Refined Phylogenetic Ortholog Inference Reveals Coevolutionary Expansion of the MAPK Signaling Network Through Finetuning of Pathway Specificity.

The evolutionary origins of the three-tier mitogen-activated protein kinase (MAPK) signaling network remain poorly understood despite its indispensable role in eukaryote physiology. Here, we develop a novel two-step method combining relaxed ortholog candidate search with iterative phylogenetic evaluation to identify orthologs across critical eukaryotic lineages. We perform a comprehensive phylogenetic analysis to delineate the history of divergence for non-human orthologs of human paralogs along the human evolutionary backbone. Our detailed evolutionary trees of MAPKs, MAP2Ks, and MAP3Ks reveal two major pulses of coevolutionary tandem expansion: one predating the divergence of fungi and animals, and the other predating the origin of animals. Our reconstruction also infers a polyphyletic origin for the atypical MAPKs. Integrating functional literature across eukaryotic taxa with our trees reveals that the two clades of MAP3K, Sterile-like (STE) and tyrosine kinase-like (TKL), had distinct trajectories and influences on downstream pathway diversification. STEs that function as MAP3Ks are conserved across extant eukaryotes. While TKL MAP3Ks are absent in many early diverging eukaryotes, their expansion aligns phylogenetically and functionally with that of downstream MAP2Ks and MAPKs. We propose that the MAPK network originated as a STE MAP3K-regulated pathway, but subsequent recruitment and radiations of TKL MAP3Ks drove downstream diversification in parallel, manifesting in top-down finetuning of pathway specificity. Our study provides an evolutionary framework for the functional diversity of this complex signaling network, demonstrating that phylogenetic insights can generate new hypotheses to understand fundamental cellular processes.