# Comparative view of DNA methylation in stramenopiles and other eukaryotes: Focus on 5-methylcytosine

Ananya Khatei, Leïla Tirichine, M Junaid Sidiq, J. Mark Cock, Alexander Juterbock

► To cite this version:

Ananya Khatei, Leïla Tirichine, M Junaid Sidiq, J. Mark Cock, Alexander Juterbock. Comparative view of DNA methylation in stramenopiles and other eukaryotes: Focus on 5-methylcytosine. Algal Research - Biomass, Biofuels and Bioproducts, 2026, 94, 10.1016/j.algal.2026.104551. hal-05522428

HAL Id: hal-05522428

https://hal.science/hal-05522428v1

Submitted on 22 Feb 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Comparative view of DNA methylation in stramenopiles and other eukaryotes: Focus on 5-methylcytosine☆

Ananya Khatei<sup>a,b,*</sup>, Leïla Tirichine<sup>c,d</sup>, M. Junaid Sidiq<sup>b</sup>, J. Mark Cock<sup>e</sup>, Alexander Juterbock<sup>a</sup>

## ARTICLE INFO

**Keywords:**

DNA methylation

DNA methyltransferases

Epigenetics

Stramenopiles

## ABSTRACT

This review provides a comparative analysis of DNA methylation patterns, enzymes, and functions in stramenopiles (diatoms, brown algae, oomycetes) and other eukaryotes. Given the ecological and evolutionary importance of stramenopiles, and the relatively limited and fragmented literature on their epigenetic regulation, a comparative synthesis of DNA methylation patterns and mechanisms in this group is both timely and necessary. By integrating current knowledge on DNA methylation in stramenopiles, this review provides a conceptual foundation for exploiting epigenetic mechanisms to enhance stress resilience, growth performance, and environmental adaptability in aquaculture species. Therefore, we synthesize genomic and epigenetic studies to examine the distribution of DNA methyltransferases (DNMTs) and the roles of DNA methylation in adaptation, phenotypic regulation, and life cycle transitions. All DNA methyltransferases (DNMT1, 2, 3, 4, 5, and 6), along with related enzymes such as the repeat-induced protein-deficient family, exhibit homology and therefore appear to share a common ancestor. Stramenopiles are reported to possess DNMT2, DNMT3, DNMT5, and DNMT1-like DNA methyltransferases. Phylogenetic analyses show that DNMT1-like enzymes in stramenopiles constitute a lineage distinct from canonical DNMT1 and DNMT4. Genomic analyses of brown algae reveal absent or very low DNA methylation levels (<3%) and the presence of DNMT2, a pattern also reported in *Drosophila*. Oomycetes, another significant group within the Stramenopiles, appear to lack cytosine methylation. Collectively, available evidence indicates that 5-methylcytosine represents the predominant and functionally validated DNA methylation mark in stramenopiles, whereas 6-methyladenine remains comparatively sparse and lineage-specific. Despite generally lower methylation levels compared to other eukaryotes, DNA methylation in stramenopiles appears to contribute significantly to phenotypic regulation by facilitating environmentally responsive gene expression, thus providing a mechanism for adaptive plasticity. This role of DNA methylation could be harnessed to confer desirable traits for aquaculture, and to mitigate climatic conditions in the natural populations through methods like epbreeding and priming.

**Highlight:** This review summarizes current knowledge on the presence and distribution of methylation within and across life cycle stages in this evolutionarily interesting and economically important group.

## 1. Introduction

Epigenetics encompasses the mechanisms that bring about heritable changes in gene expression without altering the DNA sequence [1].

Epigenetics has a profound impact on the translation of genotype into phenotype, and is thus studied across a wide range of organisms. The concept of inclusive heritability integrates both genetic and non-genetic factors to estimate the transmission of traits across generations. Genetic

**Abbreviations:** CpG, Cytosine – Phosphate- Guanine; CHH, Cytosine – H can be any nucleotide among Adenine/Cytosine/Thymine; CHG, Cytosine - Adenine/Cytosine/Thymine - Guanine; 6 mA, 6-methyl Adenine; 5mC, 5-methyl Cytosine; ApT, Adenine – Phosphate – Thymine.

<footnote><sup>a</sup> AMBIOME group, Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway</footnote>

<footnote><sup>b</sup> ICAR-Central Institute of Coldwater Fisheries Research, Bhimtal, 263136, India</footnote>

<footnote><sup>c</sup> Nantes Université, CNRS, US2B, UMR 6286, F-44000, Nantes, France</footnote>

<footnote><sup>d</sup> Institute for Marine and Antarctic Studies (IMAS), Ecology and Biodiversity Centre, University of Tasmania, Hobart, TAS, 7004, Australia</footnote>

<footnote><sup>e</sup> Sorbonne Université, CNRS, Algal Genetics Group, Integrative Biology of Marine Models Laboratory, Station Biologique de Roscoff, Roscoff, France</footnote>

<footnote>* This article is part of a Special issue entitled: 'Current Reviews 2025' published in Algal Research.</footnote>

<footnote>* Corresponding author at: AMBIOME group, Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway.</footnote>

<footnote>E-mail address: ananya.khatei@nord.no (A. Khatei).</footnote>
phenomena such as penetrance (the likelihood that a gene will produce
its associated trait) and expressivity (the extent to which a trait is
expressed) indicate that traits are influenced by more than just genotype
[2]. Current epigenetic research has been widening the conventional
concepts of genetics and evolution. In eukaryotes, the first checkpoint of
gene regulation occurs at the chromosomal level where the availability
of DNA to RNA polymerase is controlled during transcription [3,4],
supporting that adaptation and variation may arise not only through
genetic mutations but also through reversible and environmentally
responsive epigenetic modifications. These modifications include DNA
methylation, post translational modifications of histones (PTMs),
incorporation of histone variants and RNA interference. DNA methyl-
ation and PTMs operate at the chromatin level, whereas RNA interfer-
ence acts at the levels of transcriptional and post-transcriptional
processes (Fig. 1). These epigenetic modifications can be modulated by
environmental changes and epigenetic states can be maintained across
mitotic and meiotic cell divisions [5]. This is the underlying cause for
cells in multicellular organisms to differentiate into different organs
although they share the same genetic content, and for clonal organisms
to develop different characteristics under different environmental con-
ditions. Transgenerational stability of some of these epigenetic modifi-
cations can bring about immediate adaptive changes in populations that
are relevant at the evolutionary scale. Epigenetic memory is created
through the inheritance of epigenetic marks and is the basis of priming
experiments in farmed plants, animals and brown algae [6–9].

Epigenetic mechanisms have evolved in diverse ways in prokaryotes
and eukaryotes. In prokaryotes, where DNA methylation is the primary
epigenetic mechanism, N6-methyladenine (6 mA) predominates and
only a few species have C4-methylcytosine 4mC in their DNA. DNA
methylation in prokaryotes is mostly associated with Restriction-

Modification (RM) systems and the enzymes responsible are Methyl-
transferases (MTases) [10]. Well-studied methyltransferases in pro-
karyotes include dam MTase and CcrM MTase.

In eukaryotes, which show two dominant types of DNA methylation, C5-methylcytosine (5mC) has been more intensely studied than N6-methyladenine (6 mA). In general, DNA methylation causes structural changes in the chromosomal framework, thereby hindering protein-DNA interactions, ultimately affecting gene expression [10].

## 2. Evolutionary position of stramenopiles in the eukaryotic tree of life

Eukaryotes are divided into six supergroups: Opisthokonta, Amoebozoa, Excavata, Archeplastida, Rhizaria, and a diverse assemblage formerly grouped as Chromalveolata. Stramenopiles, which include both unicellular and multicellular organisms such as diatoms, oomycetes, and brown algae, were historically classified within Chromalveolata due to their plastids derived from red algal secondary endosymbiosis, along with groups like Alveolata, Haptophyta, and Cryptophyta [11].

However, advances in phylogenomic analyses, have challenged the
monophyly of Chromalveolata. These studies led to the recognition of a
new monophyletic clade known as the TSAR group—comprising Telo-
nemids, Stramenopiles, Alveolates, and Rhizaria—which together ac-
count for roughly half of the known eukaryotic diversity [11]. This
reclassification has clarified the evolutionary position of stramenopiles
as a distinct and deeply branching lineage within the eukaryotic tree of
life, separate from Archaeplastida and other primary plastid-containing
lineages.

