Cross-species dissection of saline-related genes by genetically deciphering a euryhaline microalga Chlorella sp

Domain: biology
Published: 2026-01-13
Editorial outcome: major_revision

Abstract
Deciphering adaptation to habitat shifts across the salinity boundary necessitates investigation of “lost” and “acquired” saline genes. By assembling a telomere-to-telomere genome, we propose that the euryhaline Chlorophyta Chlorella sp. MEM25 represents an early-diverging saltwater species that has evolved numerous genes essential for saltwater-freshwater transitions. By comparison with Viridiplantae genomes, we identify ancestral genes and lineage-specific genes related to salinity adaptation. Loss-of-function mutants of the proposed salt-sensitive genes in algae and plants exhibit increased salt resistance, highlighting the potential of the MEM25 genome as a breeding resource. Notably, the gene RMI1 plays an important role in salinity tolerance across species, from microalgae to higher plants.

Introduction
Life on Earth is thought to have originated 4 billion years ago in primordial, hypersaline oceans1 with salinities of ~80g·L−12. Marine-freshwater transitions and terrestrialization (the transition from water to a terrestrial environment) are generally regarded as pivotal events in the evolution and diversification of land plant flora3. The transition to a terrestrial environment by plants has been documented frequently4–7; however, habitat shifts from marine to freshwater have not been thoroughly investigated. One prevailing notion is that the transition from salty to freshwater habitats occurred in the common ancestor of Chlorophyta (commonly known as green algae) and the conquest of the land started with Streptophyta (embryophytes and their closest algal relatives, known as streptophyte algae)4,7,8.

Higher marine-freshwater transition rates than anticipated have, however, been reported for both prokaryotes9 and eukaryotes (e.g., microbial eukaryotes10). Evolutionary studies based on long-read metabarcoding across the tree of eukaryotes reveal that such habitat transitions have occurred in both directions in almost all major eukaryotic lineages10. Large-scale genome sequencing defines the extent of protein-coding and viral elements in microalgal genomes and posits a unified adaptive strategy for algal halotolerance11. Increasingly, evidence supports the suggestion that, differing from terrestrialization, ‘true land plants’ evolved uniquely within the streptophytes, and marine-freshwater transitions are prone to be lineage-specific and bidirectional4,12–14.

To shed light on the genetic and cellular adaptations that have allowed crossing over the salinity barrier, the generation of genomic data from closely related marine and freshwater lineages is crucial10. An appropriate choice of organism group is essential for such studies. Marine-type sister groups of Streptophyta have not been confirmed14. Therefore, it is essential to find a speciose group with ample genome data available, occurring in both freshwater and saline water.

Chlorophyta microalgae of the genus Chlorella seem to satisfy these criteria. Their minute cell size and resistance to environmental stress facilitate long-distance dispersal. The genus is omnipresent in terrestrial and aquatic habitats15. Although the majority inhabit freshwater16, a large number of species occur in the oceans, from the Antarctic, to temperate and tropical regions17. However, the genomes of saltwater Chlorella spp. have not previously been sequenced. The euryhaline Chlorella sp. MEM25 (hereafter MEM25) was originally isolated from the inland saline water of Hainan Island, China, in August 201618. The water is highly saline (>65‰), with a year-round high temperature (from 30 to 41°C). MEM25 grows vigorously across a broad range of environmental conditions, including in salinities ranging from 0 to 105‰. With increasing salinity, maximum biomass production was observed at 70‰18.

Here, we present genome sequences of MEM25, which represents one of the early divergences in the genus. The genome of MEM25, likely positioned at one of the bifurcation points separating saltwater and freshwater species, may reveal the suite of traits that facilitated saline adaptation. The high-quality genome assembly of MEM25 approaches the gold standard telomere-to-telomere (T2T) assembly through a meticulous blend of sequencing technologies, ensuring exceptional coverage and precision. By deciphering multi-omics data via integrated strategies, we propose that the genetic products of ancestral genes and lineage-specific genes collaboratively sculpt the adaptation of MEM25 to salinity changes. Comparison of MEM25 with freshwater green algae and halophilic Dunaliella salina underscores the intricate evolutionary dynamics entailed in transitioning between saltwater and freshwater ecosystems. With the recently established genome editing technologies19,20, it allows us to outline a list of fundamental genes related to salinity that offer potential for enhancing saline tolerance of crops.

These findings allow us to address two questions important when exploring the evolutionary history of habitat transitions. (1) How did freshwater species evolve from their saltwater ancestors, or vice versa? (2) What gene repertoires contribute to the euryhaline characteristics of MEM25?

Results
Highly contiguous genome assembly
A hierarchical assembly approach yielded a high-quality nuclear genome for MEM25 of 53,506,137 bp, distributed over 16 chromosomes (Fig. 1a), as confirmed by 4’,6-diamidino-2-phenylindole (DAPI) staining (Fig. S1). A high level of genome scaffold continuity was revealed by the long scaffold N50 (longest, 4.3 Mb; Supplementary Data 1) and a high value of Long Terminal Repeat Assembly Index (LAI) (15.33; Fig. S2), meeting the quality benchmark for reference genomes (i.e., between 10 and 20) (Supplementary Data 2)21. Notably, all sixteen chromosomes were clearly marked with centromeres (with three featured sequences; FSs in Fig. S3) and the plant-specific telomeric repeats (TTTAGGG, Supplementary Data 3; with twelve chromosomes containing telomeric repeats at both ends and Chr15 identified as a telocentric chromosome) (Fig. 1b).

An investigation into intragenomic synteny among the sixteen chromosomes of MEM25 (Fig. S4) and among three Chlorella genomes (MEM25, Chlorella pyrenoidosa FACHB-922, and Chlorella sorokiniana 123023), along with the genomes of Chlamydomonas reinhardtii24 and Arabidopsis thaliana25 (Fig. 1c), revealed no whole-genome duplications (WGDs) in the MEM25 genome. Despite the fragmented genome assembly of C. pyrenoidosa FACHB-9 (hereafter FACHB-9) and C. sorokiniana 1230, better synteny was observed between MEM25 and these strains than that between MEM25 and C. reinhardtii or A. thaliana, suggesting the sequence conservation of centromeres and telomeres in chlorella strains (Fig. 1c).

