping with 1,000 replicates and the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) with 1,000 replicates. Finally, the tree was rooted with two members of the P2-11E family (n = 2), chosen for their phylogenetic position relative to the newly added MAGs, thus providing directionality to the final phylogenomic tree (Supp. Fig. S12).

Phylogenetic diversity (PD) was calculated as the sum of all branch distances in each subtree. The gain in PD introduced by the pdCEL MAGs was then assessed as follows: PD – PD = PD . All calculations were carried out within R v.4.2.2 with the ape v.5.3 package.

Taxa-habitat associations
To assess the environmental distribution and relative abundances of Limnocylindria lineages, we utilized the Sandpiper v0.3.0 database33. Sandpiper integrates taxonomic profiles generated by the SingleM v0.20.3 software33 from over 248,000 publicly available metagenomes. The main approach of SingleM is to profile metagenomes by targeting short 20-amino-acid stretches, or ‘windows’, within single-copy marker genes. It identifies reads that cover an entire window and analyzes these further. By focusing on these short windows, SingleM can determine how novel each read is compared to known genomes33. Leveraging the fact that each analyzed gene is typically found exactly once in each genome, the software accurately estimates the abundance of each lineage. We focused on data at the family level, selecting datasets where Limnocylindria lineages achieved abundances of at least 0.5% within the total prokaryotic community in each metagenome. By using this threshold, we recovered 14,644 valid taxa-habitat matches (i.e., one of the four family-level lineages achieved a total abundance in the prokaryotic community equal to or greater than 0.5%). This threshold minimized noise and ensured robust, ecologically meaningful associations between taxa and habitats.

Protein annotations and functional redundancy analysis
Protein-coding genes were predicted, for each MAG (n = 236), using PRODIGAL75 v2.6.3. Protein domains were annotated by querying the predicted proteomes against the Pfam82 release 32 HMM database using the hmmscan-based pfam_scan.pl script. Additional domain architectures and protein annotations were obtained by running InterProScan83 13v5.24-63.0 with the CDD v3.14, SMART v7.1, and HAMAP v201701.18 databases. To further expand the protein annotation space, we conducted hmmsearch (parameters: -evalue 1E-7, -prcov 70, -hmcov 70) against the COG84 and TIGRFAM77 HMM databases. BlastKOALA v3.185 was utilized to assign KEGG Orthology (KO) identifiers to orthologous genes. Selected metabolic pathways were manually examined using the online KEGG mapping tools based on the assigned KO numbers. Rigorous manual curation was applied to ensure metabolic pathway completeness.

To assess habitat-specific patterns in functional gene content within the CSP1-4 lineage, we computed pairwise Jaccard similarity indices based on the presence or absence of KEGG Orthology (KO) identifiers. For each habitat (soil, sediment, and freshwater), KO terms detected across all associated CSP1-4 genomes were pooled into a composite gene set. The similarity between any two habitats i and j was calculated as:
∣𝐾𝑖∩𝐾𝑗∣
(1) 𝐽𝑖𝑗 = 
∣𝐾𝑖∪𝐾𝑗∣
where ∣Ki∩Kj∣ is the number of KOs shared by both habitats and ∣Ki∪Kj∣ is the total of unique KOs present in either habitat. This analysis enables a quantitative comparison of metabolic repertoire overlap across habitats and highlights functional divergence during ecological transitions within CSP1-4.

To assess functional redundancy in the proteins of selected bacterial species, we calculated the Functional Redundancy Ratio (FRR)15. For each MAG, we determined the total number of KEGG annotations from all the protein-coding genes and the number of unique KEGG annotations representing non-redundant functions. The FRR was computed as one minus the ratio of unique to total KEGG annotations per MAG. An FRR value near 0 indicates low functional redundancy, while a value near 1 indicates high functional redundancy within the genome.

Ancestral state reconstructions
To resolve the evolutionary trajectories underpinning ecological transitions and metabolic adaptation in Chloroflexota, we performed ancestral state reconstructions on a maximum-likelihood phylogeny comprising 50 representative genomes (outgroups excluded).

Habitat transitions were inferred by modeling the evolution of discrete ecological states—soil, sediment, and freshwater—using stochastic character mapping as implemented in the make.simmap() function (phytools v2.0, R). Three standard Markov (Mk) models were evaluated: the Equal Rates (ER) model, in which all transitions occur at the same rate; the All-Rates-Different (ARD) model, allowing unique rates for each transition; and the Symmetric (SYM) model, where forward and reverse transitions share identical rates. Model selection was based on log-likelihood scores and Akaike Information Criterion (AIC) values. The ER model provided the best fit and was subsequently used to generate 1,000 stochastic character maps, yielding posterior probabilities of ancestral habitat states at each node.

Trait evolution was reconstructed for 15 binary-coded pathways related to sensory perception and core metabolism, including rhodopsin, flagellar motility, chemotaxis, heme biosynthesis, and nine amino acid biosynthetic pathways (e.g., arginine, methionine, tryptophan). Each trait was modeled independently under an ER framework using fitMk() and stochastically mapped with make.simmap() (1,000 replicates per trait). Posterior node probabilities were summarized via describe.simmap() to infer the timing and directionality of trait gain or loss across major habitat transitions.

These analyses enabled the reconstruction of metabolic and sensory innovations (or regressions) associated with lineage-specific ecological specializations and are summarized in Supp. Fig. S14.

Selection force
Selection force analyses were conducted as previously described15. Briefly, gene sequences from functionally annotated protein orthologous groups—870 for Limnocylindria family-level analyses and 594 for Limnocylindrus species-level analyses—were extracted in FASTA format. To prepare the nucleotide sequence data for downstream analyses, we used a custom bash script designed to remove terminal stop codons from coding DNA sequences. For each sequence, the script checked the last three nucleotides to determine if they correspond to one of the standard stop codons (TAA, TAG, or TGA). If a terminal stop codon was detected, the script removed it from the end of the sequence. Subsequently, the preprocessed sequences were aligned using PRANK78 v.170427 in codon mode (-codon -F). Alignments containing three or more sequences were then used to construct phylogenetic trees with IQ-TREE80 v2.1.3, utilizing the codon model setting (-st CODON). Site-specific selection pressures were inferred from the codon alignments and phylogenetic trees using the Fixed Effects Likelihood86 (FEL) method implemented in HYPHY v2.5.32 within a maximum-likelihood framework, applying a p-value threshold of 0.05.

Genes from functionally annotated protein orthologous groups from the four Limnocylindrus (L. sp003670745; L. sp903937225; L. sp003670475; L. sp903864185) species were extracted in FASTA format. Orthologous groups shared among all four species were discarded, leaving only those that were unique to each species (L. sp003670745 n = 26; L. sp903937225 n = 37; L. sp003670475 n = 23; L. sp903864185 n = 69). GC content for each gene was then calculated using seqstat v1.9g (--gccomp -a). The same pipeline was run on the shared orthologous groups (n = 687) by all four Limnocylindrus species to provide a baseline for comparison.

Codon and amino acid frequency analyses
Codon frequencies were calculated using a custom Perl-based script that reads coding DNA sequences from multi-FASTA files and counts the occurrences of all 64 possible codons by iterating through the sequences in frames of three nucleotides. The total counts for each codon are then normalized by dividing by the total number of codons in each genome, resulting in codon frequency values. The script outputs a tab-delimited file where each row corresponds to a codon and each column represents the codon frequencies for a particular genome. A total of 236 genomes were analysed.

Amino acid compositions of predicted protein sequences were calculated using computational methods employing the BioPerl87 module Bio::SeqIO. Protein sequences in FASTA format were read and processed individually. For each protein sequence, the occurrences of all 20 standard amino acids were counted. These counts were then normalized to percentages relative to the total sequence length, providing the amino acid composition profiles used in our analyses.

Predicted proteome carbon and nitrogen content
The carbon and nitrogen content of proteins (n = 462,342) encoded in the genomes belonging to the studied Chloroflexota families (i.e., P2-11E, QHBO01, CSP1-4, and Limnocylindraceae) was determined by using a custom BASH script. Briefly, the script counted the occurrences of each amino acid and computed the carbon and nitrogen content using predefined molecular composition values. The carbon and nitrogen content of each protein was normalized by the corresponding predicted protein length, then averaged to obtain the mean carbon and nitrogen content per proteome.

To complement the analysis, the dataset was expanded to include additional lineages hypothesised to have transitioned to freshwater environments, respectively Planctomycetota (class Phycisphaerae28) and Gammaproteobacteria (family Methylophilaceae27) clades (3). For each lineage, freshwater (Phycisphaerae n = 260; Methylophilaceae n = 113) and soil-sediment (Phycisphaerae n = 169; Methylophilaceae n = 12) representatives were recovered from GTDB73 (release R09-RS220). Sandpiper33 database was then used to further validate their habitat distribution.

CARD-FISH
For the CARD-FISH analysis, we collected freshwater samples (10 mL) from the epilimnion (5 m depth) of Lake Zurich (406 m a.s.l., 47°18’N, 8°34’E, Switzerland). The samples were immediately fixed with formaldehyde at a final concentration of 2% and incubated at room temperature (25°C) for 1 hour. After fixation, the samples were filtered through 0.2 μm polycarbonate membrane filters (Merck Millipore, Germany) supported by 5 μm cellulose nitrate filters (Sartorius, Germany), followed by double washing with phosphate-buffered saline (PBS, 1X). The filters were air-dried and stored at -20°C until further use.

CARD-FISH was performed using a probe specific for the Limnocilindraceae lineage54 of Chloroflexota bacteria. Probes NON338 (negative control) and EUB I-III88 (positive control) were included to validate the results. All filters were in addition stained with DAPI (4′,6′-diamidino-2-phenylindole) and examined using an Axio Imager.M1 microscope (Zeiss, Germany). Micrographs of stained cells were captured with a Nikon DS-Qi2 Monochrome Microscope Camera (Nikon, Japan) and processed using NIS-Elements Advanced Research software v5.01.00.