Stramenopiles, the focus group of this review, are mostly

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** The schematic depicts multilayered gene regulation in a eukaryotic cell. In the nucleus (purple), histone modifications and DNA methylation regulate chromatin accessibility. Activating histone marks promote open chromatin, enabling transcription, whereas repressive histone modifications and DNA methylation induce chromatin compaction and transcriptional silencing. Demethylation enhances DNA accessibility and transcriptional activity. In the cytoplasm (light blue), post-transcriptional regulation is mediated by the miRNA pathway. miRNAs processed by Dicer are incorporated into the RNA-induced silencing complex (RISC), whose core component is the Argonaute (AGO) protein. The miRNA-AGO complex recognises complementary target mRNAs, leading to mRNA cleavage or translational repression. (https://mindthegraph.com/). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
photosynthetic eukaryotes (with some exceptions, including the parasitic oomycetes) and have acquired gene segments of prokaryotic origin in their genomes through horizontal gene transfer [11]. Among the SAR lineages, alveolates are considered the closest extant relatives of stramenopiles. Molecular clock analyses suggest that stramenopiles diverged approximately 1025–1077 million years ago and diversified during the early Neoproterozoic era, in agreement with putative Proterozoic stramenopile fossils [12]. Stramenopiles include brown algae (*Phaeophyceae*), golden brown algae (*Chrysophyceae*), oomycetes, and diatoms (*Bacillarophyceae*). Variations in genome size among stramenopiles indicate that the gain and loss of genetic elements have played an important role in structuring the DNA and genome architecture in these organisms (Fig. 2) [13–18].

Diatoms evolved around 190 million years ago [19,20,84] and are responsible for about 40% of the marine primary productivity, making them an ecologically important group of organisms. Whole genome sequencing has been completed for several diatom species including *Thalassiosira pseudomona*, *T. oceanica*, *Phaeodactylum tricornutum*, *Fragilariopsis cylindrus* and *Seminavis robusta* among others, while sequencing efforts for many additional species are ongoing [21–25].

Phaeophyceae, which is another distinct class of the stramenopile lineage, is taxonomically rich and has been divided into 19 orders based on both morphological and molecular data, with *Discosporangiales* identified as the earliest diverging order. The epigenetic information of stramenopiles is of interest due to the ecological and evolutionary success of this lineage, which includes species that play a significant role in aquatic ecosystems and have shown remarkable resilience over time.

### 3. Methylome of stramenopiles

DNA methylation, which refers to the addition of a methyl (-CH₃) group to a cytosine or adenine base in DNA, is the most widely studied epigenetic modification [26]. In eukaryotes, the process involves the transfer of a methyl group from S-adenyl methionine to the fifth carbon of cytosine (5mC) or the sixth carbon of adenine (6 mA), and is catalyzed by DNA methyltransferases (DNMT) [27]. Studies on DNA methylation in stramenopiles are scarce. However, advances in sequencing technologies such as nanopore and bisulfite sequencing, have made methylation detection more affordable.

Early studies reported global DNA methylation levels of approximately 2–6% in several diatom species [17,19]; however, subsequent analyses have revealed pronounced interspecific and methodological variability. In comparative terms, diatom methylation levels remain

lower than those of vertebrates (~60–80% CpG methylation). DNA methylation has been studied in the 27.4 Mbp genome of the marine model diatom *Phaeodactylum tricornutum*, providing new insights into gene expression patterns and the activity of transposable elements (TEs) [28,29]. In *P. tricornutum*, 3887 highly methylated regions were found, representing 5.16% (1,412,473 bp methylated of 27.4 Mb) of the whole genome (Table 1). A higher number of hypermethylated sites were found in the intergenic regions and TEs, in comparison to the intragenic regions. Long terminal repeat retrotransposons (LTR-RTs) showed higher methylation levels than other DNA transposons, a pattern reminiscent of TE-targeted methylation observed in land plants, although at markedly lower overall levels. DNA methylation has also been studied in other diatom species, such as *T. pseudonana* and *Fragilariopsis cylindrus*. Both species show higher levels of DNA methylation than *P. tricornutum*, with *T. pseudonana*, showing approximately 8% and *F. cylindrus* around 1 to 1.5% of cytosines methylated [24], predominantly in the CG context. This CpG-dominated methylation pattern parallels that of animals, where gene-body CpG methylation is common, but contrasts with plants, which additionally maintain extensive CHG and CHH methylation. More recently, two additional diatom species, *Cyclotella cryptica* and *T. rotula*, were found to show the highest levels of CG methylation reported in diatoms to date, with approximately 61% as global per cytosine methylation (60.1% of CG, 0.4 of CHH and 0.4 of CHG) and 69% of CpG methylated, respectively (Table 1) [30].

After an extensive survey of the available literature across diverse stramenopile lineages, we did not find any consistent or direct relationship between genome size and overall DNA methylation levels (Figs. 2, 3). Species with comparable genome sizes often exhibit markedly different methylation percentages, while both small and large genomes can display either low or relatively high levels of cytosine methylation depending on lineage, life stage, and genomic context. This observation suggests that genome size alone is not a reliable predictor of DNA methylation in stramenopiles. Instead, DNA methylation appears to be shaped by lineage-specific evolutionary histories, functional requirements, and possibly ecological adaptations. Consequently, no uniform or distinct pattern of DNA methylation can currently be inferred across stramenopiles, highlighting the heterogeneous and dynamic nature of epigenetic regulation within this diverse eukaryotic supergroup.

In brown algae, the presence of DNA methylation was first investigated in the genomes of the filamentous brown alga *Ectocarpus* sp. and kelp (*Saccharina* spp.) (Table 1). Cytosine methylation was not detected in *Ectocarpus* sp. [13]. In *S. japonica*, 1.4% of cytosines in the gametophytes were found to be methylated and methylation levels were lower

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Genome size variation mapped onto eukaryotic phylogenetic relationships.</figcaption></figure>

The phylogenetic tree (left) illustrates the evolutionary relationships among selected stramenopile taxa, including diatoms, brown algae, and the oomycete *Phytophthora infestans*. Horizontal bars (right) represent genome sizes (Mb) for each species, arranged to correspond exactly to their positions in the phylogeny. Species names are shown in italics. Colors indicate major taxonomic groupings (Blue – Oomycetes, Purple – Diatoms, Red – Brown algae), highlighting differences in genome size across evolutionary lineages. The phylogenetic tree was created with phylot (phyloT: a phylogenetic tree generator) and iphylo (iPHYLO-TREE). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
<caption>**Table 1**</caption>

Summary of reported DNA methylation levels and detection methods across representative stramenopile taxa. The table compiles approximate methylation levels derived from published studies together with the experimental approaches used for detection. Methods include whole-genome bisulfite sequencing (WGBS), antibody-based enrichment (MeDIP/MeDIP-seq), restriction enzyme-based assays (McrBC-chip, MethylRAD), and long-read nanopore sequencing. Reported methylation levels should be interpreted in light of methodological differences, as each approach varies in genomic coverage, resolution, and sensitivity to methylation context. Reported methylation values are not directly comparable across studies due to differences in detection methods, sequence-context specificity, and metric definitions (global cytosines vs CpG sites vs covered restriction sites).

<table><thead><tr><th>Group</th><th>Species</th><th>Approximate 5-mC level (%)</th><th>Detection method</th><th>Methodological notes</th></tr></thead><tbody><tr><td rowspan="5">Stramenopiles<br>Diatoms</td><td>Phaeodactylum<br>tricornutum</td><td>(<sup>Σ MethylatedC</sup>/<sub>Σ (MethylatedC + UnmethylatedC)</sub>)%<br>~5.16%</td><td>McrBC digestion followed by<br>microarray hybridization (McrBC-chip)<br>Validation by Whole-genome bisulfite<br>sequencing (WGBS)</td><td>Genome-wide 5-mC; WGBS<br>validates low global<br>methylation, Might be<br>susceptible errors due to<br>amplifications</td></tr><tr><td>Thalassiosira<br>gravida</td><td>69% (CpG context)<br>(<sup>Σ MethylatedCpG</sup>/<sub>Σ (MethylatedCpG + UnmethylatedCpG)</sub>)%</td><td>Nanopore (R9.4.1 flow cell, R10.4.1<br>flow cell)</td><td>CpG-specific estimate; long-<br>read model-dependent, higher<br>depth is required</td></tr><tr><td>Cyclotella<br>cryptica</td><td>60.9 Global cytosine methylation<br>(<sup>Σ MethylatedC</sup>/<sub>Σ (MethylatedC + UnmethylatedC)</sub>)%<br>MCpG = 60.1% MCHH = 0.4% MCHG = 0.4%<br>Total = 60.9 Global methylation</td><td>WGBS</td><td>High CpG methylation;<br>negligible CHG/CHH</td></tr><tr><td>Thalassiosira<br>pseudonana</td><td>2.57</td><td>WGBS</td><td>–</td></tr><tr><td>Fragilariopsis<br>cylindrus</td><td>8.63</td><td>WGBS</td><td>–</td></tr><tr><td rowspan="3">Brown algae<br>(Phaeophyceae)<br>Oomycetes</td><td>Ectocarpus sp.<br>Saccharina<br>japonica</td><td>Not detected<br>~1.4 (gametophyte); &lt;0.9 (sporophyte)<br>(<sup>Σ MethylatedC</sup>/<sub>Σ (MethylatedC + UnmethylatedC)</sub>)%</td><td>WGBS<br>WGBS (Bisulphite conversion rate &gt;<br>99%), Methylated DNA<br>immunoprecipitation (MeDIP), bisulfite<br>validation</td><td>–</td></tr><tr><td>Saccharina<br>latissima</td><td>~0.1–6%<br>(<sup>Number of methylated sites covered by ≥ 3 reads</sup>/<sub>Total number of potential methylation sites</sub>)%</td><td>MethylRAD</td><td>Reduced-representation;<br>underestimates CHH</td></tr><tr><td>Phytophthora<br>infestans</td><td>Dot blot (anti-6 mA); relative abundance = anti-6 mA<br>signal / input DNA</td><td>MeDIP-seq (6 mA-IP-seq)</td><td>Relative abundance only; not<br>quantitative</td></tr><tr><td rowspan="2">Vertebrate animals</td><td>Zebrafish</td><td>~75%<br>(<sup>Σ MethylatedCpG</sup>/<sub>Σ (CpGs detected by RR)</sub>)%</td><td>RRBS - Reduced representation bisulfite<br>sequencing</td><td></td></tr><tr><td>Arabidopsis<br>thaliana</td><td>~20–24%</td><td>–</td><td></td></tr><tr><td>Plants</td><td>Neurospora<br>crassa</td><td>~1.3–2.5%<br>(<sup>Σ MethylatedC</sup>/<sub>Σ (MethylatedC + UnmethylatedC)</sub>)%</td><td>WGBS</td><td></td></tr></tbody></table>