A 100 kb-resolution 3D map of the MEM25 genome (Fig. S5) and whole-chromosomal interactions (Fig. 1d) were created using HiC-GNN and visualized using UCSF Chimera. Altogether, the quality, accuracy, and completeness of the MEM25 genome assembly is higher than the available reference sequences22,26, ensuring the reliability of subsequent genomic analyses (See Supplementary note 1 for the details of genome assembly).

MEM25 appears closest to one of the branch points separating saltwater and freshwater Chlorophyta
To illustrate evolutionary transition and distribution of saline genes, comparative analyses were performed on the genomes of MEM25 and 37 species of Chlorophyta, divided into marine (n=13) and freshwater species (n=24) (Supplementary Data 4). Representative species from cyanobacteria (n=2; one saltwater and one freshwater), Rhodophyta (n=1; saltwater), Glaucophyta (n=1; freshwater), streptophyte algae (n=4; freshwater), and embryophytes (n=2) were used as outgroups. An ancient freshwater lineage Chlorokybus atmophyticus (a transitional species from streptophyte algae to land plants) and a halotolerant marine species Picochlorum spp (isolated from a brackish water pond subject to significant salinity fluctuations) were included to highlight critical transitions from green alga to land plants and from saltwater to freshwater. Cyanobacteria (i.e., Synechococcus moorigangaii and Synechocystis sp. PCC6803) were also included to provide a holistic view of genetic diversity in microalgal evolution. For the selected species, habitat information and genomic sources were thoroughly verified (See Supplementary Data 4 for details). Encompassing a diverse array of algae, spanning phylogenetically distant taxonomies, allowed for precise delineation of homologous gene families and provides insights into genetic diversity across microalgae.

To mitigate discrepancies stemming from nucleotide or transcriptional variations between prokaryotic and eukaryotic organisms, protein sequences were utilized for homologous gene family identification. Only 13 orthologs of single-copy genes were detected in the chosen Chlorophyta species (Supplementary Data 5). We thus constructed the species tree using the most closely related genes within single-copy or multi-copy orthogroups (n=199), employing the Species Tree from All Genes (STAG) algorithm27 (Fig. 2; see Fig. S6a for the tree of the chosen species with 1000 bootstrap replicates with bootstrap values above 70%). The highly credible species trees (bootstrap support exceeding 60%) align with the chloroplast phylogenetic tree (Fig. S6b), positioning MEM25 closest to the branch point separating freshwater Chlorella spp. from the marine species (Fig. 2).

The molecular clock places the emergence of MEM25 at 632 Ma, predating all examined Chlorella spp. and ranking among the oldest Chlorophyta (Fig. S7). Although certain clades diverged earlier than MEM25, their putative saltwater ancestors remain unidentified, suggesting a dispersed and independent evolutionary transition from saltwater to freshwater habitats. Unlike the swift and dramatic transition to a terrestrial environment spurred by abrupt climate shifts, the transition from saltwater to freshwater settings unfolded in localized regions, with distinct lineages evolving independently within specific niches.

To mitigate potential biases from root placement or sampling constraints, we assessed the phylogenetic connections of MEM25 within Chlorophyta exclusively (n=93). To expand our cohort, we incorporated species from the 1KP project with transcriptomic data8 and those with available genomes (Supplementary Data 6). This allowed for a better representation of Core Chlorophyta diversity, encompassing groups like Pedinophyceae, Chlorodendrophyceae, Trebouxiophyceae, Ulvophyceae, and Chlorophyceae. The unrooted tree affirmed the close evolutionary proximity of MEM25 to early marine eukaryotic microalgae, underscoring the disparities in evolutionary position and preference for saline growth conditions compared to other selected Chlorella strains (Fig. S8). While numerous green algae genera encompass both marine and freshwater species, Chlorella stands out as one of them. MEM25 potentially retains genes and traits associated with marine environmental adaptation, warranting further exploration into salt-adaptation mechanisms.

MEM25 shares both saltwater- and freshwater-featured genes
To identify the distinctive gene families of freshwater and saltwater Chlorophyta species (Supplementary Data 4), we initiated with a two-stage machine learning approach involving 86 models and model combinations. Specifically, we generated a presence-absence matrix for gene families and applied various machine learning algorithms to differentiate between freshwater and saltwater algae (see “Methods”). The RandomForest + ElasticNet [0.6], identified as the most effective combination among the 86 models, achieved a classification accuracy of 97% and pinpointed 138 critical gene families with distinct functions in freshwater versus saltwater microalgae (Supplementary Data 7). These genes were further categorized into saltwater (n=44) and freshwater-specific groups (n=94) based on their weight coefficients.

A three-dimensional PCA analysis of these genes was conducted where freshwater species clustered tightly together and MEM25 emerged as the closest saltwater species to the freshwater cluster (Fig. 3). MEM25 displays characteristics of both saltwater (housing 32 of the 44 featured saltwater genes) and freshwater (possessing 75 of the 94 featured freshwater genes) adaptation. In comparison, the genome of Chloropicon primus (a prevalent species in marine phytoplankton communities28) contains 23 saltwater-featured genes and 34 freshwater-featured genes, respectively. The findings were reinforced by a more straightforward cutoff method, using a 60% cutoff (a gene family was categorized as a saltwater gene family if it was present in over 60% of saltwater species and absent in over 60% of freshwater species, and vice versa). A number of 614 featured gene families enriched in either saltwater (n=129) or freshwater species (n=485) (Supplementary Data 8 and Fig. S9a). Pearson’s chi-square (χ²) tests confirmed the statistical significance (Pearson’s χ² (df=1)=1430.5, P=2.2e-16, Cramer’s V=0.49, 95% confidence intervals=[0.44, 0.54]), and similar results were obtained when we raised the cutoff from 60% to 80% (Fig. S9b).