Statistics and code
All statistics were performed within the R89 v4.2.2 (R Core Team, 2022) and RStudio90 v1.3.1093 (RStudio Team, 2020), Apricot Nasturtium software. Shapiro-Wilk test, followed by residues distribution visualization, was utilized for data normality assessment. Parametric datasets analysis of variance (ANOVA) was performed for normally distributed datasets. Pairwise comparisons at group level were performed with pairwise t.test whenever the dataset was normally distributed. Non-parametric datasets analysis of variance was performed with the Kruskal-Wallis tests. Subsequently, pairwise Wilcoxon rank sum test was performed for pairwise comparisons. For non-normal samples, variable relationship assessment was done by using the Pearson-rank correlation test. Discussed tests were performed using the corresponding functions within the Estats v.4.2.2 package. The stats package is part of R. To assess whether the mean values differed significantly between groups that were unbalanced in both size and variance, a permutation-based t test was performed (999 permutations) using the t.perm.R function.

No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Data availability
All genomic data utilized in this study is publicly available. 236 MAG IDs and their NCBI accession numbers are provided in Supp. Data S1. NCBI SRA IDs are provided for 14,644 metagenomes in Supp. Data S5. All additional important data supporting the study’s conclusions are included in this publication and its Supp. Data 2–15. Source data are provided with this paper.