than 0.9% in the sporophytes [26,31] (Fig. 3). A recent study using the MethylRAD method found that most 5mC DNA methylation in the *S. latissima* genome is located in repeat regions, specifically in the CpG and CHH contexts, and that the methylose varies between wild and cultured conditions and is significantly influenced by temperature [32]. However, this method does not efficiently capture CHH methylation in gene regions, which may explain the observed enrichment in repeats. In contrast, Fan et al. [26], using a method capable of detecting CHH methylation across the genome, reported higher methylation levels in gene regions than in repeats in *S. japonica*, highlighting the importance of methodological differences in interpreting methylation landscapes (Table 1).

The methylation status of animal DNA is usually altered during embryonic stages or early developmental stages through the interplay of methylases and de-methylases, resulting in life-stage-specific differential methylation [33]. A similar pattern of differential methylation has been found in the gametophytic and sporophytic stages of *S. japonica*, which may be connected to the activation of specific genes in the early development of the young sporophytic phase through morphogenesis [26]. In addition, differential methylation has also been reported in brown algae as a response to heat stress [34].

### 3.1. Adenine methylation

N6-methyladenine or 6 mA is a well-established and widespread epigenetic mark in prokaryotes, where it plays critical roles in gene regulation, DNA replication, and host-pathogen interactions. In

eukaryotes, however, the presence and function of 6 mA remain less consistent and more controversial. Low levels of 6 mA have been reported in a few model organisms such as *Homo sapiens*, *Arabidopsis thaliana*, and *Drosophila melanogaster* [35,36], while a more substantial presence of 6 mA has been observed in *Caenorhabditis elegans* [37]. *In vitro* studies indicate that 6 mA can interfere with the binding of mammalian transcription factors and chromatin regulators to DNA. Adenine methylation has also been detected in the oomycete *Phytophthora infestans*, a member of the stramenopiles [38], suggesting that this mark may be present in certain protist lineages. However, with the exception of *P. infestans*, there are very few reports documenting 6 mA in stramenopiles although there has been a report for *Nannochloropsis oceanica* [39].

## 4. Genomic distribution of 5-methylcytosine across CpG and non-CpG contexts in stramenopiles and other eukaryotes

The distribution pattern of methylation across DNA can be categorized based on nucleotide context (CpG and non-CpGs) or on functional regions (exons, regulatory regions, and transposons) and non-functional regions (intergenic regions, introns and pseudogenes). Based on the nucleotide position, the methylation types found in eukaryotes include CpG in most animals and, in addition, CHG and CHH (H = adenine, cytosine, or thymine) in plants. Diatoms show methylation mostly in CpGs, whereas the brown alga *S. japonica* mostly has methylation in a non-CpG context (Fig. 3), predominantly in CHH [26].

DNA methylation of functional elements such as exons, regulatory
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** The figure integrates evolutionary relationships with comparative genomic and epigenomic features of selected eukaryotic species. (A) A phylogenetic tree (Taxid: 3702,6239,7955,7227,74372,9606,5141,2850,4787,88149,74381,35122,590727,2035398,2185085,159749,426638,270628,35128) representing major eukaryotic groups, including Metazoa, Fungi, Streptophyta, Oomycetes, Bacillariophyta, and Phaeophyceae. Coloured background sectors highlight taxonomic affiliations of species included in the analysis. The ‘s.’ before the names represent that they are species. The phylogenetic tree was created with phylot (phyloT: a phylogenetic tree generator) and iphylo (iPHYLO-TREE) (B) Bar plot showing comparative genome size (Gb), cytosine content (%), and 5-methylcytosine (5mC) levels (%) for the same species. To enable direct comparison among variables spanning several orders of magnitude, genome size, cytosine content, and 5mC levels were log₁₀-transformed. The transformed values were subsequently shifted by subtracting the global minimum across all parameters, ensuring that the smallest value was set to zero while preserving relative differences among species and parameters. DNA methylation percentages in CpG, CHG and CHH contexts in *Saccharina japonica*, *Phaeodactylum tricornutum*, and *Arabidopsis thaliana* are represented as pie charts. (C) Pie-charts representing context (CpG, CHH and CHG) specific methylation in few representative stramenopiles.</figcaption></figure>

regions, and transposons (TEs) has been studied in several eukaryotic lineages. In land plants, the distribution of DNA methylation across exonic, intronic and repeat-regions varies across species [40]. In Stramenopiles, such as brown algae, studies have shown that genes that encode circular and long non-coding RNAs are the most methylated elements in the genome and significant methylation has also been found

at TEs [26]. For diatoms, on the other hand, the highest methylation levels were detected on TEs [41]. In invertebrates, methylation shows a mosaic pattern of distribution i.e., patches of hypermethylated regions surrounded by non-methylated regions [42]. A similar mosaic pattern was observed in 6% of the genome of *S. japonica* [26] while the remaining regions of the genome showed low levels of methylation.
Also, methylation in the brown alga *S. japonica* was more prominent in exonic regions than in intronic regions.

In animals, DNA methylation is primarily concentrated in CpG-rich regions, known as CpG islands. These CpG islands are highly conserved in vertebrates and often found in promoter regions [27], where they regulate the expression of the downstream genes [43]. CpG islands are flanked by CpG shores and display conserved methylation status. Although studies have shown that DNA hyper-methylation in the promoter regions is associated with gene silencing in animals [4], a few studies claim that methylation within the gene body could also lead to enhanced gene expression [42]. DNA methylation in the context of cytosine is absent in certain organisms, like *Caenorhabditis elegans* [43] and was detected at very low levels in *Drosophila melanogaster* [44] and *Saccharomyces cerevisiae* [45]. CpG sites within transposable elements are often heavily methylated and this DNA methylation is essential for silencing transposons and maintaining genome stability [46]. In *Drosophila melanogaster*, the low levels of DNA methylation observed are mainly found within TEs. In *C. elegans*, where CpG methylation is absent, adenine methylation has been found during embryonic stages, where it plays a role in transposon silencing [37]. Methylation in fungal genomes is generally low and is concentrated around promoter regions and TEs. In *Neurospora crassa*, 1.5% of the cytosines are methylated whereas <0.1% of cytosines are methylated in species like *Schizosaccharomyces pombe* and *Aspergillus nidulans* [47].

The correlation between DNA methylation and genome size remains a matter of ongoing debate. In plants, genome-wide DNA methylation levels are linked to the abundance of transposable elements (TEs), and this methylation likely serves to suppress TE activity to maintain proper gene expression and function [48]. However, similar conclusions have been difficult to establish in animals, where the relationship between DNA methylation and TEs remains less clear and is still under investigation [48]. In microeukaryotes, DNA methylation levels do not correlate with genome size. For instance, *Ostreococcus lucimarinus*, which has a relatively small genome, shows significantly higher levels of cytosine methylation than diatoms or *Arabidopsis thaliana*, both of which have substantially larger genomes [19].

## 4.1. ApT sites for methylation

Adenine methylation, which mostly occurs in the ApT context, ranges from 0.000006% to 0.8% in eukaryotes [49]. Three types of adenine methylation have been reported in eukaryotes: 1 mA, 3 mA and 6-mA [50]. Among these three categories of methylation, 6 mA is the most studied since it is associated with the regulation of several proteins and enzymatic activities. 6 mA is predominantly enriched in regions upstream (transcription start sites) and downstream of genes, while its levels are lowest within coding sequences [49]. In contrast to 5mC, the presence of 6 mA is often positively correlated with gene expression. In plants, about 32% of 6 mA occurs within gene bodies [51]. In *Oryza sativa*, 25% of the adenine methylation was found in coding genes and 14% in TEs [51].