This suggests that MEM25, potentially as an early-diverging saltwater species, has evolved numerous genes critical for freshwater-saltwater transitions, positioning it at one of the many pivotal junctures separating saltwater and freshwater Chlorophyta. The genome exploration of MEM25 could serve as a foundational element in unraveling the underlying molecular mechanism and processes involved in the transition in either direction between marine and freshwater.

MEM25 possesses general saline-related modules shared with freshwater species and exclusive ones that have not yet been explored
To investigate species similarities and differences in salinity adaptation mechanisms between saltwater and freshwater species, we conducted a comparative analysis of the global trancriptomic and metabolic dynamics of the euryhaline MEM25 and the neutrophilic freshwater FACHB-922 (See “Method” for growth conditions of FACHB-9). Despite the relatively high synteny observed in their genome sequences (Fig. 4a; see statistical details in Supplementary Data 9), there was a remarkably low alignment ratio of FACHB-9 transcriptomic reads on MEM25 genome sequences (0.95%), and vice versa (0.02%) (Supplementary Data 10). To mitigate biases stemming from differences in gene annotation quality between the two species, we employed a Weighted Correlation Network Analysis (WGCNA). By aggregating the expression of genes within each orthogroup to create a “metagene” expression level, we aimed to capture co-expression patterns reflective of shared functional roles, irrespective of individual gene copy numbers within each gene family.

We established a common set of annotations for 5,026 orthologous gene families, comprising 7,594 and 8,360 protein-coding genes, for comparisons between MEM25 and FACHB-9, respectively (Fig. 4b). By applying WGCNA on all 24 RNA-Seq datasets, we identified 67 gene modules (shown in various colors in Fig. 4c; |Pearson|≥ 0.75 and P≤0.005), with gene numbers ranging from 31 to 233. Subsequently, we assessed the associations of each module with four parameters: stress severity (salinity), stress duration (treatment duration), damage state (0 for algae under preferred salinity, 1 if not), and MEM25-specific mechanisms (0 if not specific to MEM25, 1 if specific to MEM25) (Supplementary Data 11). Notably, none of the modules exhibited correlations with stress duration, while 15 modules displayed significant associations with salinity, either positively (n=3) or negatively (n=12). Additionally, we identified 20 modules specifically linked to freshwater species and 13 modules associated with saltwater species, encompassing 1,841 and 1,053 orthologs, respectively (Fig. 4c).

Specifically, we found that the thistle2 module was significantly (Pearson correlation = 0.89, df = 45, P=9×10⁻⁹, 95% confidence intervals= [0.80, 0.94] P=9×10−9, Pearson = 0.89) and specifically associated with saline adaptation of MEM25, suggesting that the feature of this subfamily was lost in the lineage leading to the freshwater counterpart. Noteworthy is the distinct association of the RecQ-mediated genome instability protein 1 (RMI1; CP5g5156) with the saline adaptation of MEM25 (Supplementary Data 12). Nonetheless, the transcription of RMI1 notably decreased in both strains upon the transition from low to high salinity levels (Supplementary Data 12), indicating RMI1’s involvement in responding to heightened salinity, likely following a similar pattern in both freshwater and saltwater Chlorella species. The apparent inconsistencies could be elucidated by the considerably lower abundance of RMI1 transcripts in MEM25 relative to FACHB-9 (Supplementary Data 12). A relatively diminished dosage of RMI1 appears to facilitate MEM25’s acclimatization to saltwater. On the other hand, 69 genes of the 47 orthogroup in this module were largely unannotated (Supplementary Data 12), indicating potential salinity adaptation strategies unique to MEM25.

The metabolomic counterparts to the thistle2 module in the transcriptomic comparison were the black and pink modules (Fig. 4d). While most genes in the thistle2 module (Fig. 4c; specifically linked to MEM25’s salinity adaptation) lacked annotation, the black and pink modules in the metabolome analysis contained known osmoprotectants like D-sorbitol, D-arabitol, and glycerolipids (Supplementary Data 13). Nevertheless, the roles of most other genes and metabolites in these modules in salinity adaptation remained unexplored. The presence of lineage-specific modules may suggest specific adaptations of saltwater or freshwater Chlorella sp. to saline stresses. The identification of MEM-specific or general modules of both freshwater and saltwater species could shed light on the molecular mechanism underlying transitions between salty and freshwater environments.

Gains and expansions of saline-related genes are either exclusive to MEM25 or shared across Viridiplantae
The homolog matrix of orthogroups was analyzed to deduce ancestral genes and lineage-specific genes content within the Viridiplantae (green plants), unveiling 5, 0, and 7 significantly expanded gene families in the genomes of Streptophytina, Chlorophyta, and Chlorella spp., respectively (p<0.05; Fig. S7). For example, evolutionary analysis of gene family OG0000035 using the Computational Analysis of gene Family Evolution (CAFE) framework yielded a family-wide p value of 0.001, indicating significant deviation from the neutral birth–death model. The global evolutionary rate parameter (λ), which reflects the background rate of gene family expansion and contraction, had a mean value of 0.000414 (range: 0.0000613–0.0009377). To evaluate lineage-specific rate heterogeneity, we performed a Likelihood Ratio Test (LRT) comparing a branch-specific λ model with the global λ model (df = 9). Together with the Viterbi p values generated by CAFE (ranging from 0.0005068 to 0.950976), the results demonstrate that Streptophytina lineages exhibit λ values that significantly deviate from the global rate, indicating heterogeneous evolutionary dynamics within this clade. Comprehensive statistical outputs for additional gene families and species are provided in Supplementary Data 14.

