Lichens represent a paradigmatic case of symbiosis involving heterotrophic fungi (mycobionts) and a population of compatible photoautotrophic microorganisms (photobionts), such as algae (phycobionts) and/or cyanobacteria (cyanobionts) (Garrido-Benavent and Pérez-Ortega, 2017). They are extremely ecologically adaptable and widespread in extreme environments such as deserts, polar regions and high mountains (Nash, 1996). As ‘pioneer species’ in the ecosystem, lichens play an irreplaceable role in soil formation, carbon and nitrogen cycling, and biogeochemical processes (Honegger, 2001). Historically viewed as rigid binary partnerships between fungi and photosynthetic organisms (algae/cyanobacteria) (Schwendener, 1869), lichens are now understood to thrive through dynamic, diverse symbioses rather than fixed species-specific interactions. Lichen-forming fungi have the capacity to bind to a broad spectrum of genotypes or species of photosynthetic partners, thus deviating from established binding patterns (Allen and Lendemer, 2022). Lichen algae may look and behave quite differently in symbiosis with different lichen-forming fungi, in the free-living condition in nature and in aposymbiotic laboratory culture (Ahmadjian, 1967; Bubrick, 1988; Sanders and Masumoto, 2021). All this has hindered progress in clarifying their identities, phylogenies and life histories. Schwendener (1869) was the first to survey lichen ‘gonidia’ in a phycological context, recognizing them as organisms distinct from the surrounding fungus that correspond to known taxa of free living algae. Although there has been much research on the genetic characteristics of symbiotic fungi (Grube and Berg, 2009; Resl et al., 2022; Cometto et al., 2024), the composition and evolutionary mechanism of the chloroplast genome of symbiotic algae is still poorly understood (Spribille et al., 2022), which severely limits the in-depth analysis of lichen taxonomic relationships, host-symbiont co-evolution and ecological adaptation mechanisms.

The chloroplast genome (cpDNA), a circular double-stranded DNA molecule maintaining autonomous replication within plastids, contains genes critical for photosynthesis and organellar gene expression. Its structural evolution - particularly in gene content, repetitive elements, and nucleotide composition - reflects adaptive responses to environmental pressures (Howe et al., 2003; Spribille et al., 2022). Green algae (divisions Charophyta and Chlorophyta) serve as essential photosynthetic partners in lichen symbioses. Among these, members of the Trebouxiophyceae, Ulvophyceae, and Chlorophyceae classes (Chlorophyta) are particularly prominent as photobionts (Leliaert et al., 2012). Molecular data indicate the presence of numerous putative cryptic species within the green algae genus (e.g., approximately 30 cryptosporidia have been identified within Trebouxia, far exceeding traditional classifications) (Leavitt et al., 2015). A significant impediment to the advancement of lichen symbiotic algae research is the significantly constrained availability of axenic cultures for study, a constraint that stems primarily from the technical challenges in isolating and culturing these organisms, which has resulted in an extremely limited pool of viable research strains (Muggia et al., 2020). The existing genome sequencing efforts for symbiotic algae show pronounced taxonomic skewness, with overrepresentation of model species like Trebouxia sp. DW1 (Wang et al., 2022) at the expense of broader phylogenetic coverage. This limitation becomes particularly salient given recent molecular evidence uncovering widespread cryptic diversity in chlorophytic symbionts. In Trebouxia alone, phylogenetic analyses have detected around 30 evolutionarily distinct lineages that defy differentiation through classical taxonomic criteria (Leavitt et al., 2015), suggesting current biodiversity assessments may significantly underestimate true species richness. Genomic analyses of individual strains enable precise delineation of genetic boundaries, thus confirming the independent evolutionary origins of Asterochloris and Trebouxia. This provides molecular evidence for revising the classification system of lichen photosynthetic symbionts and resolving long-standing morphological confusions (Sanders and Masumoto, 2021). Recent studies have revealed that lichen symbiotic algal plastid genome evolution is characterised by significant symbiosis specificity. In considering the Trebouxiophyceae taxon, Puginier et al. (2024) determined that symbiotic algae have acquired the glycoside hydrolase 8 (GH8) gene via horizontal gene transfer (HGT). This gene encodes an enzyme capable of specifically degrading β-1,3/1,4-glucans (e.g., lichenin) in the cell walls of lichen-fungal symbionts (LFS). This molecular mechanism directly contributes to the formation of the symbiotic interface. It is imperative to note that Iha et al. (2021) demonstrated that the chloroplast genome of the lichen symbiotic microalgae Trebouxia exhibits distinctive features of structural remodelling. Specifically, its inverted repeat sequences (IRs) undergo a significant shortening, while key ribosomal protein genes (e.g., rps4) shift to the nuclear genome. This reorganisation of gene functions may enhance symbiotic adaptation by optimising nucleoplasmic co-regulatory mechanisms.