Comparative studies across eukaryotes show that DNA methylation varies widely in both its genomic distribution and sequence context. In vertebrates like humans and zebrafish, CpG methylation is globally high and plays a key role in regulating CpG islands. Plants such as *Arabidopsis* and rice (*Oryza*), along with fungi like *Neurospora* and *Aspergillus*, show extensive non-CpG methylation, mainly involved in silencing transposable elements and defending the genome. Stramenopiles, on the other hand, generally have lower and more variable levels of 5-methylcytosine. For example, diatoms display sparse or gene-body-associated CpG methylation, while brown algae show repeat- and context-dependent methylation. This intermediate, lineage-specific methylation pattern sets stramenopiles apart and highlights their unique position in the evolution of epigenetic regulation, making them a valuable group for studying how these mechanisms have diversified across eukaryotes.

# 5. DNA methyltransferases: key enzymes mediating cytosine (5-mC) methylation

DNA methyltransferases (DNMTs) are a family of enzymes that can transfer a methyl group from S-adenosyl-methionine (SAM) to cytosine (Fig. 4). They consist of an N-terminal regulatory region and a C-terminal catalytic domain. The presence of certain domains such as Bromodomains, ZFN-Zinc finger domains, and CXXC-domains and their ability to recognise specific DNA sequences allows them to be categorized as *de novo* DNMTs or maintenance DNMTs. The most commonly studied *de novo* DNMTs (characterized by the ability to attach a methyl group to previously unmethylated DNA) in animals are DNMT-3a and 3b (Fig. 4). During the embryonic stages of animals, DNMT3a is responsible for cellular differentiation [4]. Another *de novo* DNMT, DNMT3L (L for like) is responsible for genomic imprinting in germ cells [52]. Among the maintenance methyltransferases, DNMT1 is found in most eukaryotes. It recognises hemi-methylated regions of newly synthesized DNA (Figs. 4 & 5). In plants, Chromomethylases (CMT-2 and 3) and Methyltransferase 1 (MET1) recognise and methylate CHG and CHH sites, and “domains rearranged methyltransferase2” (DRM2) is responsible for the maintenance of methylation [53].

Another class, DNMT2, remains an enigma in epigenetics, and is widely accepted as tRNA methyltransferase. In *D. melanogaster* which shows low levels of methylation during early developmental stages, *in vitro* studies suggest that DNMT2 methylates DNA, as no other DNMT is present in the genome [54]. Although some researchers have now predicted that DNMT2 does not directly influence DNA methylation [55], there are also studies that provide evidence for DNA methyltransferase activity of this enzyme [56,57]. However, the function of DNMT2 still remains a matter of debate. In *Neurospora crassa* a completely different type of methyltransferase enzyme, known as “repeat-induced point mutation deficient” (RID) exists [58]. Unlike DNMT2 and other canonical DNA methyltransferases, RID is involved in genome defence through the repeat-induced point mutation (RIP) process. The variations in methyltransferase structures and functions across the eukaryotic tree of life suggest that the elements of DNA methylation have evolved in a diverse pattern.

## 5.1. *De novo* DNMTs

The protein structure, homology relationships and functional activities of the most common *de novo* DNMT, DNMT3, have been well established in vertebrates, such as mice [59,60]. Reports suggest that DNMT3a and DNMT3b have no role in the maintenance of imprinted methylation, which is the conserved methylated pattern during cell division [61].

The two domains, Pro-Trp-Trp-Pro (PWWP) and ATRX-DNMT3-DNMTL (ADD), that are characteristic of DNMT3 may be responsible for specific site recognition and binding [28]. In plants, *de novo* methylation is catalyzed by DRM2, which depends on RNA for DNA methylation [62]. However, in non-flowering plants, DNMT3 orthologs appear to be involved in *de novo* methylation [53]. DNMT3 is absent from fungi and *de novo* methylation is mediated by RID and MASC1 proteins [63,64]. DNMT4 has also been shown to have a similar activity to that of RIDs in fungi [63] but, so far, has not yet been shown to be exclusively a *de novo* DNMT [28]. With the exception of some diatoms, DNMT3 is mostly absent from stramenopiles (Fig. 5). Several phylogenetic studies have indicated that DNMT3 was lost from the majority of stramenopile species during evolution. A DNMT3 domain found in the diatom *P. tricornutum* lacks characteristic associated domains such as chromo domains [28]. DNMT4 is absent from *P. tricornutum* but is present in several other diatoms such as *T. miniscula*, and *T. pseudonana*. DNMT4 is closely related to DNMT3 and is mostly associated with *de novo* methylation in fungi and stramenopiles. The DNMT4-encoding gene, DIM2 (an orthologue of human DNMT), is capable of both *de novo* and maintenance methylation in fungi. Interestingly, in the
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Schematic representation of DNA methylation dynamics mediated by DNA methyltransferases and demethylases. *de novo* DNA methylation is catalyzed by DNMT3-like enzymes (DNMT3/RID/masc1), which establish new methylation marks on previously unmethylated DNA. During DNA replication, methylation patterns are partially diluted, resulting in hemimethylated DNA. Maintenance methylation is subsequently carried out by DNMT1-like enzymes (DNMT1/CMT/DNMT5/DNMT6), which restore symmetrical methylation patterns on the newly synthesized strand. Active DNA demethylation is mediated by demethylases such as MBD4, TET, and DEMET, leading to the removal of methyl groups. Dark blue circles indicate fully methylated cytosines, while light blue circles represent hemimethylated cytosines. “Me” denotes a methyl group. (https://mindthegraph.com/). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** DNA methyltransferases found in different eukaryotic lineages. Stramenopiles are reported to possess DNMT1, DNMT2, DNMT3, DNMT5, and DNMT1-type (DNMT4) DNA methyltransferases.</figcaption></figure>

ascomycete *Verticillium dahlia*, where DIM2, RID and DNMT5 are all present, DIM2 serves as the main DNA methyltransferase [64]. Notably, the same study found that DNMT5 is more active during environmental stress conditions in *V. dahlia*.

DNMT5 is another paralogue of DNMT3 and is also found in stramenopiles. This group of enzymes includes DNMT5-a, b and c [28]. DNMT5a has been reported in diatoms, *Bolidomonas* spp. and pelagophytes. Knock-out of DNMT5 in the fungus *Cryptococcus neoformans* removed CpG methylation throughout the entire genome [65]. This DNMT5 was mostly associated with transposon CpG methylation and

showed maintenance methylation specificity. In the [65] study, it was also shown that the factor responsible for *de novo* methylation had been lost and that ancestral methylation has persisted.

The major domains found in DNMT5 in the diatoms include DEXDc, SNF2/related and HelicC domains [17]. Among the family of DNMT5 genes, DNMT5c is mostly found in the dinoflagellates, lacking SNF2-related and other functional domains. DNMT5b has the longest DNMT domain in the stramenopiles. Unlike DNMT5b, DNMT5a is only found in pennate diatoms [28]. These studies suggest that it is too early to categorise these recently found DNMTs such as DNMT-4, 5 and 6 into the
strictly defined categories of *de novo* and maintenance methylation.

In brown algae, none of the well-known DNMTs responsible for *de novo* methylation (DNMT3 and related proteins) have been identified so far. Moreover, *Ectocarpus siliculosus*, a brown algal model organism, appears to lack detectable DNA methylation, which may be related to the loss of DNMT1 [13,66]. Although studies of the kelp *Saccharina japonica* have confirmed the presence of low levels of DNA methylation, no DNMT3 or other known *de novo* methyltransferases were identified in its genome. These findings suggest that the enzymes responsible for establishing and maintaining DNA methylation in this species remain unclear. In the absence of DNMT1, it is hypothesized that DNMT2 may take over its functional role in brown algae like *S. japonica*, compensating for DNA methylation maintenance. In addition to DNMT3, 4 and 5, another DNMT found in stramenopiles is DNMT6. This methyltransferase is widely found in diatoms and golden algae (*Chrysophyceae*) [65]. However, the specific functions of DNMT6 in protists and stramenopiles have yet to be determined.

### 5.1.1. *DNMT2* – the debated methyltransferase

Another methyltransferase, DNMT2, is found conserved in most of eukaryotic lineages, an exception being oomycetes [38] (Fig. 5). DNMT2 shares sequence homology with DNMTs from both eukaryotes and prokaryotes suggesting a shared common ancestor. Although DNMT2 can methylate both DNA and RNA (specifically tRNA), its substrate preference is stronger for RNA [38,67].

The presence of a conserved DNA target recognition domain (TRD) motif in the DNMT2 protein provides further evidence for its DNA methyltransferase activity [67]. Experiments on *Drosophila* spp. also challenge the previous conclusion that DNMT2 is specific for tRNA methylation [56,68]. These results not only show that DNMT2 has weak DNA methyltransferase activity but is also specific to methylation at CT/CA sites rather than CpG sites. However, experiments in mouse and *Arabidopsis thaliana* did not bring about any phenotypic or methylation change in response to silencing of DNMT2. In contrast, suppression of DNMT2 through RNA interference, removed all DNA methylation from *Drosophila* spp. genomes [56]. Moreover, the specificity of DNA methylation by DNMT2 clearly differs from that of DNMT3, i.e., the latter mostly methylates in the CpG context, whereas the former methylates in sequence contexts other than CpG.