Compared to all examined freshwater Chlorophyta, including Chlorella spp., a variety of gene families were expanded in the MEM25 genome (n=89), implying their involvement in salt adaptation. Among these expansions, only five orthogroups have been found shared in green plants and could be referred to as lineage-specific genes. Out of these, four have been annotated and are associated with oxidative stress (i.e., peroxidase, family OG0015353), osmotic adjustment (i.e., volume-regulated anion channel subunit LRRC8A, family OG0021391; amino acid permease 2, family OG0026817), and response to salty stimuli (E3 ubiquitin-protein ligase, E3L; family OG0012359)29–31 (Supplementary Data 15), suggesting these saline-related genes were already present in the last common ancestor of MEM25 and green plants prior to their divergence into either saltwater or freshwater preferred adaptations.

On the contrary, the majority of expanded groups are unique to MEM25 (84 in 89). We have designated these genes as lineage-specific genes of MEM25. The large number of MEM25-specific genes may reflect the phylogenetic distance of MEM25 from other Chlorella species with sequenced genomes. Assuming a freshwater origin of the ancestor of MEM25, portion of these genes may have been acquired during the specialization of MEM25 to a salty niche. For example, we found an exclusive presence of gene family OG0011211 in the MEM25 genome (Fig. S10). OG0011211 encodes a NAD-dependent epimerase/dehydratase family protein, absent in other Chlorophyta and plant lineages but found in three copies within MEM25 genomes (Fig. S10). This gene family is believed to have originated from bacteria where it is involved in osmotic stress32. Loss of NAD-dependent epimerase/dehydratase is known to cause osmosensitivity in bacteria32, indicating that MEM25 may have acquired specialized adaptive mechanisms from bacteria through horizontal gene transfers (HGTs), representing an evolutionary innovation associated with salinity-related genes in MEM25.

For the remaining of MEM25-specific genes, we would assume them as orphan genes which have no apparent homology to genes in other evolutionary lineages occur in all genomes. They could be candidates for the de novo evolution of genes and may contribute to evolutionary novelties, which can become relevant for lineage-specific adaptations. Consequently, MEM25 has developed a collection of expanded salinity-related genes, categorized as ancestral genes and lineage-specific genes, shaping its euryhaline nature.

Expanded genes in MEM25 are transcriptionally active in response to saline stresses
In order to unveil the molecular mechanisms of its euryhaline feature, we tracked the transcriptomic and metabolic dynamics of MEM25 under specific low and high salinity conditions. Differential gene expression analysis demonstrated that numerous genes which had undergone gene family contraction/expansion also displayed differential expression in response to changes in salinity. Among the 89 expanded orthogroups, 73% showed a significant correlation with salinity change (Fig. S11), suggesting an adaptation mechanism of MEM25 to saline environments at both genomic and transcriptional levels. For instance, all five annotated expanded orthogroups in MEM25 (i.e., family OG0012359, OG0015353, OG0011211, OG002139, OG0026817; Supplementary Data 15) were markedly upregulated following an increase in salinity (Fig. 5a). The transcriptionally enriched expanded genes also encompass those involved in fatty acid elongation (e.g., acyl-CoA thioeaterase encoding gene CP11g7958 and enoyl-[acyl-carrier-protein] reductase encoding gene CP15g9776) and antioxidant activity, such as glutathione metabolism (CP15g9764 and CP2g1989), ascorbate metabolism (L-galactose dehydrogenase CP8g6549 and monodehydroascorbate reductase CP1g297), vitamin C biosynthesis (GDP-D-mannose 3’, 5’-epimerase CP14g9420), alcohol dehydrogenase (CP9g7414), and inositol oxygenase (CP4g4403)33 (Fig. 5b and Supplementary Data 16). Therefore, the expanded genes appear to actively transcribed in the saline adaptation of MEM25.

Metabolic dynamics are synchronized with transcriptional changes
Regarding metabolism, the levels of 150 out of 451 metabolites underwent significant changes (|log2(FC)| ≥ 1 and P≤0.05). Corresponding to the upregulation of genes involved in proline biosynthesis (e.g., glutamate 5-kinase CP14g9503 and CP14g9505; delta-1-pyrroline-5-carboxylate synthetase CP8g6403; pyrroline-5-carboxylate reductase CP3g3123) and fatty acid desaturation (e.g., acyl-[acyl-carrier-protein] desaturase CP11g7888) (Supplementary Data 17), the contents of proline and unsaturated fatty acids significantly increased under high salinity conditions (Fig. S12 and Supplementary Data 18). We also noted elevated levels of metabolites with established roles in abiotic stress adaptation, including D- (+)-sucrose, gulonolactone, and vitamins (e.g., vitamin B13) (Supplementary Data 18). Following salinity changes, MEM25 tends to activate the biosynthesis of stress-related metabolites such as proline and sucrose, while the processes related to antioxidant activity were stimulated. Thus, the findings from genome expansion and omics analysis complement each other, revealing an array of saline-related genes unique to MEM25 or shared with Viridiplantae. These genes merit further validation to unveil their bona fide function and could potentially be leveraged for trait improvement in microalgae or crops.

GWAS corroborates the salinity-related role of genes identified through genome expansion analysis
To validate the salinity-related genes uncovered through genome expansion, genome-wide association studies (GWAS) was conducted. An ethyl methanesulfonate (EMS) mutant population was generated using MEM25 as the parent strain. Out of 50,000 independent mutant lines, 536 mutants with altered OD750 under either low (i.e., 35‰) or high (i.e., 70‰) salinity were selected for further phenotyping under the high-salinity conditions. Subsequently, 365 mutants with stable phenotypes after ten generations were chosen to assess variations in cell number, cell size, and dry weight (DW) under the high-salinity conditions (Fig. 6a). Lastly, the genomes of 195 saline-related mutants and three WT samples were resequenced with 30× coverage (n=198).