Code availability
The source code generated in this study is publicly available on GitHub at https://github.com/MiELevog/Cross-ecosystem-colonization [https://doi.org/10.5281/zenodo.18416940]91.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "OVERVIEW: This paper explores the genomic and phylogenetic properties of a set of related Chloroflexi lineages spanningi lineages spanning\nmultiple ecosystem types. The authors establish a robust phylogenetic framework for these lineages and undertake a series\nof analyses of genomic characteristics, genomic architecture, and to some extent, metabolic capacities to ostensibly\nilluminate the processes governing their transitions across ecological boundaries. While I commend the authors on an\nintriguing paper, I find numerous major conceptual issues that should be addressed before publication.\nThe main issue I perceive, as detailed in the below MAJOR COMMENTS, is that the paper relies far too heavily on\nspeculative statements about essentially unknowable past evolutionary events. Throughout the text, the authors confidently\nassert a particular evolutionary path for their lineage of interest, which I do not believe to be fully substantiated by the\nevidence presented. Relatedly, the authors fail to explore alternative explanations for the ecological patterns they observe,\nseemingly relying heavily on a favored hypothesis about hypermutation as a result of the loss of one particular set of DNA\nrepair genes. However, alternative repair systems, ecological pressures, or metabolic changes are not explored. To improve\nthe robustness of the work, I recommend drastically revising the text to reduce causal/speculative language (focusing more\non traits associated with habitat transition, rather than those causing it) and exploring alternative explanations for the\nintriguing correlations observed.\nUnfortunately, this may also mean less solid ground to stand on in terms of ‘reconcil[ing] adaptive and non-adaptive\nparadigms’ applicable more generally to Bacteria. While I appreciate the effort to assess other lineages, the generalizability of the study would be increased if this section was further fleshed out.\nMAJOR COMMENTS:\nl143 - do sediment-associated lineages within CSP1-4 represent coherent phylogenetically coherent clade(s)? while soil\nand sediment habitats clearly have structural similarities, the transition to (aquatic?) sediments may still entail\ngenomic/metabolic change (e.g. acclimation to low oxygen, depending on the aquatic system/sediment stratum). this habitat\ntransition is currently not really explored in the text but doing so would give us a broader sense for the processes at play\nwithin this lineage.\nfig 1 - in general, i think adding habitat affiliation to the tree on a per-species basis, if possible, would help the reader to\nbetter grasp where habitat transition events are likely to have occurred over evolutionary time (for both Limno and CSP1-4).\nline 184 - when making this claim it is important to qualify with the degree of phylogenetic novelty accounted for by the new\ngenomes. how many distinct species, genera, etc.. are included within this 68%? A large number of genomes from 1 or 2\nheavily sampled species might be less significant than a broader sampling effort in this case.\nline 207-209 - while i am (tentatively) on board with the likening of ancestral genomic features based on short branch lengths\nwithin a ribosomal tree (though i think it would be safer to just stick to overall ‘divergence’, rather than speculating about\nspecific features), inferring that modern lineages experience similar eco-evolutionary pressures to more ancient ones is too\nspeculative, at least for the results section.\nline 223 - what is meant by ‘align’? I think one could safely say correlate…\nline 226 - again, it is unclear to me how methods based on modern genome sequences allow you to speak to selection\npressures of the past.\nline 246 - is it possible that the acquired genes (or a subset of them) themselves explain the changes in GC? or is there\nevidence for GC remodeling more generally across the genome? this seems important to rule out among otherwise closely\nrelated species.\nline 298 - is there any precedent for these curve shapes/plots in the literature? I do not think there really is a justification for\nconnecting four discrete points with a trend line. if you must, then please visually indicate the points (is it just one per lineage\nper codon?)\nline 386 - while the hypothesis advanced here is interesting, it feels incomplete. are there other repair systems that could\ncompensate for the lack of dna glycosylases? it would seem important to rule this out before making the claim that this\nlineage is uniquely mutation-prone, and even further, that this mechanism may underlie genome-wide GC shifts, especially\nwithout supporting experimental evidence.\nline 405-408. What is the purpose of the DAPI stained image ? Or of the part of the graph that shows presence/absence of\nbacteriorhodopsin? As far as I can tell, neither of these are referenced in the main text and thus are confusing (also panels\nare mislettered). please either address in the main text or delete.\n410-413. Unfortunately, this sort of speculation has no place in a results section. what supports your claim that elevated\nmutagenesis led to the loss of AA biosynthetic pathways? from my perspective, the development of metabolic\ninterdependencies/interactions could just as easily explain this observation (see the CPR bacteria as an example). I would\nstrongly consider moving these sentences to the discussion section and motivating this analysis differently.\n497-99, 532-34. While an interesting hypothesis, again, I do not think you can confidently conclude this evolutionary\ntrajectory based on the evidence presented. The authors seem to focus on a favored hypothesis here (loss of DNA\nglycoyslase-> hypermutation/plasticity) but do not explore alternative hypotheses. what about the rest of the genome? What\nother metabolic differences are observed between the freshwater and soil/sediment clades? As the authors themselves note\nin the introduction, these changes too may help explain the ability of lineages to cross ecological boundaries. Without\nexploring these alternatives fully, the conclusion risks coming across as cherry-picked.\n510-516. Again, what evidence substantiates these claims about evolutionary dead ends? If there is literature you can cite in\nservice of this argument, please do so, otherwise this is entirely speculative and should be written less assertively.\nMINOR COMMENTS:\nl12 - maybe replace ‘unparalleled’ here. while of course dependent on bacteria, viruses are also present in nearly every\necosystem on earth as well.\nl17 - what is meant by ‘integrative’? perhaps clearer to phrase as ‘integrating X and Y’...\nl133 - nice use of sandpiper here!\nl194 - why are sigma factors being singled out here, among all genomic features?", "reviewer_2": "This manuscript investigated the genomes of Limnocylindria, a deep branching clade of the Chloroflexota phylum.\nThe authors aimed to illuminate the mechanisms underpinning microbial evolution during major ecosystem transitions and\nfoundations of bacterial adaptability. They used a database of genomes to determine the directionality of ecosystem\ncolonization events by this lineage, and report that during the habitat transition from soil to sediment to freshwater, non-\nadaptive processes shaped genome size and GC content. Additionally, the purging of the DNA glycosylase gene pool\nallowed for flexible genomic architecture. Meanwhile, the adaptive mechanism of proteome streamlining supported the\nsurvival of the lineage in the new ecosystem(s).\nThe research represents important evolutionary work addressing how microbial species are able to transition from one\nenvironment to others, a knowledge gap with few well-demonstrated examples in the literature. Chloroflexota is a good\nlineage with adapted lineages to a variety of environments, making it ideal to study to address associated questions. Overall,\nthe methods are generally sound and the paper is well written.\nHowever, the manuscript does at times infer and speculate on mechanisms that drive the adaptability and survival of the\nlineage during ecosystem transitions, particularly in the results section. Specific examples are noted in the line specific\ncomments, and I would recommend a revision to address some of the perhaps overly speculative correlations. Additionally,\nit may be helpful to incorporate the results of additional methods to uncover ancestral genes using ancestral state\nreconstructions. I would recommend either a rephrasing of some inferences in the results/discussion, or the demonstration of\nthese processes with additional methods.\nSpecific comments:\nThroughout the manuscript, the terms non-adaptive vs adaptive selection is used. However, negative vs. positive selection,\nand purifying vs. diversifying selection are also terms that are used. Please forgive me if there was a purpose for all these\ndifferent terms in particular usages, but it would help a reader if one set of terms was consistent throughout the manuscript.\nLine 26: Related to Figure 2D - the GC content of the freshwater adapted clade is lower than its ancestors, is this truly\n\"exceptionally high\"?\nLine 68-82: What about Actinobacteria? See work such as Ghai et al., 2013 (Metagenomics uncovers a new group of low\nGC and ultra-small marine Actinobacteria)\nLine 129-132: Was this done with an outgroup?\nLine 152: Figure 1 - the green colors transitioning from A to B are kind of confusing given the two sister clades are red and\ngreen in A, but only green in B. Maybe indicate both clades as green in A?\nLine 201-209: This section in particular seems speculative - could be tested with ancestral state reconstruction.\nLine 242: What is species defined as? (ANI/AAI?)\nLine 248: No changes in what?\nLine 251: Not able to tell what are synonymous substitutions from Figure 2, unless all of the data depicted in Figure 2 is from\ndS? If so, this needs to be stated in the caption.\nLine 257: Figure 2D - could these differences be due to noise (not true signal) due to undersampling? Also, the GC content\nof the freshwater adapted clade is still lower than its ancestors (see line comment on the abstract).\nLine 282-285: This seems speculative - is higher GC content a consequence of small size? What is the mechanism there?\nLine 285: Consider discussing implications for \"Increased mutation rate is linked to genome reduction in Prokaryotes\"\n(Bourguigon et al., 2020) within this sentence.\nLine 288-289: Again no mechanistic explanation here.\nLine 290-308: Ok I see the reasoning behind some of the mechanism for the GC content changes driving the cell shape, but\nthe size link is still unclear in the previous paragraph.\nLine 389: Figure 4 - consider matching colors with figure 1.\nLine 442-449, and throughout: This section in particular seems like it belongs in a discussion section, not results (brings in\nprevious studies, and has a defined conclusion sentence) - consider revision of the text to separate results from discussion,\nor to have one combined results and discussion section. This applies to other sections throughout the results.\nLine 450: Figure 5 - again consider matching colors with figure 1 and 4, and others."}, "author_response": "Point-by-Point Response to Reviewers\nManuscript title: “Deep-branching Chloroflexota lineages illuminate\nthe eco-evolutionary foundation of cross-ecosystem colonization”\nDear Reviewers,\nWe are sincerely grateful for the time, care, and expertise you invested in reviewing our\nmanuscript. Your detailed and constructive comments have been invaluable in helping us\nreassess our analyses, clarify our exposition, and present our findings with greater\nprecision. We recognise that several of the concerns you raised stemmed from\nshortcomings in how we originally presented certain data and framed key arguments. In\nresponse, we have comprehensively reorganised the figures, enriched the\nmethodological descriptions, and tempered speculative language so that our conclusions\nare now articulated with full transparency and evidential support. We trust that these\nrevisions address your points in full and materially strengthen the manuscript.\nIn response to the comments received, we implemented the following major changes:\n1. Enhanced clarity and tone – We thoroughly revised the manuscript for improved\nreadability and precision, softening speculative language and ensuring claims are\nconsistently supported by data or literature.\n2. Refined focus – Following reviewer suggestions, we increased emphasis on the\nCSP1-4 clade, providing a clearer parallel with Limnocylindraceae and better\ncontextualizing their contrasting evolutionary trajectories.\n3. Metabolic resolution – We expanded our metabolic analyses, including new\nreconstructions and habitat-specific comparisons, particularly emphasizing the\necological and genomic distinctiveness of CSP1-4 and Limnocylindraceae.