# Patterns and Functional Insights of DNA Methylation Variation in a South American Mayfly Across an Agriculturally Impacted Semi-Arid Watershed

## TL;DR

This study explores how DNA methylation in a Chilean mayfly changes in response to environmental stress in a semi-arid watershed affected by agriculture.

## Contribution

The study reveals novel DNA methylation patterns in mayflies linked to environmental stress and identifies their functional relevance in natural populations.

## Key findings

- Mayflies show methylation differences based on their location in the watershed.
- Methylation patterns are associated with biological functions like gene regulation and stress response.
- Non-CpG methylation is a key driver of methylation divergence in this species.

## Abstract

Climate change and human activities threaten aquatic organisms that are vital for the health and balance of rivers and streams. For protecting freshwater ecosystems, it is thus crucial to understand how these organisms respond and adapt to environmental stress. In this study, we aimed to characterize some of the molecular responses of a native Chilean mayfly, Andesiops torrens, across natural populations inhabiting a semi-arid watershed under strong agricultural pressures. We focused on DNA methylation, a natural process that can switch genes on or off. We found differences in the DNA methylation patterns of mayflies depending on their locations within the watershed. This result indicates that local environmental conditions drive molecular changes in this species. The detected responses were mainly linked to the regulation of vital biological functions and the management of environmental stress. Our results demonstrate that mayflies respond to stress through molecular mechanisms and that DNA methylation analysis can help understand the pressures faced by natural populations, which is crucial for their conservation and sustainable management.

By regulating gene expression to maintain homeostasis and enabling rapid responses to environmental change, epigenetic mechanisms can provide valuable insights into how populations respond to external pressures. Here, we examined genome-wide DNA methylation in natural populations of the mayfly Andesiops torrens from a semi-arid watershed of northern Chile exposed to intense climatic and anthropogenic stress. We analyzed 285 individuals from 30 sites using methylRAD sequencing and assembled a draft reference genome to map methylated loci and determine their associated gene functions. Discriminant analyses of principal components revealed a methylation structure among sampling sites, identifying five groups, and the coexistence within localities of individuals with distinct methylation profiles. Non-CpG methylRAD loci accounted for most methylation divergence, consistent with environmental effects. The five groups shared a broad functional spectrum dominated by regulatory processes related to cellular processes, gene regulation, morphogenesis, neurogenesis, and metabolism, and formed a continuum from core cellular regulation in small groups to more integrated developmental and adaptive stress-related control in larger groups. While the drivers of these patterns remain to be clarified, our study suggests that DNA methylation contributes to local responses in A. torrens and also reveals the potential of DNA methylation analyses as an initial approach for exploring ecological pressures in natural populations.