In total, 167,199 variations were detected in the genomes of all samples (Fig. 6b). Using the mixed linear model to analyze the associations between the three phenotypes and genetic variations, we consistently pinpointed five loci exceeding a significant threshold (−log10 P≥5.0), linked to either known or unknown genes. Specifically, we observed clusters of highly significant Single Nucleotide Polymorphisms (SNPs) proximal to S04_3657761 in Chr04 associated with the DW (−log10 P=8.22) (Fig. 6c; see the quantile-quantile plots in Fig. S13). S04_3657761 is positioned downstream of gene CP4g4326 and upstream of CP4g4327 with an intergenic region of 879 bp (Fig. 6d). The G to T transition at S04_3657761 creates a binding site for the transcriptional factor cysteine-rich polycomb-like protein which is associated with abiotic stresses, such as salinity, in a broad spectrum of species34,35. Examination of the chromatin organization unveiled topologically associating domains (TADs) and loops displaying a significant interaction between S04_3657761 and gene CP4g4237 (Fig. 6e).

Intriguingly, all three genes (i.e., CP4g4326, CP4g4327, and CP4g4237) are E3L, supporting a salinity-related role of E3L, reinforcing its salinity-related role as indicated by genome expansion analysis and transcriptomic investigations (Supplementary Data 15). While the categories of E3L genes identified through GWAS, genome expansion, and transcriptomic studies may not perfectly align, they suggest a coordinated orchestration of E3L-mediated posttranslational modifications at both the genomic and the transcriptomic levels, contributing to MEM25’s response to salinity. E3L has been demonstrated to play a crucial role in protein modification and the regulation of various biological processes and cellular responses to stress signals in plants36. Therefore, this association effectively pinpoints genes linked to functional polymorphisms, corroborating the findings of genome expansion and omics analyses.

Functional validation reinforces the utility of the newly discovered salinity-related gene repertoire
To delve deeper into the salinity-related genes, an association network was established to show transcript-metabolite correlations in MEM25 (Fig. 7a). This network included orthologous gene families (n=1231) and metabolites (n=109) that exhibited significant correlations with stress severity (i.e., salinity), with only pairs having a correlation coefficient above 90% considered in the subsequent analysis. Genes where both the transcript and the correlated metabolite displayed correlations with salinity changes were retained in the repertoire. To validate this repertoire, salt-sensitive genes were selected using three strategies. First, genes with elevated transcriptional levels (in FACHB-9 compared to MEM25) and also in the modules linked to freshwater species (not associated with the MEM25-specific mechanism) (e.g., CP4g4492 and CP1g58) were chosen. Second, genes with reduced transcriptional levels (under high salinity compared to low salinity conditions) and found in the modules associated with saltwater species (associated with the MEM25-specific mechanism) (e.g., CP5g5156 and CP8g6201) were selected. Third, genes from expanded orthologs in freshwater species (in comparison to MEM25) and present in modules associated with freshwater species (not linked to MEM25-specific mechanisms) (e.g., CP11g8082 and CP10g8454) (Supplementary Data 19) were used.

A gene inventory (including genes largely unannotated) was compiled based on these strategies and we expected improved salt tolerance by knocking out individual genes in the repertoire. The homologs of five chosen genes have not been functionally investigated while one gene has been linked to DNA repair and meiosis in plants37. None of them have ever been associated with salinity tolerance.

MEM25 thrives best at 70‰ salinity, while FACHB-9 prefers 0‰ salinity. Recognizing the challenge of conferring salinity resistance to a freshwater species through single gene manipulation, we utilized a “buffering” strain, Nannochloropsis oceanica, adapted to a “medium” salinity level of 35‰ to evaluate the biological significance of the selected genes. Knockout mutants of all six candidate genes were created for N. oceanica. In comparison to the wild type, all mutants exhibited significantly increased biomass (Fig. 7b) and cell numbers (Fig. 7c) under the high salinity conditions (i.e., 70‰).

The successful validation of gene function has inspired us to explore the potential application of these genes for trait improvement of higher plants. CP1g58 of MEM25 encodes a protein homologous to the Arabidopsis zinc knuckle (CCHC-type) family protein (AT3G42860), which has not been experimentally validated. On the other hand, the Arabidopsis counterpart of the protein encoded by Chlorella CP5g5156 is RMI1 (AT5G63540), with the Arabidopsis mutant rmi1 associated with DNA damage repair37. CP1g58 in MEM25 (Fig. S14a) and the homologous gene g3721 in freshwater FACHB-9 (Fig. S14b) exhibited significantly decreased expression levels under the elevated salinity. For CP5g5156 in MEM25 (Fig. S14c), two homologous genes were identified in FACHB-9 (g1423 and g1502), all showing decreased expression levels with increasing salinity (Fig. S14d). These findings suggest that lower expression of CP1g58 and CP5g5156 homologs in Arabidopsis may confer enhanced resistance to salt. Thus, we choose these genes for further validation in higher plants.

In line with our expectations, two independent knockout plant lines of AT5G63540 (RMI1; homologs of CP5g5156) exhibited increased root growth under salt stress (Fig. 7d), indicating RMI1 as a negative regulator in salinity tolerance in plants. RMI1 may exemplify one of the numerous ancestral genes contributing to saline adaptation specific to Chlorophyta in freshwater or saltwater. In contrast, compared to the wild type, the knockout mutant of gene AT3G42860 in Arabidopsis (homologs of CP1g58) exhibited marginal difference in root growth under salt stress (Fig. 7d). Our findings suggest that the proteins encoded by AT5G63540 and its homologs play relevant roles in salinity tolerance across various species, from microalgae to plants. To our knowledge, this is the first report to document the effect of these genes on salinity adaptation, reinforcing the value of the gene repertoire identified here for pinpointing candidate genes linked to salinity resistance that could enhance plant agronomic traits.