To date, there are no reports of the presence of DNMT2 nor cytosine methylation in oomycetes. DNMT2 has been found in diatoms and brown algae but there are few studies of its DNA methylation activity in these species. In brown algae, the presence of DNMT2 has been confirmed in *S. japonica* [26] and it was later confirmed that DNMT2 genes are found throughout the brown algae [66]. As DNMT2 is the sole DNA methyltransferase in *S. japonica*, methylation of 2% of cytosines suggests that this enzyme may methylate DNA in this species.

### 5.2. Maintenance of 5mC

Maintenance of DNA methylation refers to the transfer of DNA methylation marks from the parental to the daughter DNA strand after DNA replication during the process of mitotic cell division. The “maintenance methyltransferase (MMT) group” of enzymes recognises hemimethylated DNA sites. DNMT1, which is the most common MMT, performs methylation maintenance in metazoans [28]. Other MMTs, like chromomethyltransferase (CMT) and DIM2 and MASC1 perform MMT activity in plant and fungi, respectively [28]. All the above MMTs belong to the same subfamily [69]. DNMT1 is associated with bromo-adjacent homology (BAH) domains and chromodomains, which are required for interactions with DNA and other proteins [69]. DNMT1 enzymes mostly target CpG sites, except for CMTs, which methylate at CHH and CHG sequence contexts. CHH methylation takes place in the absence of DNMT1, but CpG methylation requires this enzyme [70]. These specificities explain the methylation status at non-CpG sites in diatoms and microalgae, which lack DNMT1.

In the SAR (stramenopiles, alveolates, and rhizaria) group, DNMT1-like enzymes have been observed in the Raphidophyceae (*Heterosigma akashiwo* and *Chatonella subsalsa*) [28]. However, they have never been reported in diatoms and Phaeophyceae. Studies in mouse models have shown that DNMT1 cannot function alone. It functions only in the presence of DNMT3a/3b. In contrast, other DNMTs, including DNMT3a/3b, are capable of functioning in the absence of DNMT1. This could explain the persistence of DNA methylation in species like diatoms that lack DNMT1 but possess DNMT3 and DNMT/5.

### 5.3. DNA demethylases

Epigenetic reprogramming of DNA methylation marks is the result of an interplay between methylation and demethylation. Two different pathways for DNA demethylation have been reported in humans [71]. The first pathway involves hydrolysis of 5-methylated cytosine into cytosine and water, while the second involve DNA glycosylase activity. DNA glycosylases, which are known for their DNA demethylation activity in most animals, possess a MBD4 (Methyl CpG-binding) domain [71]. DNA glycosylases are highly conserved proteins responsible for DNA demethylation and transcriptional regulation. The ten-eleven translocation (TET) enzyme also demethylates DNA [72]. This enzyme induces the oxidation of 5mC to 5hmC (5-hydroxy-methyl-cytosine), which is poorly recognised by maintenance methyltransferases. In land plants, DNA demethylation is mediated by a different set of DNA glycosylases to that found in animals. These land plant DNA glycosylases include DEMETER (DME) and REPRESSOR OF SILENCING (ROS1). Few studies have looked at DNA demethylation in stramenopiles. The MBD4 domain has been detected in *S. latissima* [73] and *P. tricornutum* [74], but a role in DNA demethylation has not been confirmed yet.

### 5.4. Methyltransferases facilitating Adenine methylation

In eukaryotes, 6 mA deposition is linked to the MT-A70 protein family. In *Caenorhabditis elegans*, the MT-A70 homolog DAMT-1 is known to control genomic 6 mA levels [35,37], but other MT-A70 proteins such as METTL3 and METTL14 mainly methylate RNA and have only weak activity on DNA. Meanwhile, AlkB family proteins, originally discovered for their role in DNA repair, also help remove methyl groups from both DNA and RNA. Although MT-A70 and AlkB homologs are found throughout eukaryotes, their exact roles in regulating DNA 6 mA are still unclear. This suggests that the pathways for 6 mA methylation and demethylation might differ between lineages and could involve diverse mechanisms.

## 6. Role of DNA methylation in gene regulation

DNA methylation has been studied in many stramenopiles including diatoms, brown algae and oomycetes. Although DNA methylation was found at only low levels (2–6%) in these organisms, its contribution to their environmental adaptation has been found to be correlated (Fig. 6) [75]. In *P. tricornutum*, DNA methylation (CHG and CHH) in the body of genes associated with nitrate starvation, was positively correlated with their expression [76]. The transcription of transposable elements has been shown to be negatively correlated with the DNA methylation level in stramenopiles [46]. Genes that were highly methylated, included those encoding signalling functions and were expressed under certain conditions. Gene-body methylated genes included transporters, transferases and other genes carrying out general metabolic processes. Another study in *P. tricornutum* has shown that CHH hypermethylation in the gene body in response to elevated pCO₂ levels resulted in increased gene expression [77].

Differential methylation in response to heat stress has been studied in *S. japonica* [34]. Genes related to heat-stress, such as the Heat shock proteins (HSP) and ubiquitin-proteasome pathway related genes, were analyzed to test for differentially methylated regions (DMRs) under heat
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Conceptual overview of the role of DNA cytosine methylation in environmental adaptation, phenotypic plasticity, and life-cycle regulation in stramenopiles. Environmental cues (e.g., temperature, nutrition, and stress through pollution) can induce changes in DNA methylation patterns through the action of DNA methyltransferases, leading to the addition of methyl groups (CH₃) in different contexts (such as CpG, CHH and CHG) on genomic DNA. These methylation changes influence transcriptional regulation by modulating chromatin structure and gene accessibility, thereby affecting gene expression programs. Dynamic DNA methylation is associated with key biological processes, including regulation of developmental genes during life-cycle transitions, and phenotypic expression. Together, these processes contribute to phenotypic plasticity and adaptive responses, allowing stramenopiles to adjust growth, development, and morphology in response to environmental variation. (https://mindthegraph.com/).</figcaption></figure>

stress conditions [34]. The gene bodies showed higher numbers of DMRs than the upstream and downstream region of the genes. Highly expressed genes were mostly regulated by methylation of gene body rather than methylation of the upstream or downstream regions. Also, a higher number of gene-body DMRs was found in the CHH compared to the CG context. As observed in diatoms, gene body methylation in this kelp was positively correlated with gene expression [34]. In contrast, methylation of the upstream region was negatively correlated with gene expression. This might be due to the presence of more CGs in the upstream region, thereby correlating the lower expression with methylation in CG context rather than CHH context. However, the distribution of CG and CHH across the genome remains unstudied. This study by Liu et al. [34], concluded that the stramenopiles show overall lower DNA methylation in comparison to other eukaryotes and most of these methylations are in the context of CHH and more concentrated in transposable elements. This is similar to the methylation patterns found in plants.

While several DNMTs such as DNMT1 and DNMT3 are not present in many stramenopiles, DNMT1-like enzymes have recently been identified

in *Raphidophyceae* [28] and also in the *Discosporangiales* (which is the only order in the *Phaeophyceae* with DNMT1 methyltransferase) [66]. In conclusion, the patterns of DNA methylation in stramenopiles resemble those in plants but must be catalyzed by a different set of DNMTs. The resemblance of the methylome of brown algae to that of *Drosophila* is noteworthy. Both taxa show low levels of DNA methylation and possess only DNMT2. In different eukaryotic lineages, DNMT2 has evolved to show substrate specificity for either DNA or RNA. In the oomycete *P. infestans*, 5mC has not been reported but 6 mA occurs and affects gene expression [38]. Like 5mC in other eukaryotes, the distribution of 6 mA is bimodal around the TSS in oomycetes [38] i.e. methylation levels are higher in the flanking regions of the transcriptional start site (TSS) and there is a drop in the methylation peak at the TSS. This suggests a negative correlation of 6 mA with gene expression, which means hypermethylation in the context of 6 mA is associated with reduced gene expression. Hence, DNA methylation could potentially play a role in gene regulation in response to changing environmental conditions in some Stramenopile species (Fig. 6).
## 6.1. Role of DNA methylation in transposable elements and repetitive sequences

Another important element that structures the genomes are transposable elements. These are genetic elements that have the potential to become mobile and transpose from one location to another. Transposable elements represent a variable portion of eukaryotic genomes and can regulate gene expression by altering regulatory mechanisms. To avoid negative effects of transposons on genome function, they need to be controlled by epigenetic strategies such as RNAi mechanisms, DNA methylation, and histone modification. The genome of the diatom *P. tricornutum* consists of 6.4% of TEs [77], with the most abundant being long terminal repeat-retrotransposons (LTR-RT). A diatom-specific Copia-type LTR-RT found in *P. tricornutum* constitutes about 5.8% of the genome. In the case of brown algae, studies on *Ectocarpus* sp. showed that 22.7% of the genome consists of repeat regions and transposable elements [13]. Oomycete genomes consist of up to 50% transposable elements [78].