In a comparative genomics study, Lemieux et al. (2014) found that Coccomyxa subellipsoidea in the free-living state maintains an intact quadripartite plastid genome structure (IR/LSC/SSC), with a genome size of 160–180 kb encoding about 100–110 genes, including a complete cluster of photosynthesis-related genes (PSA, PSB, etc.), ribosomal RNA (rrn) and transfer RNA (trn) genes. In contrast, the symbiotic Coccomyxa viridis studied by Muggia et al. (2020) exhibited partial or complete loss of the IR region, a phenomenon that may be related to genomic reduction due to symbiotic selection pressure exerted by the host fungus. A comparative analysis by Wang et al. (2022) revealed that the Coccomyxa chloroplast genome exhibited a higher functional gene conservation, particularly concerning key functional genes involved in carbon fixation (rbcL) and photosystem II assembly (psbA), in comparison to the symbiotic Trebouxia. This observation was corroborated by Sanders and Masumoto (2021). This discrepancy may be indicative of the distinct metabolic plasticity exhibited by Coccomyxa in its free-living and symbiotic states.

Peltigera Willd. is one of the earliest lichen genera described (Willdenow, 1787). Subsequent studies have shown that there are two different types of symbionts within the Peltigera: (1) a binary symbiosis consisting of a cyanobacterium (e.g., Nostoc) and a fungus (2) a green alga (e.g., Coccomyxa); as the main photosynthetic symbiont (Miadlikowska et al., 2000). Previous research has mainly focused on traditional taxonomy, mitochondrial genome analysis and biological activity and component synergy (Wei et al., 2009). Traditional classification of lichen symbionts has primarily relied on morphological characteristics and short molecular markers (e.g., ITS, rbcL) (Howe et al., 2003; Wei et al., 2009; Guo et al., 2021; Resl et al., 2022; Spribille et al., 2022; Pushpavathi and Krishnamurthy, 2024; Cometto et al., 2024; Li et al., 2025a). However, these approaches frequently result in ambiguous species delimitation due to phenotypic plasticity and high sequence conservation among closely related taxa (Armaleo et al., 2019). Recent advances in genomic sequencing have provided new taxonomic insights, exemplified by the complete chloroplast DNA (cpDNA) assembly of Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea (Martínez-Alberola et al., 2020). Comparative genomic analyses of this strain with other Trebouxiophyceae species have demonstrated the utility of whole-organelle genomes in refining lichen systematics. Building upon these findings, our laboratory successfully sequenced and assembled the complete cpDNA of Trebouxia sp. DW1, a photobiont isolated from Peltigera rufescens, and conducted comparative genomic analyses with related Trebouxiophyceae species (Wang et al., 2022).

In this study, the green alga symbiont (Chloroidium sp. W5) of P. elisabethae was selected as the study subject, and the following work was systematically carried out: (1) Chloroplast genome composition analysis: high-throughput sequencing and comparative genomics were used to reveal the structural characteristics of the symbiotic algae chloroplast genome (such as gene content, repetitive sequence distribution and GC content) and to identify specific variations driven by the symbiotic environment. (2) Repetitive sequence analysis: This analysis reveals the dynamics of the Chloroidium sp. W5 genome structure and provides important clues for studying coevolutionary mechanisms in lichen symbiotic systems. (3) Codon preference analysis: The codon usage preference of Chloroidium sp. W5 was calculated. By analysing the frequency of codon usage, it reflects the evolutionary pressure and adaptive changes of the chloroplast genome. (4) Synteny Analysis: Revealing the conserved and dynamically evolving features of Chloroidium sp. W5 genomic structure and emphasizing the relevance of its structural rearrangement to symbiotic adaptation through synteny analysis. (5) Phylogenetic reconstruction and taxonomic determination: A highly supported phylogenetic tree was reconstructed using both whole-genome and chloroplast genomic data from 18 Trebouxiophycean species. Here, we discuss the structure, organization, gene content of Chloroidium, a common terrestrial coccoid green alga, and comparison analysis with other chloroplast genomes reported for Trebouxiophyceae. We also provide a fairly resolved phylogenetic reconstruction on the basis of well-conserved chloroplast genes coding for proteins. It is evident that the study methodology can be applied to non-model lichen symbionts.