Methods
Growth conditions
Chlorella sp. strain MEM25 was maintained in dim light at 4°C on solid modified F2 medium, which was prepared as the early report40. C. pyrenoidosa FACHB-9 was maintained in BG11 medium (1500 mg L−1 NaNO3, 30 mg L−1 K2HPO4, 75 mg L−1 MgSO4·7H2O, 36 mg L−1 CaCl2·2H2O, 6 mg L−1 ferric citrate, 6 mg L−1 ammonium citrate, 1 mg L−1 EDTA, 20 mg L−1 Na2CO3, 2.86 mg L−1 H3BO3, 1.81 mg L−1 MnCl2·4H2O, 0.222 mg L−1 ZnSO4·7H2O, 0.390 mg L−1 NaMoO4·5H2O, 0.079 mg L−1 CuSO4·5H2O, 0.0494 mg L−1 Co(NO3)2·6H2O)22, at 25°C under a 12-h light/12-h dark photoperiod (50 μmol·photons·m−2·s−1). For inoculation, cells were transferred into the liquid medium and were maintained under illumination of 50 μmol photons m−2 s−1 at 25°C with a light/dark cycle (12h/12h).

Chromosome staining
Algal cells in mid-logarithmic phase were collected and treated with 0.1% w/v colchicine for 6 hours, followed by fixation in Carnoy’s solution for 12 h. Thereafter, the cells were washed in 70% methanol and fresh F2 medium. They were then treated with a softening solution containing 5% EDTA with 0.1% Tween 20 for 1 h, followed by hydrolysis with 30 mM KOH and 500 mM HCl at 50°C for 1 h, respectively. The cells were collected and suspended in 0.1 M citrate buffer (pH=5) with Cellic® Htec2 (Novoenzymes) at 37 °C for 1 h. Next, the cells were immediately stained with 0.5 mg mL−1 4’, 6-diamino-2-phenylindole (DAPI) for 5 min and were examined under a microscope41.

Genome assembly
Single molecule real time sequencing (SMRT) libraries and Illumina short read libraries were constructed using fresh mid-log phase algae (BioMarker, China), followed by sequencing with the PacBio Sequel II and HiSeq 2000 platforms. The Hi-C library and sequencing were conducted with the Illumina HiSeq platform (Novogene, China). Multiple approaches have been employed to guarantee the quality of genome sequences. PacBio and Hi-C data, Hifiasm (version 0.16.1-r375) was used for subgenome splitting. SMRT sequencing data were assembled using Falcon (version 0.3.0) and CANU (version 2.1.1). 3D-DNA (version 180922) and ALLHiC (version 0.9.8) were used for draft genome correction and genome assembly, respectively. Finally, Juicebox (version 1.11.08) and Pilon (version 1.24) were used to correct genome fragments (based on the chromosome contact frequency matrix) and base errors of the genome with Illumina and RNA-seq data, respectively. The Hi-C interaction frequency matrix was analyzed by Homer, FitHic (version 2.0.8), HiCExplorer (version 3.7.2), and HiCCUPS (version 5.14). The 3-D chromosome coordinate location was calculated using HiC-GNN and was visualized using UCSF Chimera (version 1.15).

Genome annotation and analyses
For genome annotation, various references were used, including A. thaliana, Auxenochlorella protothecoides UTEX25, Chlamydomonas reinhardtii, Chlorella sorokiniana 1602, Chlorella variabilis NC64A, Coccomyxa subellipsoidea C-169, Dunaliella salina CCAP1918, and Micractinium conductrix SAG241. Augustus (version 3.3.3), GlimmerHMM (version 3.0.4), SNAP (version 2013.11.29), Geneid (version 1.4), and Genscan were used to predict gene structure ab initio. Cufflinks (version 2.2.1) was used to predict novel genes and correct gene structure. EVidenceModeler (version 1.1.1) and PASA (version 2.4.1) were used to remove redundancy and correct the integrated annotation results.

Gene function was inferred using BLASTP (with a cutoff of e value<1×10−5) by comparing with known protein sequence databases, such as Evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), National Center for Bio-technology Information (NCBI), International Protein Resource Information System (InterPro)42, The Protein Families Database (Pfam), SwissPro, and KEGG.

Conserved non-coding DNA sequence annotation
The repeat sequences were annotated using RepeatMasker (version 4.1.0) while conserved non-coding DNA sequences (CNSs) were identified using dCNS (version 0.4). For CNSs annotation, the reference genomes included those of Chlorella sp. A99, A. thaliana, A. protothecoides UTEX-25, C. reinhardtii, D. salina, C. sorokiniana 1230, C. pyrenoidosa FACHB-9, M. conductrix SAG241, C. variabilis NC64A, Ostreococcus tauri, and Picochlorum soloecismus DOE101. The short segment tandem repeat sequences were eliminated and the adjacent CNSs with regional overlap or with spans less than 50 bp were merged. Finally, a total of 721 CNSs was obtained.

Telomere and centromere scanning
Tandem repeats finder (TRF, version 4.09) was used to search for tandem repeat sequences and identify telomere sequences. For centromere annotation, minimap2 (version 2.2.26) was used to mask the functional regions, followed by removal of redundancy and searching and extraction of the repeated units. The genome sequences were segmented into fragments with a length of 500 bp. CD-hit (version 4.8.1) was used to find the sequences with similarities above 70%. MEME SUITE (version 5.3.0) and Homer were used to extract the featured fragments of these sequences, which were subsequently aligned to the individual chromosome. The sequence similarity was visualized by Circoletto.

Genome quality assessment and collinearity analysis
LTR_FINDER (version 1.07) was used to identify Long terminal repeat retrotransposons (LTR-RT), this comprises a non-redundant LTR-RT library, with LTR Assembly Index (LAI) values calculated. Gene annotation and assembly were evaluated via BUSCO (version 5.2.2) using the single copy Homeotic gene database of Chlorophyta (https://busco-data.ezlab.org/v5/data/lineages/chlorophyta_odb10.2020-08-05.tar.gz). The genome synteny and collinearity were detected by JCVI (version 1.3.5) and the results were visualized by the R package Rideogram (version 0.2.2).

