Evolutionary Insights into the Relationship of Frogs, Salamanders, and Caecilians and Their Adaptive Traits, with an Emphasis on Salamander Regeneration and Longevity

**Background:** Modern amphibians (frogs, salamanders, caecilians) possess remarkable adaptive traits, including salamanders' ability to regenerate entire limbs, tail, brain, eye, and heart, as well as their exceptional longevity relative to body size. Frogs are known for frequent vocalization, and caecilians have degenerative vision due to their fossorial lifestyle. The genetic basis of these traits is poorly understood. This study aimed to investigate the genetic changes underlying these unique traits, especially salamander regeneration and longevity, by comparing amphibian genes to those of other vertebrates.

**Methods:** The author collected transcriptome sequences from five amphibian species (Yunnan caecilian, Baoxing tooth toad, oriental fire-bellied toad, stream salamander, Chinese fire-bellied newt) and combined these with genomic/transcriptomic data from five additional amphibian species and 12 other vertebrates (including human, green anole, naked mole rat, Brandt's bat, zebrafish, etc.). Phylogenomic analyses were performed using maximum likelihood (RAxML) and Bayesian inference (PhyloBayes) on filtered supermatrices of 369 coding sequences and 772 protein sequences. Divergence times were estimated using MCMCTREE with four calibration points. Selection analyses included lineage-specific dN/dS estimation (CODEML), identification of positively selected genes (PSGs) using branch-site models, fast-evolving genes (FEGs), and rapidly evolving GO categories (REGOs). Parallel evolution was tested by identifying shared amino acid changes between amphibians and distantly related species with similar traits (e.g., tail-regenerating lizard, long-lived naked mole rat). Functional impact of variants was predicted using SIFT, PROVEAN, and PolyPhen-2. Quantitative real-time PCR (qPCR) was performed on healing limb blastemata of Chinese fire-bellied newts at 0 h, 1 day, 5 days, 10 days, and 20 days post-amputation to validate expression of two candidate genes (OGFR and SERPINI1).

**Key Results:** The phylogenomic analyses strongly supported the Batrachia hypothesis (frog–salamander sister relationship) and a monophyletic origin of Lissamphibia. Divergence time estimates placed the origin of modern amphibians at ~309 million years ago (MYA) and Batrachia at ~290 MYA, consistent with the fossil record. Salamanders exhibited significantly higher dN/dS ratios than any other vertebrate group (mean dN/dS significantly higher; Wilcoxon rank sum test p < 0.03 for all pairwise tests), despite having extremely low synonymous substitution rates (dS approximately one-third of frogs and caecilians; p < 2.9 × 10^-40). This indicates accelerated adaptive evolution driven by selection, not mutation. Salamanders had the highest proportion of development-related REGOs (33%) compared to other vertebrates (13–19%). Development-related genes in salamanders evolved significantly faster than other genes (p = 0.039). Several genes under positive selection or parallel evolution were identified: EEF1E1 (apoptosis), PAFAH1B1 (fibroblast migration), OGFR (cell proliferation), SERPINI1 (neural development), PCNA (DNA repair), and SIRT1 (histone deacetylation). OGFR and SERPINI1 showed parallel evolution between salamanders and tail-regenerating lizards. qPCR confirmed that both OGFR and SERPINI1 expression changed significantly during limb regeneration, with a decrease at day 5 and recovery by day 20. For longevity, PCNA evolved more than two times faster in salamanders than in any other vertebrate, with seven unique amino acid mutations, three predicted deleterious. SIRT1 showed a parallel change (K375R) with the long-lived naked mole rat. For frogs, ALDH3A2 (associated with Sjögren–Larsson syndrome and dysarthria) was under positive selection, with sites in the NAD-binding domain. For caecilians, RLBP1 (visual cycle) showed parallel changes with echolocating bats and naked mole rats, and AKR1B1 (retinal disease) had positively selected sites in the NADP-binding motif.

**Clinical Implications:** This study identifies key genes potentially involved in regeneration and longevity, such as PCNA, SIRT1, OGFR, and SERPINI1, which may serve as targets for future functional studies. Understanding the genetic mechanisms of salamander regeneration could inform strategies to enhance wound healing and tissue regeneration in humans. The identification of genes associated with aging (e.g., SIRT1, PCNA) may provide insights into delaying age-related decline. The parallel evolution findings suggest that some genetic pathways are conserved across species with similar traits, offering potential avenues for translational research in regenerative medicine and gerontology.