\n4. Ancestral state reconstruction – We incorporated new ancestral state\nreconstructions for both habitat and metabolic traits, adding mechanistic support\nfor our hypotheses on gene gain, loss, and adaptation across transitions.\n5. Strengthened mechanistic inference – We added literature-supported biochemical\nevidence (e.g., from DNA glycosylase loss and hypermutator phenotypes) to\nbolster our proposed model of mutation-driven genome reduction in\nLimnocylindraceae.\nBelow, we respond to each of your comments in sequence, explaining how your feedback\nhas been incorporated and where additional analyses or clarifications have been added.\nWe quote the reviewers’ remarks in italics and present our replies in regular text (rendered\nin dark blue). All line numbers cited correspond to the revised manuscript version\nwith tracked changes.\nComment 1: “OVERVIEW: This paper explores the genomic and phylogenetic properties\nof a set of related Chloroflexi lineages spanning multiple ecosystem types. The authors\nestablish a robust phylogenetic framework for these lineages and undertake a series of\nanalyses of genomic characteristics, genomic architecture, and to some extent, metabolic\ncapacities to ostensibly illuminate the processes governing their transitions across\necological boundaries. While I commend the authors on an intriguing paper, I find\nnumerous major conceptual issues that should be addressed before publication.\nThe main issue I perceive, as detailed in the below MAJOR COMMENTS, is that the paper\nrelies far too heavily on speculative statements about essentially unknowable past\nevolutionary events. Throughout the text, the authors confidently assert a particular\nevolutionary path for their lineage of interest, which I do not believe to be fully\nsubstantiated by the evidence presented. Relatedly, the authors fail to explore alternative\nexplanations for the ecological patterns they observe, seemingly relying heavily on a\nfavoured hypothesis about hypermutation as a result of the loss of one particular set of\nDNA repair genes. However, alternative repair systems, ecological pressures, or metabolic\nchanges are not explored. To improve the robustness of the work, I recommend drastically\nrevising the text to reduce causal/speculative language (focusing more on traits associated\nwith habitat transition, rather than those causing it) and exploring alternative explanations\nfor the intriguing correlations observed.\nUnfortunately, this may also mean less solid ground to stand on in terms of ‘reconcil[ing]\nadaptive and non-adaptive paradigms’ applicable more generally to Bacteria. While I\nappreciate the effort to assess other lineages, the generalizability of the study would be\nincreased if this section was further fleshed out.”\nResponse 1: We sincerely thank Reviewer 1 for this comprehensive overview. Your\ncomments have been instrumental in refining both the presentation and the clarity of our\nstudy. In response, we have undertaken four overarching improvements:\n1) Calibrated interpretation. We have replaced definitive assertions with conditional\nwording—explicitly stating assumptions and using qualifiers (e.g., “likely,”\n“suggests,” “plausible”) wherever inferences derive from indirect evidence.\n2) Robustness evaluations. We confirmed that our principal findings remain\nconsistent when phylogenies are inferred under alternative substitution models\nand when analyses are repeated on jack-knifed genome subsets (see Suppl. Fig.\nS11).\n3) Ancestral-state reconstructions. We performed stochastic character mapping of\nhabitat affiliations and metabolic traits to test alternative evolutionary scenarios\nand reinforce statistical support for the soil → sediment → freshwater transition.\n4) Trait-focused narrative. Following your suggestion, we now emphasize\nobservable phenotypic and ecological traits over speculative causal mechanisms\nand have expanded the comparative analysis of the CSP1-4 sister clade to\ncontextualize transitional patterns.\nWe will address each specific point you raised in turn below, citing revised line numbers\nand figures to illustrate the exact changes. We hope these overarching revisions, together\nwith the detailed, point-by-point responses that follow, fully address your concerns.\nphylogenetically coherent clade(s)? while soil and sediment habitats clearly have structural\nsimilarities, the transition to (aquatic?) sediments may still entail genomic/metabolic\nchange (e.g. acclimation to low oxygen, depending on the aquatic system/sediment\nstratum). this habitat transition is currently not really explored in the text but doing so\nwould give us a broader sense for the processes at play within this lineage.”\nResponse 2: We appreciate the reviewer’s thoughtful feedback regarding the CSP1-4\nlineage. We concur that habitat transitions, particularly from soil to sediment, are likely\naccompanied by genomic and metabolic adaptations within CSP1-4 lineages. Although\ninformation pertaining to taxonomy and habitat specificity was previously presented in the\nSupplementary Information (Supp. Data S1), we acknowledge that these findings were not\nexplicitly discussed in the main text.\nIn response to the reviewer’s suggestion, we conducted additional metabolic and\ngenomic analyses specifically targeting CSP1-4 members. These new analyses reveal that\nsoil-, sediment-, and freshwater-associated species form phylogenetically coherent\nsubclades (i.e., species), each characterized by habitat-dependent genomic and\nmetabolic adaptations. To more prominently present these findings, we have substantially\nexpanded the main text (lines 241–266), introduced a new figure (Fig. 2), and added a new\nsupplementary information file (Supp. Data S12). The following text has been incorporated\ninto the manuscript:\n“The CSP1-4 family comprises 16 genera and 51 strictly habitat-specific species—35 from\nsoil, 4 from sediment, and 11 from freshwater. Genome-scale metabolic reconstructions,\nevaluated via a pairwise similarity matrix, revealed the greatest metabolic overlap between\nsoil and sediment lineages (Fig. 2a,b). Soil genomes uniquely encoded enzymes for\ndepolymerising plant polysaccharides and polyols (e.g., sucrose phosphorylase, 6-\nphospho-β-glucosidase, inosose dehydratase), along with nutrient-stress regulators (cstA,\nphoH/phoL, and phoB), reflecting adaptation to complex carbon inputs and episodic\ncarbon–phosphate scarcity (Supp. Data S12). In contrast, sediment lineages harboured the\ncatalytic α- and β-subunits of the nitrate reductase/nitrite oxidoreductase complex, a\ncomplete nitrate/nitrite ABC transporter, and the high-affinity cytochrome d terminal\noxidase, consistent with respiration in oxygen-poor, nitrate-rich sediments. Freshwater\nrepresentatives were distinguished by expanded flagellar and chemotaxis loci—traits rare in\nChloroflexota—suggesting motility-driven navigation of heterogeneous aquatic niches\n(Supp. Data S12). Genomic architecture differed consistently among habitats, reflecting\ndistinct ecological constraints (Fig. 2c-f). Freshwater genomes had the largest estimated\nsizes and encoded markedly fewer sigma factors than those from soil or sediment,\nindicative of regulatory streamlining in planktonic environments. GC content and coding\ndensity were elevated in both sediment and freshwater lineages relative to soil\nrepresentatives, suggesting habitat-specific selection for more compact and information-\ndense genomes outside terrestrial ecosystems. Together, these habitat-specific patterns in\ngene content and genome structure support distinct ecological strategies shaped by\ncarbon availability, nutrient dynamics, and redox conditions across terrestrial and aquatic\nenvironments (further details on Limnocylindria metabolism are provided in the\nSupplementary Discussion).”\nbasis, if possible, would help the reader to better grasp where habitat transition events are\nlikely to have occurred over evolutionary time (for both Limno and CSP1-4).”\nResponse 3: We thank the reviewer for this valuable suggestion. In response, we have\namended Fig. 1 to incorporate per-species habitat annotations and expanded our\nanalytical framework to infer ancestral habitat transitions:\nA. Tip-level habitat mapping\n• Each of the 50 terminal nodes is now marked with a habitat-specific color: grey\n(soil), purple (sediment), or orange (freshwater), making extant ecosystem\naffiliations immediately visible.\nB. Habitat ancestral-state reconstruction\n• We employed the fit.mk() function in phytools v2.0 package to fit three discrete-\nstate Markov models—Equal-Rates (ER), All-Rates-Different (ARD), and Symmetric\n(SYM)—to our maximum-likelihood tree (outgroup excluded).\n• Model selection based on log-likelihood and ΔAIC identified the ER model as the\nbest-supported.\n• Under the ER framework, we performed 1,000 stochastic mappings (with the\nmake.simmap() function) to estimate posterior probabilities of soil, sediment, and\nfreshwater states at each internal node.\n• Nodes in Fig. 1 are now additionally color-graded according to their mean\nposterior probabilities, providing a visual representation of ancestral habitat states.\nC. Materials and Methods\n• We have added a new subsection, “Ancestral State Reconstruction,” detailing\nhabitat-assignment criteria, model fitting, model selection procedures, and the\nstochastic mapping approach.\nD. We expanded the Fig. 1 legend (lines 200-204) to include the following: “Node insets\nrepresent pie charts. At each internal node, the inset pie chart shows the marginal posterior\nprobability of each ancestral habitat occupancy (inferred under the Equal-Rates model with\n1,000 stochastic mappings); slice areas are proportional to these probabilities, and colours\ncorrespond to habitat categories. Tip labels are coloured according to the habitat from\nwhich each genome was recovered.”\nWe believe that these enhancements substantially improve the manuscript by enabling\nreaders to trace habitat-transition events both visually and quantitatively, thereby\nproviding a deeper understanding of the evolutionary dynamics of environmental\nspecialization within Limnocylindria.\nComment 4: “line 184 - when making this claim it is important to qualify with the degree of\nphylogenetic novelty accounted for by the new genomes. how many distinct species,\ngenera, etc.. are included within this 68%? A large number of genomes from 1 or 2\nheavily sampled species might be less significant than a broader sampling effort in this case.”\nResponse 4: We thank the reviewer for emphasizing the importance of qualifying\nphylogenetic novelty beyond the number of genomes alone. Although these data were\npreviously included in Supp. Data S1, we agree they should be clearly stated in the main\n4\ntext. In response, we have now specified both (i) the number of newly sampled species\n(five) and (ii) the corresponding gain in phylogenetic diversity (Faith’s PD = 48.14%).\nThe revised sentence (lines 230-233) now reads: “…and Limnocylindraceae ( n = 67\ngenomes, representing five novel species), which increases the family’s genomic coverage\nby 68% and expands its phylogenetic diversity by 48.14%.”\nThe method used to calculate Faith’s PD was introduced into the Methods section. The\nphylogenetic reconstruction underlying this analysis is presented in Supp. Fig. S12. We\nbelieve this revision makes the scope of taxonomic and phylogenetic novelty explicit and\nfully addresses the reviewer’s concern.\nComment 5: “line 207-209 - while i am (tentatively) on board with the likening of ancestral\ngenomic features based on short branch lengths within a ribosomal tree (though i think it\nwould be safer to just stick to overall ‘divergence’, rather than speculating about\nspecific features), inferring that modern lineages experience similar eco-evolutionary\npressures to more ancient ones is too speculative, at least for the results section.”\nResponse 5: We thank the reviewer for this careful observation. We fully agree that the\noriginal phrasing overreached in linking short branch lengths to specific ancestral\ngenomic features, and in drawing parallels between ancient and contemporary eco-\nevolutionary pressures. These interpretations were speculative and not sufficiently\nsupported by the available data.