Comparative genomic and phylogenetic analysis
Genome sequences for the selected species were retrieved from the NCBI and IMG-M databases (https://img.jgi.doe.gov/cgi-bin/m/main.cgi) (Supplementary Data 4). OrthoFinder2 (version 2.5.2) was used for orthogroup inference, with 39,321 orthogroups obtained for the 46 selected species. Among them, 199 orthogroups were present in all the selected species, and these were used to infer species trees by using the STAG algorithm. Dollo parsimony was implemented for Count to infer family- and lineage-specific characteristics across the evolutionary tree.

Chloroplast genome sequences were retrieved from the CGIR database (https://ngdc.cncb.ac.cn/cgir/). Parsed hits for all species were aligned using MAFFT7 (version 7.480). Gaps and ambiguously aligned sites were removed using gBlock (version 0.91b). Sequences that caused aberrant alignments and whose real identity could not be confirmed were removed manually. Phylogenetic analyses were performed with a maximum likelihood method using IQ-TREE (version 2.1.4-beta).

The R8S script was employed to estimate temporal divergence based on the molecular evolution rate and stable fossil nodes. A strict clock model was used to avoid horizontal gene transfer and other events that affected divergence times. Multiple time constraints (fossil records for no less than three species within the same genus) were incorporated to evaluate our results using fossil cross-validation. The fossil records used in this study are relevant ones that have previously been applied to estimate the divergence times of eukaryotes, including those for (1) A. thaliana to Oryza sativa (115-308 Mya), (2) Mesostigma viride to Chlorokybus atmophyticus (174-631 Mya), (3) Ostreococcus lucimarinus to Micromonas pusilla (333-639 Mya), (4) Synechocystis sp. PCC 6803 to Synechococcus moorigangaii (1580-2557 Mya), and (5) Porphyra umbilicalis to Cyanophora paradoxa CCMP329 (1386–1680 Mya). The fossil records are available at Timetree (http://www.timetree.org/).

Genome-wide association
A mutant pool for MEM25 was generated by EMS mutagenesis following our previous report. In brief, the mutagenesis conditions were optimized by treating log-phase cells with different concentrations of EMS for varying durations, followed by assessing the number and morphology of the resulting colonies. A mutant pool was constructed using the optimized conditions and candidate mutants were further assessed for growth in either the low salinity (i.e., 35‰) or the high salinity (i.e., 70‰) conditions. The turbidity was monitored by recording OD750 at indicated intervals. A total of 536 mutants with altered OD750 were selected for further phenotyping under the high-salinity conditions. The 365 mutants with stable phenotypes after ten generations were assessed further for growth in the high salinity conditions (i.e., 70‰). Dry weight, cell size, and cell number were recorded. Finally, genomes of 195 salinity-related mutants and three WT samples were resequenced using the second-generation sequencing Illumina platform for PE150 sequencing (Novogene Company, China).

Fastp (version 0.20.0) was used for filtration and quality control of original sequencing data (fastp -t 10 -f 10 -F 10 -T 10 -W 6 -u 20 -n 10 -c). Genome alignment was conducted using BWA-MEME (version 0.7.17-r1188) and SAMtools (version 1.12) (bwa mem -R ‘@RG	ID:{sample}	SM:{sample}	PL:Illumina’\index fq1 fq2 | samtools sort -@ 2 -m 1G | samtools view -h -b -q 30 > q30.sort.bam), followed by quality control using Qualimap (version 2.2.2). PCR duplicates and optical duplicates were removed using Picard’s MarkDuplicates program (https://broadinstitute.github.io/picard/). Only high-quality sequencing data were used for the subsequent analysis. Variations were detected using Genome Analysis Toolkit (GATK) (version 4.2.1). A value of 0.00743 was obtained for the genomic heterozygosity of MEM25 by using a combination of Jellyfish (version 2.3.0) and GenomeScope (version 2.0). Genome alignment on the sequences around INDEL (Insertion and Deletion) was performed to detect variations for each sample. The results of all samples were merged when population variations, SNPs, and INDEL were obtained. Quality control on SNPs (QUAL<30 || MQ<40.00 || SOR>4.000 || QD<2.00 || FS>60.000 || MQRankSum<-10.000 || ReadPosRankSum<-10.000 || ReadPosRankSum > 10.000) and INDEL (QUAL<30 || MQ<40.00 || SOR>10.000 || QD<2.00 || FS>200.000 || ReadPosRankSum<-20.000 || ReadPosRankSum > 20.000) was conducted separately. Variations with abnormal sequencing depth were removed. GWAS analysis was performed using TASSEL, employing a Mixed linear model and Bonferroni correction. Variations were annotated via SnpEff. R package CMplot (https://github.com/g-insana/CMPlot.jl) was used to draw a Manhattan plot, Quality-Quality plot, and genome variation distribution maps.

Sample preparation for transcriptome and metabolome analysis
Cells were cultured into log phase under optimal conditions. Salt stress was induced by transferring microalgae to high-salinity conditions (that is 35 g·L−1 for FACHB-9 and 105 g·L−1 for MEM25). Aliquots of cells were collected following the salinity shifts for either transcriptome (after 3h and 24h) or metabolome (after 24h) analysis. Triplicates were used for each treatment.

Transcriptome analysis
Total RNA extraction, sequencing, and assembly were performed according to previous reports40. Transcriptome sequencing was completed by BioMarker Company (China, Beijing). Quality control and removal of low-quality fragments was conducted by FastQC (version 53.0) and fastp, followed by the trimming of low-quality regions via Trimmomatic (version 0.39). The RNA seq data were compared with the reference genomes using GSNAP (version 2021-07-23), followed by quantification of the values of FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) using StringTie (version 2.1.7), R package Ballgown (version 2.22.0), and DESeq2. Differentially expressed genes were defined as those with FDR-adjusted p-value ≤ 0.05 and |log2 FoldChange| ≥ 1. Function and pathway enrichment analysis was conducted using KEGG and the R package clusterProfiler (version 3.18.1).