Information from evolutionary and phylogenetic analyses, including in-depth molecular and genomics analyses, suggests that evolution is not only a gradual incremental process, but also involves bursts of morphological alterations and speciation. This theory is commonly referred to as “punctuated equilibrium” [79,80]. Transgenerational inherited epigenetic marks, such as DNA methylation and histone modifications, may partly explain such drastic evolutionary shifts. The introduction of new traits into eukaryotic organisms, is often attributed to the expression of TEs and HGTs (Horizontal Gene Transfers) that remained silent through epigenetic mechanisms such as DNA methylation, and start being expressed when demethylation releases their transcriptional repression under adverse environmental cues. As mentioned in the earlier sections, TEs constitute 5–15% of stramenopile genomes. Most of the 5mC is located in transposable elements in both land plants and Stramenopiles and most TEs are in a methylated state. The balance between DNA methylation and demethylation determines whether they remain silent or become active and insert into new genes, promoters and enhancers. *P. tricornutum* carries piggyBac transposable elements in HSP70 proteins [29]. These elements are hypermethylated and affect HSP70 expression.

A significant number of transposable elements and repetitive sequences have also been discovered in the diatom *Cyclotella cryptica*, suggesting a recent genomic expansion [81]. Methylated repetitive elements make up most of the methylome in this species and are associated with an alteration in the expression of genes in their proximity. In fact, in diatoms, most DNA methylation affects TEs rather than genes. Most diatom genomes encode DNMT5, which is mostly associated with the methylation of transposable elements. The overexpression of DNMT5 explains the hypermethylation of TEs in *P. tricornutum*. In *S. latissima*, differential methylation was observed in strains from different origins [32]. This study showed that most of the methylated regions were found in the repeated regions. An interesting observation in this study was that field kelp showed higher methylation levels than the laboratory-grown kelp. However, more sites were methylated in laboratory-grown samples. Methylation levels rose with increasing temperature and were predominantly associated with transposable elements. A decrease in methylated sites under high temperature showed that methylation was concentrated in some specific genomic regions under altered conditions. In kelp, methylation is most prominent in CHH contexts making its role particularly relevant for silencing transposable elements.

## 6.2. Role of DNA methylation in life cycle changes

In stramenopiles, brown algae, with the exception of *Fucales*, show a distinct alternation of generations. The only cue we have for a functional role of DNA methylation during the life cycle is the differential methylation status observed between gametophytes and sporophytes in

*S. japonica*, where methylation in the gametophyte was found to be higher [26]. As in bryophytes [82], kelp also show higher CHH methylation in transposable elements. Single-celled eukaryotes like *Chlamydomonas reinhardtii* also express several genes that are gamete specific and zygote specific, suggesting the presence of silencing mechanisms that regulate the life cycle [83]. These transitions in *C. reinhardtii* are further accompanied by extensive CHH methylation which occurs in both TEs and gene bodies highlighting the epigenetic regulation of developmental changes during life cycle transitions (Fig. 6).

In the study by Yang et al. [31], it was observed that DNA methylation level is higher in the gametophyte than in the sporophyte. This was an indication of demethylation during the life cycle transition to the sporophyte. As discussed earlier, DNA methylation is mostly negatively correlated with gene expression, which could be a possible reason for demethylation at this stage in order to activate genes that help in the development of sporophytes. In contrast, the higher methylation in the gametophyte may reflect the repression of sporophytic or stress-responsive genes that are not required in this stage, thereby maintaining stage-specific gene expression. In the same study, it was observed that the methylation during the sporophyte generation was highest in the holdfast and least in the stipe. Most of the DMRs that were observed included the region in the genome responsible for cell motility, cell cycle and cell-membrane biogenesis which gives evidence of regulation of genes by DNA methylation.

## 7. Conclusions

In conclusion, the study of DNA methylation in stramenopiles offers valuable insights into the dynamic processes of adaptation and phenotypic regulation within these unique eukaryotes. By influencing how genes are expressed without altering the underlying DNA sequence, epigenetics contributes to phenotypic variation and adaptability (Fig. 6). This makes it a key area of study for understanding how organisms respond to environmental pressures and for informing climate resilience strategies. While stramenopiles exhibit lower methylation levels compared to other eukaryotes, particularly in the CpG context, their methylation patterns remain significant. However, CHH methylation is higher in most stramenopiles as compared to other eukaryotes. These organisms, despite the absence of some commonly found DNMTs, demonstrate an ability to modulate gene expression and adapt to changing environments, particularly under stress conditions such as heat or nutrient limitations.

From a comparative perspective, these findings highlight distinct features of 5-methylcytosine (5-mC) methylation in stramenopiles relative to other eukaryotic lineages. While 5-mC is typically enriched in the CpG context in many eukaryotes, stramenopiles display comparatively lower overall 5-mC levels and a broader sequence-context distribution, suggesting lineage-specific regulatory strategies. This divergence underscores the evolutionary plasticity of 5-mC-mediated epigenetic regulation and indicates that conserved epigenetic marks can be deployed differently across eukaryotic groups to meet distinct ecological and developmental demands. The findings so far suggest that the methylation systems in stramenopiles may be more significant and varied than previously thought, despite their overall lower methylation levels.

The future exploration of DNA methylation in stramenopiles, especially in relation to life cycle transitions and environmental adaptability, holds great promise. A deeper understanding of how these epigenetic mechanisms influence phenotypic plasticity and evolutionary processes could provide valuable strategies for improving agricultural practices, mitigating climate change effects, and better understanding the molecular evolution of this ecologically important group. Further studies in a broader range of stramenopiles will undoubtedly enhance our comprehension of their epigenetic landscape and its evolutionary implications.
CRediT authorship contribution statement

**Ananya Khatei:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. **Leïla Tirichine:** Writing – review & editing, Supervision. **M. Junaid Sidiq:** Writing – review & editing, Visualization. **J. Mark Cock:** Writing – review & editing, Validation, Supervision. **Alexander Juterbock:** Writing – review & editing, Validation, Supervision, Project administration, Investigation, Conceptualization.

Funding

This work was supported by funding from Nord University, Bodo, Norway.

Declaration of competing interest

No conflict of interest.

Acknowledgements

Not applicable.

Data availability

No data was used for the research described in the article.

References

[1] J.P. Hamilton, Epigenetics: principles and practice, Dig. Dis. 29 (2) (2011) 130-135.

[2] D.P. Snustad, M.J. Simmons, Principles of Genetics, John Wiley & Sons, Hoboken, NJ, 2015.

[3] A.J. Bannister, T. Kouzarides, Regulation of chromatin by histone modifications, Cell Res. 21 (3) (2011) 381-395.

[4] L.D. Moore, T. Le, G. Fan, DNA methylation and its basic function, Neuropsychopharmacology 38 (1) (2013) 23-38.

[5] E. Prokhortchouk, P.A. Defossez, The cell biology of DNA methylation in mammals, Biochim. Biophys. Acta, Mol. Cell Res. 1783 (11) (2008) 2167-2173.

[6] E. Sani, P. Herzyk, G. Perrella, V. Colot, A. Amtmann, Hyperosmotic priming of *Arabidopsis* seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome, Genome Biol. 14 (2013) R59.

[7] I. Kakoulidou, E.V. Avramidou, M. Baránek, S. Brunel-Muguet, S. Farrona, F. Johannes, E. Kaiserli, M. Lieberman-Lazarovich, F. Martinelli, V. Mladenov, P. S. Testillano, Epigenetics for crop improvement in times of global change, Biology 10 (8) (2021) 766.

[8] C. Gauci, A. Juterbock, A. Khatei, G. Hoarau, I. Bartsch, Thermal priming of *Saccharina latissima*: a promising strategy to improve seaweed production and restoration in future climates, Mar. Ecol. Prog. Ser. 745 (2024) 59-71.

[9] A. Juterbock, B. Duarte, J. Coyer, J.L. Olsen, M.E.L. Kopp, I. Smolina, S. Arnaud-Haond, Z.M. Hu, G. Hoarau, Adaptation of temperate seagrass to Arctic light relies on seasonal acclimatization of carbon capture and metabolism, Front. Plant Sci. 12 (2021) 745855.

[10] H.J. Seong, S.W. Han, W.J. Sul, Prokaryotic DNA methylation and its functional roles, J. Microbiol. 59 (2021) 242-248.

[11] F. Burki, A.J. Roger, M.W. Brown, A.G. Simpson, The new tree of eukaryotes, Trends Ecol. Evol. 35 (1) (2020) 43-55.

[12] H.S. Yoon, R.A. Andersen, S.M. Boo, D. Bhattacharya, Stramenopiles, in: Encyclopedia of Microbiology, Third Edition, Elsevier, 2009, pp. 721-731.

[13] J.M. Cock, L. Sterck, P. Rouzé, D. Scornet, A.E. Allen, G. Amoutzias, V. Anthouard, F. Artiguenave, J.M. Aury, J.H. Badger, et al., The *Ectocarpus* genome and the independent evolution of multicellularity in brown algae, Nature 465 (7298) (2010) 617-621.

[14] N. Ye, X. Zhang, M. Miao, X. Fan, Y. Zheng, D. Xu, J. Wang, L. Zhou, D. Wang, Y. Gao, Y. Wang, *Saccharina* genomes provide novel insight into kelp biology, Nat. Commun. 6 (2015) 6986.