\nIn response, we have removed the relevant sentences and revised the paragraph to focus\nsolely on empirical observations of branch lengths, positive selection, and genomic\nsignatures within CSP1-4. The revised text now reads as follows (lines 294-318):\n“Phylogenetic analyses reveal that Limnocylindraceae shares a common ancestry with the\nCSP1-4 clade (Fig. 1a). The latter occupies an exceptionally short branch stemming from its\nmost recent common ancestor—the shortest in our phylogeny—indicating reduced\nevolutionary change from the ancestral state (0.087 substitutions per site since\ndivergence). To further understand the evolutionary landscape that may have favoured the\nemergence of Limnocylindraceae, we conducted clade-specific selection force analyses on\na set of approximately 800 genes conserved across the four clades. We observed that\nCSP1-4 exhibits a 60% increase in the number of genes under diversifying (positive)\nselection compared to its ancestral lineage (i.e., QHBO01), along with a 25% increase in\nthe number of sites under positive selection (Supp. Table S1). The elevated levels of\npositive selection observed in CSP1-4, the highest among the four clades, suggest that this\nlineage is undergoing adaptive evolution, characterized by an accumulation of non-\nsynonymous substitutions. Additionally, the relaxation of selective pressure in CSP1-4 is\ncomplemented by an increased transposase density, a higher number of redundant\nproteins, and larger intergenic spacer regions—the longest among the four clades (Fig. 1b,\nSupp. Fig. S3). Together, these factors indicate decreased negative (purifying) selection\nand an enhanced potential for adaptive evolution15,33,34. These observations align with the\nhabitat expansion of CSP1-4 in sediment and aquatic ecosystems.”\nComment 6: “line 223 - what is meant by ‘align’? I think one could safely say correlate…”\nReply 6: We thank the reviewer for this helpful clarification. In response, we have replaced\n“align” with “correlate” in line 317.\nsequences allow you to speak to selection pressures of the past.”\nReply 7: We thank the reviewer for this important point. As noted in our response to\nComment 5, we have revised the relevant section to remove speculative interpretations\nabout historical selection pressures. The updated text now focuses exclusively on\nobserved genomic signatures in extant lineages, without extending inferences to ancestral\nconditions.\nComment 8: “line 246 - is it possible that the acquired genes (or a subset of them)\nthemselves explain the changes in GC? or is there evidence for GC remodeling more\ngenerally across the genome? this seems important to rule out among otherwise closely\nrelated species.”\nResponse 8: We agree with the reviewer that horizontally acquired genes—or a subset\nthereof—could plausibly contribute to the observed changes in GC content. To assess this,\nwe compared the GC content of species-specific genes with that of the shared core\ngenome. This analysis did not reveal substantial differences between the two categories,\nsuggesting that the increase in GC content is not driven by the acquisition of genes with\natypical base composition.\nAs this analysis did not yield additional insight beyond confirming genome-wide trends,\nwe now present it in Supp. Fig. S13. We have also updated the main text to clarify that\nspecies-specific gene sets do not differ in GC content from conserved genes (lines 349-\n350): “no substantial differences in GC content were observed between species-specific\ngenes and the shared core genome.”\nComment 9: “line 298 - is there any precedent for these curve shapes/plots in the literature? I do not think there really is a justification for\nconnecting four discrete points with a trend line. if you must, then please visually indicate the points (is it just one per lineage\nper codon?)”.\nResponse 9: We thank the reviewer for this thoughtful observation and agree that our\noriginal presentation may have inadvertently implied a level of continuity not supported\nby the underlying data. Our intention was not to depict a formal statistical trend, but rather\nto illustrate a conceptual pattern—specifically, the funnel- or wine-bottle-shaped\ndistributions frequently observed across Limnocylindria lineages, as shown in Supp. Figs.\nS6 and S7.\nTo address the reviewer’s concern, we revised the figure (Fig.4) to explicitly display\nindividual data points and replaced the continuous lines with dotted curves to minimize\nthe risk of visual overinterpretation. Additionally, the figure legend has been updated to\nclarify that the plotted curves are conceptual representations, derived from the amino acid\ncodon frequency distributions shown in Supp. Figs. S6 and S7 (lines 442-444): “The dotted\ntrendlines are conceptual representations, derived from the amino acid codon frequency\ndistributions shown in Supp. Figs. S6 and S7.”\nWe believe these revisions improve clarity and more accurately convey the nature of the\nunderlying data, and we thank the reviewer for their helpful suggestion.\nComment 10: “line 386 - while the hypothesis advanced here is interesting, it feels\nincomplete. are there other repair systems that could compensate for the lack of dna\n6\nglycosylases? it would seem important to rule this out before making the claim that this\nlineage is uniquely mutation-prone, and even further, that this mechanism may underlie\ngenome-wide GC shifts, especially without supporting experimental evidence.”\nResponse 10: We appreciate this critical point and agree that a robust mechanistic\nfoundation is needed. In response, we have clarified the lesion-specific role of the missing\nDNA glycosylases and added comparative evidence from model systems and natural\nlineages where similar losses have been studied.\n(i) Non-redundancy of base excision repair (BER) enzymes:\nThe lesions handled by Tag (K01246), AlkA (K01247) and MPG (K03652) (small N-alkyl\nadducts), family-4 UDG (TIGR03914), Ung (K03648) and Mug (K03649) (uracil in DNA),\nNei (K05522) (oxidised pyrimidines) and the nucleotide-sanitising enzyme MutT\n(K03574) (pre-emptive hydrolysis of 8-oxo-dGTP) are all small, helix-stable\nmodifications that are poor substrates for the general repair systems. Bacterial nucleotide-\nexcision repair (UvrABC) excels at bulky distortions and can remove thymine glycol, but\ndisplays negligible activity toward 3-methyl-guanine, uracil, or uracil-containing\nmismatches. DNA mismatch repair corrects a subset of U·G mispairs yet cannot process\nU·A or excise damaged bases directly, and therefore still depends on a DNA glycosylase\nfor final lesion removal. Direct-reversal enzymes such as AlkB oxidatively demethylate only\na limited subset of alkyl lesions (e.g. 1-methyl-A, 3-methyl-C) and do not compensate for\nthe broader specificity of Tag/AlkA/MPG. Homologous recombination and translesion\nsynthesis promote survival at stalled forks but leave the original chemical modification\nintact, requiring subsequent base-excision repair. As the authors are aware, no other\npathway—alone or in combination—restores the full spectrum of activities provided by the\nsimultaneous presence of these eight BER genes, underscoring why their concerted loss\ncannot be fully compensated in bacterial genomes.\n(ii) Empirical evidence for mutagenesis and GC erosion upon glycosylase loss:\n• In E. coli, loss of the two 3-methyladenine DNA glycosylases Tag (K01246) and\nAlkA (K01247) produces an ≈10- to 20-fold rise in the spontaneous mutation rate\nand pronounced hypersensitivity to methyl-methanesulphonate (MMS), consistent\nwith unrepaired 3-methyl-guanine that yields G → A transitions (Grzesiuk et al.,\n2001).\n• Deletion of ung (K03648) increases G·C → A·T transitions about 30-fold because\nuracil generated by cytosine deamination is left in the genome (Duncan & Weiss,\n1982).\n• A deficiency of the nucleotide-sanitising phosphohydrolase MutT (K03574)\nelevates overall mutation frequencies by 10²–10⁴-fold, with a diagnostic A·T → C·G\ntransversion spectrum that stems from misincorporation of 8-oxo-dGTP (Bessman\net al., 1996).\n• Although the oxidised-pyrimidine glycosylase Nei (K05522) partially overlaps with\nEndoIII/Nth, nei single mutants (with nth still present) are still measurably more\nsensitive to H₂O₂ and γ-irradiation and show a modest (≈2–3-fold) increase in GC\n→ TA transitions, indicating that lesions such as thymine glycol and 5-hydroxy-\ncytosine are not fully repaired (Jiang et al., 1997).\ncases eroding GC by the following —G → A, G·C → A·T, A·T → C·G. Their documented\nmagnitudes demonstrate that, even with Nth present, the simultaneous absence of Tag,\nAlkA, MPG (K03652), family-4 UDG (TIGR03914), Ung, Mug (K03649), Nei and MutT would\nleave no bacterial repair pathway capable of fully compensating for the resulting load of\nalkylated, deaminated and oxidised bases.\n(iii) Relevance to Limnocylindraceae:\nIn this lineage, the simultaneous loss of DNA glycosylases that target the three principal\nlesion classes—alkylation, deamination, and oxidation—constitutes a rare convergence of\nvulnerabilities. The attendant rise in G → A and G → T substitutions, together with the\nmarkedly reduced GC content, extensive genome reduction, and accelerated sequence\nevolution, all point to a mutational landscape dominated by unrepaired base damage.\nElevated mutation rate has repeatedly been linked to genome shrinkage in prokaryotes\n(Bourguignon et al., 2020), and experimental-evolution work with Salmonella typhimurium\nhas shown that strains lacking these repair activities can, by virtue of their underlying\nmutational bias, very rapidly lose GC content (Lind & Andersson, 2008).\nWe have expanded the Discussion accordingly (lines 653-665):\n“In Limnocylindraceae, the concerted loss of Tag-/AlkA-/MPG-mediated alkyl repair, UDG-\ndriven uracil repair, Nei-directed oxidation repair, and MutT nucleotide sanitising strips the\nlineage of its chief defences against the three most pervasive small-base lesions. Because\nnucleotide-excision repair, mismatch repair, homologous recombination and AlkB-type\ndioxygenases cannot excise 3-methyl-guanine, uracil, thymine glycol or 8-oxo-dGTP with\ncomparable efficiency, these lesions will likely persist into replication. Laboratory strains\ndeprived of the same activities manifest 10- to 10⁴-fold mutator phenotypes and strongly\nGC-eroding spectra—G→A (tag alkA), G·C→A·T (ung) and GC→TA (nei) (Bessman et al.,\n1996; Duncan & Weiss, 1982; Grzesiuk et al., 2001; Jiang et al., 1997). Such biases furnish\na direct mechanistic path to the lineage’s AT-enrichment and gene loss, echoing broad\nphylogenomic evidence that links elevated mutation pressure to bacterial genome\nreduction (Bourguignon et al., 2020) and the rapid GC erosion produced in BER-deficient\nSalmonella typhimurium evolution experiments (Lind & Andersson, 2008).”\nReferences:\nBessman, M. J., Frick, D. N., & O’Handley, S. F. (1996). The MutT proteins or “Nudix”\nhydrolases, a family of versatile, widely distributed, “housecleaning” enzymes. Journal of\nBiological Chemistry, 271(41), 25059–25062. https://doi.org/10.1074/jbc.271.41.25059\nDuncan, B. K., & Weiss, B. (1982). Specific mutator effects of ung (uracil-DNA\nglycosylase) mutations in Escherichia coli. Journal of Bacteriology, 151(2), 750–755.\nhttps://doi.org/10.1128/JB.151.2.750-755.1982\nGrzesiuk, E., Gozdek, A., & Tudek, B. (2001). Contribution of E. coli AlkA, TagA\nglycosylases and UvrABC-excinuclease in MMS mutagenesis. Mutation\nResearch/Fundamental and Molecular Mechanisms of Mutagenesis, 480–481, 77–84.\nhttps://doi.org/10.1016/S0027-5107(01)00171-3\nJiang, D., Hatahet, Z., Blaisdell, J. O., Melamede, R. J., & Wallace, S. S. (1997).\nEscherichia coli endonuclease VIII: Cloning, sequencing, and overexpression of the nei\n8\nstructural gene and characterization of nei and nei nth mutants. Journal of Bacteriology,\n179(11), 3773–3782. https://doi.org/10.1128/JB.179.11.3773-\n3782.1997;JOURNAL:JOURNAL:JB;WGROUP:STRING:PUBLICATION\nLind, P. A., & Andersson, D. I. (2008). Whole-genome mutational biases in bacteria.\nProceedings of the National Academy of Sciences of the United States of America,\n105(46), 17878–17883.\nhttps://doi.org/10.1073/PNAS.0804445105/SUPPL_FILE/0804445105SI.PDF\nComment 11: “line 405-408. What is the purpose of the DAPI stained image ? Or of the\npart of the graph that shows presence/absence of bacteriorhodopsin? As far as I can tell,\nneither of these are referenced in the main text and thus are confusing (also panels are\nmislettered). please either address in the main text or delete.”\nResponse 11: We thank the reviewer for drawing attention to this oversight. We agree\nthat these elements were confusing and insufficiently integrated into the main text. This\nissue likely arose during a reshuffling of content between the main manuscript and the\nSupplementary Material. In response, we have removed the bacteriorhodopsin\npresence/absence panel and the DAPI-stained microscopy image from the main figure.\nThe microscopy image is now included in the Supplementary Material (Supp. Fig. S10),\nwhere it is briefly described. We have also corrected the panel lettering accordingly.\nComment 12: “410-413. Unfortunately, this sort of speculation has no place in a results\nsection. what supports your claim that elevated mutagenesis led to the loss of AA\nbiosynthetic pathways? from my perspective, the development of metabolic\ninterdependencies/interactions could just as easily explain this observation (see the CPR\nbacteria as an example). I would strongly consider moving these sentences to the\ndiscussion section and motivating this analysis differently.”