Chloroidium sp. W5, the photosynthetic symbiont of the lichen P. elisabethae, exhibits several remarkable chloroplast genomic features that provide novel insights into the evolutionary adaptation mechanisms of lichenized algae. The absence of the canonical quadripartite structure (LSC/SSC/IR) in this chloroplast genome is particularly noteworthy. This structural simplification aligns with observations in other symbiotic algae such as Trebouxia and Coccomyxa (Poquita-Du et al., 2024), suggesting potential adaptive advantages in symbiotic systems. The loss of inverted repeats (IRs), known to maintain chloroplast genome stability (Turmel et al., 2017), may indicate relaxed selective pressures in the protected symbiotic environment. Similar genome reduction patterns have been documented in other obligate symbionts (Smith and Keeling, 2015), supporting the hypothesis that symbiotic lifestyles promote genomic streamlining. In this study, comparative genomics analysis revealed significant genomic rearrangements in the chloroplast genome of Chloroidium sp. W5, including multiple types of structural variants such as inversions, deletions, insertions and duplications. These rearrangement events may affect gene function through multiple mechanisms. First, structural variants may alter promoter regions, particularly due to the absence of the typical chloroplast tetrameric structure (LSC/IR/SSC) in this species, which could significantly impact transcription initiation efficiency and gene expression levels (Daniell et al., 2016). Second, gene rearrangements may lead to the loss of gene function or the acquisition of novel functions, a phenomenon previously reported in chloroplast genome studies of other plants (Jansen and Ruhlman, 2012). Notably, the present study found that the chloroplast genome of Chloroidium sp. W5 contains a high abundance of repetitive sequences and SSR sites. These repetitive elements may promote genomic rearrangement through homologous recombination mechanisms (Wicke et al., 2011), which in turn affects gene function. From an evolutionary perspective, the stability of the chloroplast genome is critical for maintaining the stability of endosymbiotic relationships. Our results suggest that genome rearrangements may disrupt this stability, which in turn affects the symbiotic interface between chloroplasts and host cells, particularly in terms of the efficiency of material and energy exchange (Smith and Keeling, 2015). Of particular interest, the abundance of scattered repeat sequences in the Chloroidium sp. W5 genome may provide a genetic basis for the adaptation of this species to extreme environments by promoting genomic variation. This finding echoes the results of Chumley et al. (2006) in Pelargonium x hortorum, suggesting that repetitive sequence-mediated genome rearrangement may be an important mechanism for plant adaptation to environmental stress. Therefore, we hypothesize that the genomic plasticity feature observed in Chloroidium sp. W5 may be one of the key factors for its ability to survive in extreme environments. Tuller et al. (2010) demonstrated that in systems where there is high expression, such as in bacteria and yeast, natural selection tends to optimise codon usage patterns. This means that highly expressed genes tend to use the optimal codons (especially A/U ending-type codons) that match the host tRNA library. This minimises ribosomal stalling during translation and increases the efficiency of protein synthesis. It is noteworthy that the Chloroidium sp. W5 chloroplast genome exhibited a significant A/U-type codon preference (RSCU > 1) in this study, a finding that is highly consistent with the theoretical predictions of Tuller et al. (2010). Moreover, this codon preference demonstrated a synergistic evolutionary pattern with the significantly high AT content (56.1%) characterising this genome, strongly suggesting that this may be a genome-level adaptive strategy developed during the long-term adaptation of the lichen symbiosis system. This adaptive evolution may be achieved through the following mechanisms: (1) optimising the translation efficiency of photosynthesis-related genes to adapt to the symbiotic environment; and (2) reducing energy consumption to cope with the nutrient-limited conditions typical of lichen symbionts. In the present study, the chloroplast genomes of Chloroidium sp. W5 were compared with those of extremophilic red algae. The results demonstrated structural simplification (IR loss, gene reduction), repetitive sequence expansion, and functional gene specialisation. However, the adaptive mechanisms differed between the two groups. Symbiotic algae rely on host interactions and maintain symbiotic efficiency through codon optimisation and genome plasticity. In contrast, extremophilic algae respond directly to physical stresses (e.g., retention of heat- and salt-tolerance genes). Collectively, these findings lend support to the hypothesis that environmental stresses drive adaptive genome evolution, and they provide molecular evidence for understanding the evolutionary strategies of organisms in extreme or symbiotic environments.

Phylogenetic reconstructions based on chloroplast gene sequences have contributed to resolve deep level relationships within the Trebouxiophyceae (Lemieux et al., 2014). Phylogenomic analysis definitively placed Chloroidium sp. W5 within the Watanabeales clade, resolving previous taxonomic uncertainties (Darienko et al., 2015). Its distant relationship with Chlorella species supports recent revisions in Trebouxiophyceae classification (Bock et al., 2011). The moderately supported relationship between Watanabeales and the Prasiolales + Trebouxiales clade suggests these taxa may share ancestral adaptive features for symbiotic lifestyles (Wang et al., 2022). Synteny analysis (Figure 4) revealed extensive genomic rearrangements, indicating a dynamic evolutionary history for Chloroidium sp. W5. These structural variations may represent adaptive responses to the symbiotic environment, with similar patterns reported during niche specialization in other algal lineages (Leliaert et al., 2012). These findings confirm that lichen symbiosis imposes unique evolutionary pressures on chloroplast genomes, driving: (i) structural simplification through gene loss and relocation; (ii) genomic plasticity via repeat expansion; and (iii) nucleotide-level adaptation in codon usage and base composition.