[15] T. Shan, J. Yuan, L. Su, J. Li, X. Leng, Y. Zhang, H. Gao, S. Pang, First genome of the brown alga *Undaria pinnatifida*: chromosome-level assembly using PacBio and Hi-C technologies, Front. Genet. 11 (2020) 140.

[16] K. Nishitsuji, A. Arimoto, Y. Higa, M. Mekaru, M. Kawamitsu, N. Satoh, E. Shoguchi, Draft genome of the brown alga, *Nemacystus decipiens*, Onna-1 strain: fusion of genes involved in the sulfated fucan biosynthesis pathway, Sci. Rep. 9 (1) (2019) 4607.

[17] F. Maumus, P. Rabinowicz, C. Bowler, M. Rivarola, Stemming epigenetics in marine stramenopiles, Curr. Genomics 12 (5) (2011) 357-370.

[18] W.R. Roberts, K.M. Downey, E.C. Ruck, J.C. Traller, A.J. Alverson, Improved reference genome for *Cyclotella cryptica* CCMP332, a model for cell wall

morphogenesis, salinity adaptation, and lipid production in diatoms (*Bacillariophyta*), G3: Genes|Genomes|Genetics 10 (9) (2020) 2965-2974.

[19] A. Rastogi, X. Lin, B. Lombard, D. Loew, L. Tirichine, Probing the evolutionary history of epigenetic mechanisms: what can we learn from marine diatoms, AIMS Genet. 2 (3) (2015) 173-191.

[20] K. Brylka, M.P. Ashworth, A.J. Alverson, D.J. Conley, The Cretaceous Diatom Database: a tool for investigating early diatom evolution, J. Phycol. 60 (5) (2024) 1090-1104.

[21] T. Mock, M.P. Samanta, V. Iverson, C. Berthiaume, M. Robison, K. Holtermann, C. Durkin, S.S. BonDurant, K. Richmond, M. Rodes, T. Kallas, Whole-genome expression profiling of the marine diatom *Thalassiosira pseudonana* identifies genes involved in silicon bioprocesses, Proc. Natl. Acad. Sci. 105 (5) (2008) 1579-1584.

[22] C. Bowler, A. Vardi, A.E. Allen, Oceanographic and biogeochemical insights from diatom genomes, Annu. Rev. Mar. Sci. 2 (1) (2010) 333-365.

[23] A. Rastogi, F.R.J. Vieira, A.F. Deton-Cabanillas, A. Veluchamy, C. Cantrel, G. Wang, P. Vanormelingen, C. Bowler, G. Piganeau, H. Hu, L. Tirichine, A. genomics approach reveals the global genetic polymorphism, structure, and functional diversity of ten accessions of the marine model diatom *Phaeodactylum tricornutum*, ISME J. 14 (2) (2020) 347-363.

[24] T. Mock, R.P. Otillar, J. Strauss, M. McMullan, P. Paajanen, J. Schmutz, A. Salamov, R. Sanges, A. Toseland, B.J. Ward, A.E. Allen, Evolutionary genomics of the cold-adapted diatom *Fragilariopsis cylindrus*, Nature 541 (7638) (2017) 536-540.

[25] C.M. Osuna-Cruz, G. Bilcke, E. Vancaester, S. De Decker, A.M. Bones, P. Winge, N. Poulsen, P. Bulankova, B. Verhelst, S. Audoor, D. Belisova, The *Seminavis robusta* genome provides insights into the evolutionary adaptations of benthic diatoms, Nat. Commun. 11 (1) (2020) 3320.

[26] X. Fan, W. Han, L. Teng, P. Jiang, X. Zhang, D. Xu, C. Li, M. Pellegrini, C. Wu, Y. Wang, M.J.S. Kaczuowski, Single-base methylene profiling of the giant kelp *Saccharina japonica* reveals significant differences in DNA methylation to microalgae and plants, New Phytol. 225 (1) (2020) 234-249.

[27] P. Sarkies, Encyclopaedia of eukaryotic DNA methylation: from patterns to mechanisms and functions, Biochem. Soc. Trans. 50 (3) (2022) 1179-1190.

[28] A. Hoguin, F. Yang, A. Groisillier, C. Bowler, A. Genovesio, O. Ait-Mohamed, F.R. J. Vieira, L. Tirichine, The model diatom *Phaeodactylum tricornutum* provides insights into the diversity and function of microeukaryotic DNA methyltransferases, Commun. Biol. 6 (1) (2023) 253.

[29] A. Veluchamy, A. Rastogi, X. Lin, B. Lombard, O. Murik, Y. Thomas, F. Dingli, M. Rivarola, S. Ott, X. Liu, Y. Sun, An integrative analysis of post-translational histone modifications in the marine diatom *Phaeodactylum tricornutum*, Genome Biol. 16 (1) (2015) 102.

[30] F. Di Costanzo, M. Di Marsico, I. Orefice, J.B. Kristoffersen, P. Kasapidis, T. Chaumier, L. Ambrosino, M. Miralto, R. Aiese Cigliano, F. Verret, L. Tirichine, High-quality genome assembly and annotation of *Thalassiosira rotula* (synonym of *Thalassiosira gravida*), Sci. Data 12 (1) (2025) 310.

[31] X. Yang, X. Wang, J. Yao, D. Duan, Genome-wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with sporophyte development of *Saccharina japonica*, Int. J. Mol. Sci. 22 (18) (2021) 9877.

[32] L. Scheschonk, K. Bischof, M.E.L. Kopp, A. Juterbock, Differences by origin in methylome suggest eco-phenotypes in the kelp *Saccharina latissima*, Evol. Appl. 16 (2) (2023) 262-278.

[33] M. Breton-Larrivée, E. Elder, S. McGraw, DNA methylation, environmental exposures and early embryo development, Anim. Reprod. 16 (2019) 465-474.

[34] F. Liu, P. Zhang, Z. Liang, Y. Yuan, Y. Liu, Y. Wu, The global dynamic of DNA methylation in response to heat stress revealed epigenetic mechanism of heat acclimation in *Saccharina japonica*, J. Phycol. 59 (1) (2023) 249-263.

[35] K. Boulias, E.L. Greer, Means, mechanisms and consequences of adenine methylation in DNA, Nat. Rev. Genet. 23 (7) (2022) 411-428.

[36] Z. Liang, L. Shen, X. Cui, S. Bao, Y. Geng, G. Yu, F. Liang, S. Xie, T. Lu, X. Gu, H. Yu, DNA N6-adenine methylation in *Arabidopsis thaliana*, Dev. Cell 45 (3) (2018) 406-416.

[37] E.L. Greer, M.A. Blanco, L. Gu, E. Sendinc, J. Liu, D. Aristizábal-Corrales, C.H. Hsu, L. Aravind, C. He, Y. Shi, DNA methylation on N6-adenine in *C. elegans*, Cell 161 (4) (2015) 868-878.

[38] H. Chen, H. Shu, L. Wang, F. Zhang, X. Li, S.O. Ochola, F. Mao, H. Ma, W. Ye, T. Gu, L. Jiang, *Phytophthora* methylomes are modulated by 6mA methyltransferases and associated with adaptive genome regions, Genome Biol. 19 (1) (2018) 181.

[39] Y. Gong, Q. Wang, L. Wei, W. Liang, L. Wang, N. Lv, X. Du, J. Zhang, C. Shen, Y. Xin, L. Sun, Genome-wide adenine N6-methylation map reveals epigenetic regulation of lipid accumulation in *Nannochloropsis*, Plant Commun. 5 (3) (2024).

[40] J.D. Hollister, B.S. Gaut, Epigenetic silencing of transposable elements: a trade-off between reduced transposition and deleterious effects on neighboring gene expression, Genome Res. 19 (8) (2009) 1419-1428.

[41] A. Veluchamy, X. Lin, F. Maumus, M. Rivarola, J. Bhavsar, T. Creasy, K. O'Brien, N. A. Sengamalay, L.J. Tallon, A.D. Smith, E. Rayko, Insights into the role of DNA methylation in diatoms by genome-wide profiling in *Phaeodactylum tricornutum*, Nat. Commun. 4 (1) (2013) 2091.

[42] T. Phillips, The role of methylation in gene expression, Nature Educ. 1 (1) (2008) 116.

[43] V. Haberle, A. Stark, Eukaryotic core promoters and the functional basis of transcription initiation, Nat. Rev. Mol. Cell Biol. 19 (10) (2018) 621-637.

[44] S. Deshmukh, V.C. Ponnaluri, N. Dai, S. Pradhan, D. Deobagkar, Levels of DNA cytosine methylation in the *Drosophila* genome, PeerJ 6 (2018) e5119.

[45] F. Capuano, M. Mülleder, R. Kok, H.J. Blom, M. Ralser, Cytosine DNA methylation is found in *Drosophila melanogaster* but absent in *Saccharomyces cerevisiae*,
Schizosaccharomyces pombe, and other yeast species, Anal. Chem. 86 (8) (2014) 3697-3702.

[46] W. Zhou, G. Liang, P.L. Molloy, P.A. Jones, DNA methylation enables transposable element-driven genome expansion, Proc. Natl. Acad. Sci. U. S. A. 117 (32) (2020) 19359-19366.