\nResponse 12: We thank the reviewer for this valuable feedback and agree that the causal\nlink between elevated mutagenesis and loss of amino acid biosynthesis was too\nspeculative for the Results section. We have now restructured the text accordingly. In the\nrevised Results (lines 538-549), we present the observed biosynthetic deficiencies in\nLimnocylindraceae descriptively and without interpretation as follows:\n“Limnocylindraceae genomes are missing the core enzymes for six canonical essential-\namino-acid pathways (Fig. 4a), indicating a substantial, though not complete, contraction of\ntheir biosynthetic repertoire. This partial auxotrophy likely heightens dependence on the\nexternal amino acids or peptide-rich organic matter. Freshwater lakes, which often contain\ndissolved amino acids and oligopeptides, provide a potential source of these essential\nsubstrates(Krempaska et al., 2021). However, it remains unclear the extent to which reliance\non external amino acids shaped the proteomic composition of Limnocylindraceae.”\nComment 13: “497-99, 532-34. While an interesting hypothesis, again, I do not think you\ncan confidently conclude this evolutionary trajectory based on the evidence presented. The\nauthors seem to focus on a favored hypothesis here (loss of DNA glycoyslase->\nhypermutation/plasticity) but do not explore alternative hypotheses. what about the rest of\nthe genome? What other metabolic differences are observed between the freshwater and\nsoil/sediment clades? As the authors themselves note in the introduction, these changes\ntoo may help explain the ability of lineages to cross ecological boundaries. Without\nexploring these alternatives fully, the conclusion risks coming across as cherry-picked.”\nResponse 13: We thank the reviewer for this thoughtful and constructive comment. We\nagree that our previous framing placed disproportionate weight on the hypermutation\nhypothesis and underrepresented other mechanisms relevant to ecological adaptation. In\nresponse—building on Comment 2 and integrating material from the Supplementary\nDiscussion—we have revised the Discussion section to present a more balanced view of\nevolutionary trajectories within the Limnocylindraceae and CSP1-4 clades. Specifically:\n1. We have expanded our treatment of adaptive processes in CSP1-4, highlighting\nhabitat-specific gene acquisitions, increased positive selection, and genome\nexpansion as key features of their transition into sediment and aquatic\nenvironments.\n2. We now present elevated mutagenesis in Limnocylindraceae not as the primary\ncause of ecological success, but as a mechanism that may have accelerated\ngenome shrinkage, enabling more rapid loss of biosynthetic functions\ndispensable in freshwater environments.\nThis revised framing emphasizes that niche-directed genome evolution is likely the\nunifying theme, with CSP1-4 and Limnocylindraceae representing two contrasting\nevolutionary states: one based on functional innovation and expansion, the other on\nmutational pruning and metabolic simplification. Ancestral state reconstructions\n(Supp. Fig. S14) reinforces this dichotomy, showing CSP1-4 gains in flagellar assembly,\nchemotaxis and lysine biosynthesis, versus Limnocylindraceae gains of\nbacteriorhodopsins accompanied by losses of eight amino-acid biosynthetic pathways.\nWe believe these revisions offer a more integrative and accurate interpretation of our\nfindings. We have added the following paragraph to the discussion section (lines 637-657)\n:\n“Ecological transitions in Limnocylindraceae and CSP1-4 exemplify niche-directed genome\nevolution28, yet the sister clades appear to sit at opposite ends of that continuum. CSP1-4\npresents genomic signatures consistent with diversifying selection accompanied by net\ngene acquisition. Soil representatives retain a broad plant-polymer catabolic arsenal;\nsediment-dwelling taxa carry additional nitrate-respiratory modules and microaerobic\nterminal oxidases; and freshwater representatives uniquely encode flagellar and\nchemotaxis loci—features uncommon in other Chloroflexota33 (Supp. Data S12). By\ncontrast, Limnocylindraceae shows only modest habitat-linked gene gains, most notably\nthe acquisition of proton-pumping rhodopsins (Supp. Discussion).\nThis asymmetry may reflect divergent responses to relaxed selective constraint. In\nLimnocylindraceae, such relaxation appears to have permitted the accumulation of loss-of-\nfunction mutations in DNA repair genes—including Tag/AlkA/MPG, UDG-family, Nei, and\nMutT (Fig. 4a)—leading to the degradation of base excision repair (BER) pathways that\ndefend against alkylated, deaminated, and oxidized bases. The resulting hypermutator\nstate likely accelerated GC content erosion and contributed to the loss of multiple\nbiosynthetic pathways (Fig. 5b). Thus, while CSP1-4 seems to have navigated habitat\ntransitions through adaptive gene gain and genome expansion, Limnocylindraceae\nappears to have undergone reductive genome evolution and metabolic simplification\nunder sustained mutation pressure following BER collapse.”\nevolutionary dead ends? If there is literature you can cite in service of this argument, please\ndo so, otherwise this is entirely speculative and should be written less assertively.”\nResponse 14: We thank the reviewer for this important observation. We agree that the\noriginal wording was too assertive and suggestive of determinism. In response, we have\nrevised this section of the text to frame the concept of evolutionary constraint more\ncautiously and to clarify that the “cul-de-sac” metaphor refers to a potential reduction in\nfuture adaptive flexibility, rather than a definitive evolutionary impasse.\nWe have also added citations to studies in microbial systems where genome reduction\nand hypermutator phenotypes have been associated with diminished long-term\nevolvability (e.g., Prochlorococcus, Pseudomonas aeruginosa), and now emphasize that\nsuch outcomes are context-dependent and probabilistic rather than certain.\n“Having shed a large part of its metabolic repertoire (Fig. 4bc) and accumulated extensive\nsequence changes (Fig. 2), Limnocylindraceae exhibits a highly reduced genomic\narchitecture. While this streamlining likely facilitated niche specialization, the combination\nof genome shrinkage (Fig. 1b) and elevated mutation pressure (Fig. 2a) may also limit the\nlineage’s capacity to explore new adaptive landscapes. Similar trade-offs have been\ndocumented in other microbial systems: in Prochlorococcus, genome reduction restricts\nphysiological plasticity and niche range (Marais et al., 2008; Ulloa et al., 2021), while\nhypermutator strains of Pseudomonas aeruginosa lose fitness when introduced to new\nenvironments (Hall et al., 2022). Thus, although Limnocylindraceae remains evolutionarily\nviable, its current genomic configuration may reduce future evolutionary flexibility—a\npossible example of the tension between hyperadaptability and hyperspecialization\n(Wielgoss et al., 2013).”\nComment 15: “l12 - maybe replace ‘unparalleled’ here. while of course dependent on\nbacteria, viruses are also present in nearly every ecosystem on earth as well.”\nResponse 15: We thank the reviewer for this helpful suggestion. We have replaced\n“unparalleled” with “remarkable” to provide a more accurate and balanced description of\nbacterial adaptability.\nComment 16: “l17 -what is meant by ‘integrative’? perhaps clearer to phrase as ‘integrating X\nand Y’...”\nResponse 16: We thank the reviewer for this helpful suggestion. In line with this and other\nrelated comments, we have revised the abstract to replace the term “integrative” with\n“comparative genomics”, which more precisely reflects our methodological approach\n(lines 15-16).\nComment 17: “l133 - nice use of sandpiper here!”\nResponse 17: We thank the reviewer for the kind remark.\nComment 18: “l194 - why are sigma factors being singled out here, among all genomic\nfeatures?”\nResponse 18: We thank the reviewer for this thoughtful question. Sigma factors are\nhighlighted here because they are a well-established proxy for transcriptional regulatory\ncomplexity and ecological versatility. A reduction in sigma factor number often reflects\n11\ndiminished regulatory plasticity and is associated with specialization to narrow or stable\nniches. This trend has been documented in multiple genome-reduced bacteria, including\nCandidatus Pelagibacter ubique and Buchnera aphidicola, which retain only two sigma\nfactors compared to the broader sets found in their free-living relatives. The loss of\nalternative sigma factors frequently parallels the broader elimination of accessory and\nstress-response genes during genome reduction. In this context, we believe that including\nsigma factor counts is both informative and relevant to the broader patterns of metabolic\nstreamlining and ecological adaptation discussed in the manuscript.\nWe added the following at lines 258-259: “...sigma factors (proxies for transcriptional\nregulatory complexity and ecological versatility)...”\nREVIEWER 2\nComment 1: “The research represents important evolutionary work addressing how\nmicrobial species are able to transition from one environment to others, a knowledge gap\nwith few well-demonstrated examples in the literature. Chloroflexota is a good lineage with\nadapted lineages to a variety of environments, making it ideal to study to address\nassociated questions. Overall, the methods are generally sound and the paper is well\nwritten.”\nResponse 1: We thank the reviewer for their thoughtful and encouraging comments. We\nare pleased that the evolutionary scope of our study, the selection of Chloroflexota, and\nthe overall execution of the work were well received.\nComment 2: “However, the manuscript does at times infer and speculate on mechanisms\nthat drive the adaptability and survival of the lineage during ecosystem transitions,\nparticularly in the results section. Specific examples are noted in the line specific\ncomments, and I would recommend a revision to address some of the perhaps overly\nspeculative correlations. Additionally, it may be helpful to incorporate the results of\nadditional methods to uncover ancestral genes using ancestral state reconstructions. I\nwould recommend either a rephrasing of some inferences in the results/discussion, or the\ndemonstration of these processes with additional methods.”\nResponse 2: We thank the reviewer for this constructive and thoughtful feedback. We\nagree that some interpretations in the original manuscript may have been stated too\nstrongly, particularly within the Results section. In response, we have carefully revised the\nrelevant passages throughout the manuscript, rephrasing or softening inferences where\nthey relied on indirect evidence. In addition, we have followed the reviewer’s suggestion\nto incorporate additional methods, including ancestral state reconstruction (see Methods;\nlines 933-959), to better support claims related to habitat transitions and gene loss. We\naddress each specific instance flagged in the line-by-line comments below and have\nrevised the manuscript accordingly.\nComment 3: “Throughout the manuscript, the terms non-adaptive vs adaptive selection is\nused. However, negative vs. positive selection, and purifying vs. diversifying selection are\nalso terms that are used. Please forgive me if there was a purpose for all these\n12\ndifferent terms in particular usages, but it would help a reader if one set of terms was consistent throughout the manuscript.”\nResponse 3: We thank the reviewer for this helpful observation. We agree that the use of\nmultiple terms to describe selective processes (e.g., adaptive vs. non-adaptive, positive vs.\nnegative, purifying vs. diversifying) may be confusing to readers. While we note that both\npositive (diversifying/Darwinian) and negative (purifying) selection can be adaptive\ndepending on context, we appreciate the need for terminological clarity. To simplify and\nstandardize our usage throughout the manuscript, we have adopted the terms\npositive/negative selection to describe the direction of selective pressure at the\nnucleotide/gene level, and adaptive/non-adaptive processes when referring to broader\nevolutionary frameworks. We believe this revision improves consistency and readability.\nComment 4: “Line 26: Related to Figure 2D - the GC content of the freshwater adapted\nclade is lower than its ancestors, is this truly \"exceptionally high\"?”\nResponse 4: We thank the reviewer for pointing this out. At line 26, our use of\n“exceptionally high” refers to the GC content of Limnocylindraceae (median 63.15%; n =\n(median estimated genome size ~1.31 Mbp).