DNA methylation site loss for plasticity-led novel trait genetic fixation

Phenotypic plasticity allows organisms to adapt traits in response to environmental changes, yet the molecular basis by which such plastic traits become genetically fixed remains unclear. Here, we investigated gut length plasticity in medaka fish (Oryzias latipes) through genome-wide methylation profiling, CRISPR/Cas9-mediated deletion, and population genomic analyses. We found that seasonal methylation of CpG sites upstream of the Plxnb3 is correlated with gut length plasticity, and deletion of this region abolishes plasticity. Additionally, standing variation in Ppp3r1 is associated with genetically fixed longer gut length in populations lacking plasticity. These results suggest that loss of epigenetic regulation via CpG site reduction triggers the genetic fixation of novel traits. Our findings provide molecular evidence linking epigenetic plasticity and genetic assimilation, advancing understanding of plasticity-led evolution in natural populations.
, Sampling and measurement of gut length
To detect the seasonal plasticity of gut length, we collected 228 medaka individuals from three rivers in Kagawa prefecture from January 2014 to August 2015. The details are in supplementary table S1. To explore the genetically fixed gut length in medaka populations, we sampled 400 individuals from 40 wild-derived laboratory stocks that originated from wild populations and consisted of seven genetic backgrounds (SJPN1-4, NJPN1, NJPN2, and KOR) (1). Each stock is a descendant of populations collected from each wild habitat in 1983 (2) and has been maintained by taking offspring every 1-2 years, close to the generation time in the field (3) in independent tanks in common environments (i.e., the same food and feeding times) at our outdoor breeding facility at The University of Tokyo (1, 4) (see fig. S9 in Katsumura et al. [2014] (4)). The sampled stocks were: SJPN1 (PO.NEH): Ichinoseki, Toyota, Mishima, Iwata, Sakura, Shingu; SJPN2 (San..., , # Data from: DNA methylation site loss for plasticity-led novel trait genetic fixation

[Access this dataset on Dryad](https://doi.org/10.5061/dryad.cz8w9gjb3)

This dataset supports the analyses reported in Katsumura et al. (2026), *PNAS* 123(13), e2534817123 ([https://doi.org/10.1073/pnas.2534817123](https://doi.org/10.1073/pnas.2534817123)). It comprises morphometric measurements (standard length and gut length) of Japanese medaka (*Oryzias latipes*) from three origins — wild-caught individuals, laboratory-reared fish derived from wild populations, and CRISPR/Cas9-generated *plxnb3* upstream-deleted (∆upPlxnb3) mutants — along with immunohistochemistry images of medaka gut tissue, genome-wide SNP genotypes obtained by RAD-seq, and geographic coordinates of the sampling regions. These resources underpin Fig. 1, Fig. 3, Fig. 4, Fig. 5, and Fig. S6 of the manuscript.

## Description of the data and file structure

Morphometric and geographic data are provided as CSV files (comma-delimit..., ,