[47] C. He, Z. Zhang, B. Li, S. Tian, The pattern and function of DNA methylation in fungal plant pathogens, Microorganisms 8 (2) (2020) 227.

[48] F. Lucibelli, M.C. Valoroso, S. Aceto, Plant DNA methylation: an epigenetic mark in development, environmental interactions, and evolution, Int. J. Mol. Sci. 23 (15) (2022) 8299.

[49] L.Y. Beh, G.T. Debelouchina, D.M. Clay, R.E. Thompson, K.A. Lindblad, E. R. Hutton, Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization, Cell 177 (7) (2019) 1781-1796.

[50] M. Bochtler, H. Fernandes, DNA adenine methylation in eukaryotes: enzymatic mark or a form of DNA damage? BioEssays 43 (3) (2021) e2000243.

[51] S. Kumar, T. Mohapatra, Dynamics of DNA methylation and its functions in plant growth and development, Front. Plant Sci. 12 (2021) 596236.

[52] J.M. SanMiguel, M.S. Bartolomei, DNA methylation dynamics of genomic imprinting in mouse development, Biol. Reprod. 99 (1) (2018) 252-262.

[53] R. Yaari, A. Katz, K. Domb, K.D. Harris, A. Zemach, N. Ohad, RdDM-independent de novo and heterochromatin DNA methylation by plant CMT and DNMT3 orthologs, Nat. Commun. 10 (1) (2019) 1613.

[54] V. Krauss, G. Reuter, DNA methylation in *Drosophila*—a critical evaluation, Prog. Mol. Biol. Transl. Sci. 101 (2011) 177-191.

[55] S. Takayama, J. Dhahbi, A. Roberts, G. Mao, S.J. Heo, L. Pachter, D.I. Martin, D. Boffelli, Genome methylation in *Drosophila melanogaster* is found at specific short motifs and is independent of DNMT2 activity, Genome Res. 24 (5) (2014) 821-830.

[56] N. Kunert, J. Marhold, J. Stanke, D. Stach, F. Lyko, A Dnmt2-like protein mediates DNA methylation in Drosophila, Development 130 (21) (2003) 5083-5090.

[57] A. Jeltsch, A. Ehrenhofer-Murray, T.P. Jurkowski, F. Lyko, G. Reuter, S. Ankri, W. Nellen, M. Schaefer, M. Helm, Mechanism and biological role of Dnmt2 in nucleic acid methylation, RNA Biol. 14 (9) (2017) 1108-1123.

[58] M.R. Rountree, E.U. Selker, DNA methylation and the formation of heterochromatin in *Neurospora crassa*, Heredity (Edinburgh) 105 (1) (2010) 38-44.

[59] T. Yokomine, K. Hata, M. Tsudzuki, H. Sasaki, Evolution of the vertebrate DNMT3 gene family: a possible link between existence of DNMT3L and genomic imprinting, Cytogenet. Genome Res. 113 (1-4) (2006) 75-80.

[60] M. Okano, D.W. Bell, D.A. Haber, E. Li, DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development, Cell 99 (3) (1999) 247-257.

[61] T. Dahlet, A. Argüeso Lleida, H. Al Adhami, M. Dumas, A. Bender, R.P. Ngondo, M. Tanguy, J. Vallet, G. Auclair, A.F. Bardet, M. Weber, Genome-wide analysis in the mouse embryo reveals the importance of DNA methylation for transcription integrity, Nat. Commun. 11 (1) (2020) 3153.

[62] X. Cao, S.E. Jacobsen, Role of the *Arabidopsis* DRM methyltransferases in de novo DNA methylation and gene silencing, Curr. Biol. 12 (13) (2002) 1138-1144.

[63] E. Gladyshev, Repeat-induced point mutation and other genome defense mechanisms in fungi, in: The Fungal Kingdom, 2017, pp. 687-699.

[64] H.M. Kramer, D.E. Cook, G.C. van den Berg, M.F. Seidl, B.P. Thomma, Three putative DNA methyltransferases of *Verticillium dahliae* differentially contribute to DNA methylation that is dispensable for growth, development and virulence, Epigenetics Chromatin 14 (1) (2021) 21.

[65] S. Catania, P.A. Dumesic, H. Pimentel, A. Nasif, C.I. Stoddard, J.E. Burke, J. K. Diedrich, S. Cooke, T. Shea, E. Gienger, R. Lintner, Evolutionary persistence of DNA methylation for millions of years after ancient loss of a de novo methyltransferase, Cell 180 (2) (2020) 263-277.

[66] F. Denoeud, O. Godfroy, C. Cruaud, S. Heesch, Z. Nehr, N. Tadrent, A. Couloux, L. Brillet-Guéguen, L. Delage, D. Mckeown, T. Motomura, Evolutionary genomics of the emergence of brown algae as key components of coastal ecosystems, Cell 187 (24) (2024) 6943-6965.

[67] V.M. Del Castillo Falconi, K. Torres-Arciga, G. Matus-Ortega, J. Díaz-Chávez, L. A. Herrera, DNA methyltransferases: from evolution to clinical applications, Int. J. Mol. Sci. 23 (16) (2022) 8994.

[68] G.C. Vieira, M.F. D'Ávila, R. Zanini, M. Deprá, V.L. da Silva Valente, Evolution of DNMT2 in drosophilids: evidence for positive and purifying selection and insights into new protein interactions, Genet. Mol. Biol. 41 (2018) 215-234.

[69] L. Ponger, W.H. Li, Evolutionary diversification of DNA methyltransferases in eukaryotic genomes, Mol. Biol. Evol. 22 (4) (2005) 1119-1128.

[70] Nasrullah, A. Hussain, S. Ahmed, M. Rasool, A.J. Shah, DNA methylation across the tree of life, from micro to macro-organism, Bioengineered 13 (1) (2022) 1666-1685.

[71] R. Prasad, T.J. Yen, A. Bellacosa, Active DNA demethylation—the epigenetic gatekeeper of development, immunity, and cancer, Adv. Genet. 2 (1) (2021) e10033.

[72] X. Wu, Y. Zhang, TET-mediated active DNA demethylation: mechanism, function and beyond, Nat. Rev. Genet. 18 (9) (2017) 517-534.

[73] G.A. Pearson, N. Martins, P. Madeira, E.A. Serrão, I. Bartsch, Sex-dependent and -independent transcriptional changes during haploid phase gametogenesis in the sugar kelp *Saccharina latissima*, PLoS One 14 (9) (2019) e0219723.

[74] L. Aravind, V. Anantharaman, S. Abhiman, L.M. Iyer, Evolution of eukaryotic chromatin proteins and transcription factors, in: Protein Families: Relating Protein Sequence, Structure, and Function, 2013, pp. 421-502.

[75] M. Ferrari, A. Muto, L. Bruno, R. Cozza, DNA methylation in algae and its impact on abiotic stress responses, Plants 12 (2) (2023) 241.

[76] X. Lin, L. Tirichine, X. Zhang, The dynamic duo: how DNA methylation and gene transcription help diatoms thrive in modern oceans, J. Exp. Bot. 74 (14) (2023) 3879-3882.

[77] R. Huang, J. Ding, K. Gao, M.H. Cruz de Carvalho, L. Tirichine, C. Bowler, X. Lin, A potential role for epigenetic processes in the acclimation response to elevated pCO₂ in the model diatom *Phaeodactylum tricornutum*, Front. Microbiol. 9 (2019) 3342.

[78] H.S. Judelson, Dynamics and innovations within oomycete genomes: insights into biology, pathology, and evolution, Eukaryot. Cell 11 (11) (2012) 1304-1312.

[79] D.W. Zeh, J.A. Zeh, Y. Ishida, Transposable elements and an epigenetic basis for punctuated equilibria, BioEssays 31 (7) (2009) 715-726.

[80] S.J. Gould, N. Eldredge, Punctuated equilibria: the tempo and mode of evolution reconsidered, Paleobiology 3 (2) (1977) 115-151.

[81] J.C. Traller, S.J. Cokus, D.A. Lopez, O. Gaidarenko, S.R. Smith, J.P. McCrow, S. D. Gallaher, S. Podell, M. Thompson, O. Cook, M. Morselli, Genome and methylome of the oleaginous diatom *Cyclotella cryptica* reveal genetic flexibility toward a high lipid phenotype, Biotechnol. Biofuels 9 (1) (2016) 258.

[82] J. Vigneau, M. Borg, The epigenetic origin of life history transitions in plants and algae, Plant Reprod. 34 (4) (2021) 267-285.

[83] J. Neupert, S.D. Gallaher, Y. Lu, D. Strenkert, N.A. Segal, R. Barahimipour, S. T. Fitz-Gibbon, M. Schroda, S.S. Merchant, R. Bock, An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga *Chlamydomonas*, Nat. Commun. 11 (1) (2020) 6269.

[84] A. Falciatore, M. Jaubert, J.P. Bouly, B. Bailleul, T. Mock, Diatom molecular research comes of age: model species for studying phytoplankton biology and diversity, Plant Cell 32 (3) (2020) 547-572.