\nWe believe this is notable because low GC content is commonly associated with reduced\ngenome size in free-living bacteria, making the high GC content of Limnocylindraceae\n(~1.31 Mbp genome) an exception to this general trend. For comparison:\n• Ca. Actinomarinales (~1.1 Mbp; GC ~32–33%) (López-Pérez et al., 2020)\n• Ca. Pelagibacter ubique HTCC7211 (1.46 Mbp; GC 30.8%) (Stingl et al., 2007)\n• Prochlorococcus marinus MED4 (1.66 Mbp; GC 30.8%) (Dufresne et al., 2003)\n• SAR86 (~1.0 Mbp; GC 33–34%) (Roda-Garcia et al., 2023)\n• Ca. Nitrosopelagicus brevis CN25 (1.23 Mbp; GC 33.2%) (Santoro et al., 2015)\n• OM43 clade (Methylophilales HIMB624; 1.33 Mbp; GC 35.4%) (Huggett et al.,\n2012)\n• Ca. Fonsibacter ubiquis (1.16 Mbp; GC 29.02%) (Henson et al., 2018)\nRegarding Fig. 2d, we note that all Limnocylindrus (the only genus of Limnocylindraceae)\nspecies depicted are freshwater-adapted. While some internal variation is evident (e.g.,\nthe basal species at 66.8% vs. others at ~63.1%), the key point is that all members of the\ngenus maintain a GC content significantly higher than typically seen in genome-reduced\nbacteria.\nTo further clarify evolutionary patterns and address potential confusion, we have added\nancestral state reconstructions of habitat (Supp. Fig. S14; main nodes Fig. 1a) and now\nannotate habitat affiliations for all genomes in Fig. 1a. Additionally, Supp. Fig. S9 plots\ngenome size versus GC content for freshwater Limnocylindraceae in comparison with\nother genome-reduced bacterial lineages, providing broader context for the unusually\nhigh GC content observed in this family.\nReferences:\nLópez-Pérez, M., Haro-Moreno, J. M., Iranzo, J., & Rodriguez-Valera, F. (2020).\nGenomes of the “ Candidatus Actinomarinales” Order: Highly Streamlined Marine\nEpipelagic Actinobacteria . MSystems, 5(6).\nculturing yielded novel SAR11 strains and other abundant marine bacteria from the\nOregon coast and the Bermuda Atlantic Time Series study site. The ISME Journal, 1(4),\n361–371.\nDufresne, A., Salanoubat, M., Partensky, F., Artiguenave, F., Axmann, I. M., Barbe, V.,\nDuprat, S., Galperin, M. Y., Koonin, E. V., Le Gall, F., Makarova, K. S., Ostrowski, M., Oztas, S.,\nRobert, C., Rogozin, I. B., Scanlan, D. J., De Marsac, N. T., Weissenbach, J., Wincker, P., …\nHess, W. R. (2003). Genome sequence of the cyanobacterium Prochlorococcus marinus\nSS120, a nearly minimal oxyphototrophic genome. Proceedings of the National Academy\nof Sciences of the United States of America, 100(17), 10020–10025.\nRoda-Garcia, J. J., Haro-Moreno, J. M., Rodriguez-Valera, F., Almagro-Moreno, S., &\nLópez-Pérez, M. (2023). Single-amplified genomes reveal most streamlined free-living\nmarine bacteria. Environmental Microbiology, 25(6), 1136–1154.\nSantoro, A. E., Dupont, C. L., Richter, R. A., Craig, M. T., Carini, P., McIlvin, M. R.,\nYang, Y., Orsi, W. D., Moran, D. M., & Saito, M. A. (2015). Genomic and proteomic\ncharacterization of “Candidatus Nitrosopelagicus brevis”: An ammonia-oxidizing archaeon\nfrom the open ocean. Proceedings of the National Academy of Sciences of the United\nStates of America, 112(4), 1173–1178.\nHuggett, M. J., Hayakawa, D. H., & Rappé, M. S. (2012). Genome sequence of strain\nHIMB624, a cultured representative from the OM43 clade of marine Betaproteobacteria.\nStandards in Genomic Sciences, 6(1), 11–20.\nHenson, M. W., Lanclos, V. C., Faircloth, B. C., & Thrash, J. C. (2018). Cultivation and\ngenomics of the first freshwater SAR11 (LD12) isolate. The ISME Journal, 12(7), 1846.\nComment 5: “Line 68-82: What about Actinobacteria? See work such as Ghai et al., 2013\n(Metagenomics uncovers a new group of low GC and ultra-small marine Actinobacteria)”\nResponse 5: We thank the reviewer for drawing our attention to the work by Ghai et al.\n(2013) and for highlighting the role of Actinobacteria. Based on Fig. 1b of that study, a\nclose phylogenetic relationship is suggested between marine and freshwater clades\nwithin the Acidimicrobiia. To explore this further, we examined the habitat distribution of\nseveral orders within Acidimicrobiia as represented in the GTDB database (including\nActinomarinales, ATN_3, MCC26256, UBA2766, and UBA5794). While Actinomarinales\nappears to be restricted to marine environments, other related clades are found across a\nrange of aquatic and terrestrial habitats, including freshwater, sediments, and wastewater\nsystems. Although the precise directionality of the habitat transition (freshwater-to-marine\nor vice versa) remains unclear, we have included Actinomarinales as a potential example\nof a freshwater-marine transition in the revised manuscript, denoted as\n“…..Actinomarinales (freshwater to marine?)” to reflect this uncertainty (lines 84-85).\nComment 6: “Line 129-132: Was this done with an outgroup?”\nResponse 6: We thank the reviewer for this helpful observation. The phylogenomic\nreconstruction discussed in lines 129–132 was indeed rooted using an outgroup. To make\nthis clearer, we have revised the text at line 127 to read: “We built a rooted phylogenomic\ntree using maximum-likelihood methods…”. Additionally, while the legend of Fig. 1a\nalready indicated that the tree is rooted, we have now made this more explicit by adding:\n“Outgroup rooting was performed using members of the Eremiobacterota phylum.”\nprovided in the Methods section under the subheading “Evolutionary history\nreconstructions.”\nComment 7: “Line 152: Figure 1 - the green colors transitioning from A to B are kind of\nconfusing given the two sister clades are red and green in A, but only green in B. Maybe\nindicate both clades as green in A?”\nResponse 7: We thank the reviewer for this helpful suggestion. As advised, we have\nupdated Fig. 1a so that both sister clades are now depicted in green, ensuring visual\nconsistency with panel B. Additionally, we have enhanced Fig. 1a by including habitat\naffiliations at the tips of each branch and by annotating ancestral habitat states at major\nphylogenetic nodes. Full details of the ancestral state reconstruction are now provided in\nSupp. Fig. S14.\nComment 8: “Line 201-209: This section in particular seems speculative - could be tested\nwith ancestral state reconstruction.”\nResponse 8: We agree with the reviewer that the original phrasing was speculative. In\nresponse, we have revised this section to remove unsupported inferences. Additionally,\nwe performed an ancestral state reconstruction of key metabolic pathways, the results of\nwhich are now presented in Supp. Fig. S14. A detailed discussion of these findings is\nincluded in the Supplementary Discussion (lines 127-152 in the supplementary\ninformation).\nComment 9: “Line 242: What is species defined as? (ANI/AAI?)”\nResponse 9: We thank the reviewer for pointing this out. Species were defined as MAGs\nsharing >95% average nucleotide identity (ANI) and >70% conserved DNA, consistent\nwith widely accepted thresholds for bacterial species delineation (Jain et al., 2018). We\nhave now clarified this in the manuscript (lines 336–340), which reads:\n“Selection-pressure analyses of 52 MAGs representing four Limnocylindrus species—\ndefined by >95% average nucleotide identity and >70% conserved DNA—indicate that the\nbasal lineage contains both fewer genes and sites per gene under negative selection than\nits evolutionary younger, lower-GC relatives (Fig. 2a,c).”\nReference:\nJain, C., Rodriguez-R, L. M., Phillippy, A. M., Konstantinidis, K. T., & Aluru, S. (2018).\nHigh throughput ANI analysis of 90K prokaryotic genomes reveals clear species\nboundaries. Nature Communications 2018 9:1, 9(1), 1–8. https://doi.org/10.1038/s41467-\n018-07641-9\nComment 10: “Line 248: No changes in what?”\nResponse 10: We thank the reviewer for pointing out this ambiguity. By “no changes,” we\nwere referring to genes for which no nucleotide substitutions were detected across the\nalignments—i.e., genes that appear fully conserved within the dataset and are inferred to\nbe under strong purifying selection. To improve clarity, we have revised the sentence to\nexplicitly state this in the manuscript (lines 343-348).\n15\n“Thus, the approximately 3% decrease in GC content within Limnocylindrus is associated\nwith an increase in both the number of genes and the number of sites under negative\nselection, along with a reduction in the number of fully conserved (invariable) genes—\ndefined here as genes with no detectable nucleotide substitutions across the alignment,\nconsistent with strong purifying selection.”\nComment 11: “Line 251: Not able to tell what are synonymous substitutions from Figure 2,\nunless all of the data depicted in Figure 2 is from dS? If so, this needs to be stated in the\ncaption.”\nResponse 11: We thank the reviewer for this comment. The sentence has been revised to\nrefer specifically to Fig. 2c, where genes under negative selection are used to indicate the\npredominance of synonymous substitutions (i.e., nucleotide changes that do not alter the\nencoded amino acid and are typically interpreted as markers of negative selection). We\nalso replaced “purifying selection” with “negative selection” for consistency in\nterminology.\nThe sentence now reads as follows (lines 350-355): “This indicates that negative selection\naffects a larger portion of the genome, while an elevated mutation rate introduces more\nsequence variation, as evidenced by the predominance of synonymous substitutions—\nnucleotide changes that do not alter the amino acid sequence and are typically retained\nunder negative selection—in most genes (Fig. 2c).”\nComment 12: “Line 257: Figure 2D - could these differences be due to noise (not true signal) due to undersampling? Also, the GC content\nof the freshwater adapted clade is still lower than its ancestors (see line comment on the abstract).”\nResponse 12: To provide further clarification, Fig. 2d illustrates GC content dynamics\nwithin Limnocylindrus, the only currently known genus of the Limnocylindraceae family.\nBoth the genus and family are freshwater-adapted, as more clearly indicated in the revised\nFig. 1a.\nOne of the main purposes of Fig. 2d is to show that:\n(i) GC content decreases within Limnocylindrus lineages after genome reduction\nhas occurred. All clades depicted in Fig. 2d are part of the genome-reduced, freshwater-\nadapted Limnocylindraceae, and comparisons are made within this group.\n(ii) The decrease in GC content is more pronounced in genic regions than in\nintergenic spacers, likely due to the higher mutational target size of genes. In other words,\nlonger genic regions accumulate more GC-reducing mutations simply by probability.\nFig. 3 further supports this interpretation by showing that these GC-reducing mutations\nare enriched at third codon positions—typically synonymous sites—indicating that they\naccumulate without affecting protein function and likely contribute to the observed GC\ndecrease (Fig. 2a,c further substantiate this by showing how the number of sites/genes\nand genes under negative selection—mutations that do not alter amino acid identity—are\nincreasing in the 3 species with lower GC content).\nTo improve clarity for readers, we have revised the manuscript to explicitly label the\ntaxonomic level (\"genus\", \"family\", \"class\") and the ecological context (\"freshwater-\nadapted\") when referring to Limnocylindrus and Limnocylindraceae.\ndifferences in GC content between shared and species-specific genes, we conducted a\ntargeted comparison. As no substantial differences were found, this result is now included\nin Supp. Fig. S13.\nFinally, we note that our analysis is based on GC content measurements across\napproximately 36,000 genes and intergenic regions (from >10 MAGs per species), and\ntherefore, we do not believe that undersampling is a significant concern in this case.\nComment 13: “Line 282-285: This seems speculative - is higher GC content a\nconsequence of small size? What is the mechanism there? ”\nResponse 13: We thank the reviewer for this insightful comment. Our intention was not to\nsuggest that higher GC content is a direct consequence of small genome size, but rather\nto highlight an unexpected pattern in how GC content is distributed between coding and\nintergenic regions in freshwater-adapted Limnocylindraceae. As shown in Fig. 2d, the GC\ncontent decrease in this lineage is more pronounced in protein-coding regions than in\nintergenic spacers.\nThis is counterintuitive, as intergenic regions—generally being non-coding and under\nweaker selective pressure—are typically more susceptible to mutation-driven GC erosion\n(Ochman, 2003; Raghavan et al., 2012). This observation holds for the other clades: CSP1-\n4, QHBO01, and P2-11E (Supp. Fig. S5a).\nHowever, in Limnocylindraceae, we observe that intergenic regions are both extremely\nshort (median length: 12 bp, Supp. Fig. S4b) and higher in GC content than coding\nregions (median gene length: 810 bp). We interpret this pattern as a consequence of\nmutational pressure (Fig. 2a,c) acting more strongly on the larger coding regions, simply\ndue to their greater sequence length and thus greater probability of accumulating GC-\nreducing mutations. This contrasts with patterns observed in CSP1-4, QHBO01, and P2-\n11E (Supp. Fig. S5a), where coding regions are not disproportionately affected in this way.\nTo avoid confusion, we have revised the text accordingly and added the intergenic spacer\nlength to support our explanation.