Integrative bioinformatics analysis of potential convergent evolution in tardigrade stress-response proteins

Abstract Tardigrades, known for their extraordinary resilience to extreme environmental conditions, employ specialized stress-response proteins to maintain cellular integrity under stress. These proteins, including SAHS, CAHS, MAHS, and Dsup, play critical roles in protecting cells from dehydration, radiation, and other environmental stressors. Previous work has characterized many of these proteins in Ramazzottius varieornatus , but the taxonomic breadth, copy-number variation, and structural diversity of these families across other tardigrade lineages remain poorly understood. The recent expansion of genomic resources for additional species motivated a comprehensive multi-species analysis to map ortholog diversity and structural features across tardigrade taxa. We investigated the genetic, structural, and evolutionary features of these stress-response proteins using an integrative bioinformatics approach, analyzing gene structure, orthologous clustering, molecular phylogeny, conserved motifs, and 3D protein structures across four tardigrade species— R. varieornatus, Hypsibius exemplaris, Paramacrobiotus metropolitanus, and H. henanensis . Our results reveal significant variation in predicted gene copy numbers and sequence conservation that reflect species-specific adaptations to environmental stress. Orthologous clustering showed shared evolutionary patterns across species, and conserved motifs were identified within the SAHS, CAHS, and MAHS families. Notably, we identified a new Dsup protein (H.Henanensis.Chr5.66), a potential ortholog in H. henanensis , thereby expanding the known diversity of Dsup proteins. The Dsup protein family exhibited notable sequence diversity across species, yet structural analyses revealed conserved α-helix regions and hydrophobicity patterns, suggesting a flexible conformation that aids DNA protection during extreme stress. Phylogenetic analyses revealed patterns consistent with parallel clustering patterns among stress-response proteins. This study advances our understanding of the molecular adaptations that underpin tardigrades' resilience and offers insights into potential applications for enhancing stress tolerance in other organisms.