Extremophilic Adaptations Across the Archaeal Domain Are Governed by Lineage Clustering

<title>Abstract</title> <p>Archaeal adaptations to high salinity, temperature, acidity, and anoxia are often evaluated by counting marker genes across genomes. However, this approach can mistake shared ancestry for repeated evolutionary events and can produce circular associations when ecological traits are defined using the same genes being tested. 2,919 annotated archaeal genomes in NCBI RefSeq were screened, representing 1,532 taxa and approximately 7.96 million proteins for methanogenesis, the Wood–Ljungdahl pathway, reverse gyrase, and microbial rhodopsins using profile hidden Markov models. Halophily, methanogenesis, thermophily, and acidophily were scored independently from physiological measurements and taxonomy rather than from marker presence. Marker performance was evaluated against an independent annotation-derived reference set, absence claims were repeated in 2,018 high-quality genomes, and trait–marker associations were tested using phylogenetic logistic regression and Pagel’s correlated-evolution model on a species-representative archaeal tree. Marker F1 scores ranged from 0.86 to 1.00 for the four principal systems. Among 1,406 halophilic genomes, 119 were also methanogens and 107 contained a complete Wood–Ljungdahl pathway. The apparent exclusion between salt adaptation and methanogenesis was confined to the class Halobacteria, in which 1,070 halophilic genomes lacked methanogenesis, the Wood–Ljungdahl pathway, and reverse gyrase. In contrast, these systems were frequent among halophiles outside Halobacteria. A naive association between halophily and methanogenesis was extremely significant, but disappeared after phylogenetic correction using both phylogenetic logistic regression and Pagel’s test. These results show that archaeal extremophilic adaptations are primarily lineage-clustered rather than intrinsically incompatible. The apparent salt–methane exclusion is therefore best explained by lineage-specific evolutionary history and phylogenetic pseudoreplication, not a domain-wide biochemical constraint</p>