\nThe text at lines 386–392 now reads as follows:\n“By examining four representative species, we found that in freshwater-adapted\nLimnocylindraceae, intergenic regions—despite their short median length (12 bp)—\nconsistently exhibited higher GC content than coding regions (Fig. 2d). This pattern may\nreflect a reduced likelihood of mutation accumulation in these shorter sequences, as their\nlimited length presents a smaller mutational target compared to longer coding regions.”\nReferences:\nOchman, H. (2003). Neutral Mutations and Neutral Substitutions in Bacterial\nGenomes. Molecular Biology and Evolution, 20(12), 2091–2096.\nhttps://doi.org/10.1093/MOLBEV/MSG229\nRaghavan, R., Kelkar, Y. D., & Ochman, H. (2012). A selective force favoring\nincreased G+C content in bacterial genes. Proceedings of the National Academy of\nSciences of the United States of America, 109(36), 14504–14507.\nlinked to genome reduction in Prokaryotes\" (Bourguigon et al., 2020) within this sentence.”\nResponse 14: We thank the reviewer for this valuable suggestion. In response, we have\nrevised the relevant paragraph to more explicitly connect our findings with the framework\nproposed by Bourguignon et al. (2020). While that study does not specifically examine GC\ncontent, it establishes that genome reduction in prokaryotes is frequently linked to\nincreased mutation rates. Our observation of GC content reduction in coding regions of\nLimnocylindraceae is consistent with this broader mutational model and suggests that\nbase composition erosion may represent an additional outcome of the same underlying\nprocesses. We believe this framing better contextualizes our result within current\nevolutionary theory.\nWe have added the following paragraph to the manuscript (lines 401–406): “Although GC\ncontent was not directly analyzed in Bourguignon et al. (2020), their finding that genome\nreduction in prokaryotes is frequently associated with elevated mutation rates supports a\nbroader mutational framework. Our results align with this model and suggest that\nmutation-driven genome erosion may also influence base composition—manifesting as GC\ncontent reduction—in genome-reduced, free-living lineages such as Limnocylindraceae.”\nComment 15: “Line 288-289: Again no mechanistic explanation here. ”\nResponse 15: We thank the reviewer for pointing this out. In response, we have revised\nthe relevant section to better clarify the proposed mechanism. Specifically, we now\nexplain that the observed GC reduction in Limnocylindraceae likely results from mutation-\ndriven processes, where longer coding regions serve as larger mutational targets\ncompared to short intergenic spacers. This asymmetry in sequence length may account\nfor the disproportionate accumulation of GC-lowering substitutions in genes. We have\nupdated the paragraph accordingly (lines 386-392) to more clearly present this\nmechanistic rationale.\nComment 16: “Line 290-308: Ok I see the reasoning behind some of the mechanism for\nthe GC content changes driving the cell shape, but the size link is still unclear in the\nprevious paragraph.”\nResponse 16: We thank the reviewer for this comment. In the previous paragraph, our\nreference to “size” pertains to the relative lengths of coding (genic) and non-coding\n(intergenic) regions within the genome. As clarified in our response to Comment 13, we\nhave revised the text to explicitly state this distinction and to better explain how (and\nwhere) the differing lengths influence the accumulation of GC-reducing mutations. We\nhope the updated version improves clarity and avoids further misunderstanding.\nComment 17: “Line 389: Figure 4 - consider matching colors with figure 1.”\nResponse 17: We thank the reviewer for this helpful suggestion. We have updated Fig. 4\nto align the color scheme with that used in Fig 1, improving visual consistency across the\nmanuscript. We agree that this change enhances clarity and makes the figures easier to\ninterpret.\nComment 18: “Line 442-449, and throughout: This section in particular seems like it\nbelongs in a discussion section, not results (brings in previous studies, and has a defined\nconclusion sentence) - consider revision of the text to separate results from discussion,\n18\nor to have one combined results and discussion section. This applies to other sections\nthroughout the results.”\nResponse 18: We thank the reviewer for the observation. We opted for a combined\nResults and Discussion format, which allows interpretation alongside data presentation. In\nlight of the comment, we reviewed the relevant sections to ensure all conclusions are\ndirectly supported by results and that speculative language has been minimized.\nComment 19: “Line 450: Figure 5 - again consider matching colors with figure 1 and 4,\nand others.”\nResponse 19: We thank the reviewer for this consistent and helpful suggestion. In\nresponse, we have updated the color scheme in Fig. 5 to match that used in Fig. 1 and 4,\nensuring visual consistency throughout the manuscript. We agree that this improves the\noverall clarity and cohesion of the figures.\nManuscript title: “Deep‑branching Chloroflexota lineages illuminate the\neco‑evolutionary foundation of cross‑ecosystem colonization”.\nREVIEWER 1\nComment 1: “The abstract is still too strongly worded for what is essentially an\nevolutionary hypothesis that cannot / is not proven definitively in the text. I suggest\nphrasing as 'we postulate' or 'we speculate' that.. based on X or Y evidence. As written, it is\nstill far too deterministic.”\nResponse 1: We thank the reviewer for this important point and agree that portions of the\nprevious abstract overstated causal certainty for inferences that are necessarily indirect. In\nresponse, we have thoroughly revised the abstract to ensure that (i) mechanistic\nstatements are explicitly framed as hypotheses consistent with the genomic evidence,\n(ii) deterministic or teleological wording is removed, and (iii) the overall take-home message\nis expressed in probabilistic terms, consistent with comparative genomics–based\ninference.\nConcretely, we made the following changes:\n1. Mechanistic inferences are now explicitly conditional and evidence-linked. For\nexample, we replaced causal phrasing implying certainty (e.g., “dismantled… likely\nfostering… accelerated… enabled”) with language that directly reflects what is\nsupported by the genomic patterns, i.e., “coincides with,” “is consistent with,” and\n“may have.” In the revised abstract, the DNA repair signal is framed as: “loss of key\nDNA glycosylases coincides with degradation of base excision repair and is\nconsistent with a hypermutator state that may have accelerated genomic\nerosion…”.\n2. We avoided teleology and deterministic endpoints. We replaced trajectory\nlanguage that implied a predetermined outcome with neutral evolutionary\nphrasing (e.g., “gave rise to”), and maintained probabilistic interpretation\nthroughout, in line with our general approach elsewhere in the revision (context-\ndependent, non-deterministic framing).\n3. We softened the take-home message to an inference rather than a demonstration,\nand clarified causal direction. The conclusion now reads: “our findings are\nconsistent with mutation-driven genome reduction and proteome optimization\nacting in concert to support cross-ecosystem boundary crossing and freshwater\nspecialization…” This replaces the previous “Our findings demonstrate…” framing\nComment 2: “Please incorporate citations to recent reviews covering microbial habitat\ntransitions, including from/to freshwater environments. You may find these interesting\npoints of comparison for your own work:\nhttps://doi.org/10.1111/1462-2920.16313\nhttps://www.annualreviews.org/content/journals/10.1146/annurev-micro-041320-032304”\nmanuscript’s framing within recent syntheses on microbial habitat transitions.\nA. Incorporation of the suggested Environmental Microbiology review\nIn response, we have added the review (https://doi.org/10.1111/1462-2920.16313) to the\nrevised manuscript. It is now cited at lines 75 and 77.\nB. Annual Review of Microbiology article already included\nThe second suggested review (https://doi.org/10.1146/annurev-micro-041320-032304)\nwas already included in the submitted version and has been retained in the revision. It is\ncited at lines 56, 69, and 75.\nREVIEWER 3\nComment 1: “Reviewer #3 (Remarks to the Author):\nI am reviewing a revised version of the manuscript. Overall, the manuscript reads well, and I\nconsider that the authors have satisfactorily addressed most of Reviewer-2’s comments.\nOne point I think should be discussed further is whether the quality of the MAGs could\nhave influenced estimates of gene loss or genome reduction. As short-read (Illumina)\nMAGs typically miss genes from the accessory genome, I wonder whether the absence of\ngenes (e.g., DNA glycosylases) could have been overestimated in specific lineages. In\naddition, were these MAGs manually curated to ensure that all contigs belong to the\nMAGs?\nResponse 1: We thank the reviewer for these comments. We agree that short-read\n(Illumina) MAGs can be incomplete and may under-represent the flexible (accessory)\ngenome, which can, in principle, affect presence/absence inference. We therefore\ndesigned our analyses to minimize the risk of overestimating gene loss or genome\nreduction and to interpret “absence” conservatively at the lineage level.\nA. MAG quality control and contig-level curation\nAll MAGs included in downstream analyses were subjected to stringent quality filtering\n(completeness/contamination thresholds; see Methods). To further reduce the risk of\nmisbinning, we performed bin-curation steps that evaluate contig consistency (e.g.,\ncoverage, sequence composition, and taxonomic signal) and remove incongruent contigs.\nImportantly, the pdCEL-derived genomes used here are curated within the pdCEL\nframework, and our analyses prioritize these curated bins.\nB. Why the focal genes are unlikely to be “missed accessory” genes\nThe reviewer is correct that accessory genes can be missed in fragmented MAGs.\nHowever, the genes at the centre of this discussion (e.g., DNA glycosylases and other core\nDNA maintenance functions) are typically not pangenome-restricted traits in closely\nrelated bacteria; they are generally part of the conserved cellular toolkit. Conversely,\ngenes in the pangenome are, by definition, present only in subsets of\nstrains/subpopulations, meaning they are absent from a substantial fraction of individuals.\nThus, even if one were to posit rare strain-specific occurrences (e.g., via occasional HGT),\nthis would still support our key interpretation: these functions are not broadly conserved\n2\nand are depleted from the lineage’s core gene repertoire, rather than being systematically\npresent but missed by assembly.\nRelatedly, no single genome type (MAG, SAG, or isolate) can “resolve” the accessory\ngenome on its own—capturing pangenome variation requires population-level sampling\nacross multiple representatives. In this context, MAGs are advantageous because they are\nreconstructed from environmental populations and, in practice, tend to reflect the majority\ngenomic signal present in those populations rather than a single idiosyncratic isolate.\nC. Replication and dense sampling argue against a MAG artifact\nOur inference does not rely on isolated absences in one or a few genomes. We assembled\ngenomes independently from multiple time points, habitats, and ecosystems, and we\nanalysed a densely sampled dataset for Limnocylindraceae: 67 pdCEL genomes (curated)\nplus 31 publicly available genomes (total n = 98). The relevant genes were not detected\nacross these 98 independent representatives, making it unlikely that the pattern is driven\nby sporadic MAG incompleteness or assembly dropout in specific samples.\nD. Conservative conclusion\nBased on the consistent absence across a large, curated, and multi-source genome set, we\ninfer that these genes are generally absent (i.e., not conserved) in the lineage. We\nexplicitly acknowledge that we cannot exclude the possibility that individual strains could\nsporadically acquire such functions via HGT, but such rare occurrences would not change\nthe central result that these genes are not part of the lineage’s conserved genomic\nrepertoire.\nWe have clarified these points in the revised manuscript's Methods section:\n“Because short-read MAGs can under-recover parts of the flexible (accessory) genome, we\napplied conservative criteria for interpreting gene presence/absence. Specifically, we\nrestricted downstream analyses to quality-filtered, curated MAGs and inferred lineage-level\ngene depletion only when a gene was consistently undetected across multiple\nindependent MAGs, rather than from single-genome non-detections. Accordingly,\n“absence” is interpreted as loss from the conserved (core) gene repertoire at the lineage\nlevel, while rare strain-specific occurrences (e.g., via horizontal transfer) cannot be formally\nexcluded.”\n3"}]

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