Metabolome analysis
~500 mg of algal biomass was collected, followed by metabolite extraction. Algal cells were suspended in 1 mL mixture of methanol and water (7/3, vol/vol) and kept at −80°C for 2 min. The internal standard used was 2-chloro-l-phenylalanine (0.3 mg mL−1). The mixture was vortexed at 60 Hz for 2 min, followed by ultrasonication at ambient temperature for 30 min. Samples were then centrifuged at 13,000 g, 4 °C for 15 min. The supernatants were collected using crystal syringes, filtered through 0.22 μm microfilters and transferred to LC vials, followed by storage at −80 °C. LC–MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies).

Metabolite-transcript correlation analysis
Pearson correlation coefficients (PCCs) were calculated for metabolite and transcript profiles as described previously43. The mean of all the biological replicates for individual metabolites and the normalized mean value of the transcriptional levels of each gene were evaluated. The coefficients were calculated using the log2 (fold change) values, with PCC > 0.90 and PCC P value (PCCP) < 0.001 used as the cutoff. The connection network between orthogroups and metabolites was built via Cytoscape (version 3.8.2).

Weighted gene co-expression network analysis
Each orthogroup, out of the 5026 shared by MEM25 and FACHB-9 (including 3101 single copy orthogroups), was designated as a metagene. Taking into account both the transcriptional level and the gene copy number’s impact, the metagenes’ transcriptional levels were quantified by summing the transcriptional levels of all genes within each orthogroup. The WGCNA version 1.70-3 was used to analyze the transcriptome and metabolome data, with the orthogroups categorized into different modules, correlated with specific trait(s). A cutoff was set with the correlation coefficient > 0.75 and a P value < 0.005. For the transcriptome, the WGCNA soft threshold of the expression matrix was set at 7, with the scale-free topological model fitting R2>0.9. Regarding the metabolome, the values were 9 and 0.9, respectively.

Principal component analysis
Principal component analysis of featured genes was performed using the R package ggord (https://zenodo.org/badge/latestdoi/35334615). The packages prcomp and factoextra were used for the analysis and ggplot2-based visualization. The abscissa of the PCA score chart represents the first principal component, namely PC1, and the ordinate represents the second principal component, namely PC2. The confidence interval level for PCA analysis was set to 0.95.

Machine learning analysis
A machine learning analysis was conducted on a gene family presence-absence matrix across the 46 species to discern crucial genetic traits that differentiate freshwater and marine algae. Each species was represented in binary form, with “1” denoting the presence of a gene family and “0” indicating its absence. Our method involved a two-stage machine learning approach encompassing 86 models and model combinations, including RandomForest, Lasso, glmboost, Ridge, SVM, KNN, Naive Bayes, GBM, DecisionTree, and Enet (with various alpha values). Initially, models performed feature selection to pinpoint potential gene families, followed by additional filtering or classification to enhance classification accuracy. A 5-fold cross-validation method was employed across all models to ensure robust performance evaluation. For models like Ridge Regression without inherent feature constraints, accuracy was incrementally tested with feature sets in batches of 50 genes to determine the optimal feature count for peak performance. This meticulous approach ensured that each model combination achieved its highest classification accuracy, enabling precise identification of genetic traits crucial for distinguishing between freshwater and marine species. The most effective model combination, RandomForest + ElasticNet [0.6], boasting a classification accuracy of 97%, was utilized to highlight featured genes.

Cas9-mediated gene knockout and phenotype assays
Cas9-mediated target gene disruption in Nannochloropsis was conducted using an episomal CRISPR system44 following a protocol described previously40. Two sgRNAs were designed for each gene. For each transformation reaction, 109 log-phase cells were mixed with 1 µg cassette in an electroporation cuvette (2 mm gap). Electroporation was performed using the GenePulse XcellTM (Bio-Rad) with 11 kV cm−1 field strength. After the pulse, the cells were immediately mixed with 5 ml F2 medium for recovery. Then, the cells were plated on the F2 agar plate with 2 μg·mL-1 zeocin until transformant colonies appeared. Mutations with target disruption were screened by PCR with specific primers (Supplementary Data 20). The growth of microalgae was monitored by measuring the turbidity or cell number at specified intervals with a GeneQuant 1300 Spectrophotometer (GE) or LUNA-II™ Automated Cell Counter (Logos Biosystems). Dry cell weight of a 10-ml algal culture was determined simultaneously.

A. thaliana mutants and phenotype
A. thaliana Col-0 (WT) and SALK_202085C (cnbp) T-DNA insertion mutant lines were obtained from The Nottingham Arabidopsis Stock Center (NASC). The T-DNA insertion position and homozygous lines were verified according to instructions from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/help/genotyping). The genomic sequence of the mutated gene was used to design the forward primer while the T-DNA left border sequence was used to design the reverse primer (LBa1, 5’-tggttcacgtagtgggccatcg-3’). Seeds were surface sterilized and germinated on half-strength MS medium containing 3% sucrose and 0.8% phytoagar (pH 5.7) in a growth chamber at 22°C with 100 μmol·photons·m−2·s−1 light and a 16h light/8h dark cycle. Two-week-old seedlings were used for NaCl treatments (150 mM). Seeds of the wild-type and mutant plants were grown on the same plates containing MS medium with or without NaCl. Plants were grown at 22°C with 100 μmol·photons·m−2·s−1 light and a 16h light/8h dark cycle. The root length was measured with a ruler at the specified times.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled \"Cross-species dissection of saline-genes by genetically deciphering a euryhaline microalga\" demonstrates promising research but requires several major revisions. First, the genus name should be incorporated into the title. While the study effectively compares multiple species, it lacks detailed analysis of saline-genes and their evolutionary patterns across species. The introduction needs strengthening, particularly regarding the rationale for selecting Chlorella genome and its ecological significance in freshwater/seawater environments.\nThe manuscript's structure and organization require substantial modification. The supplementary materials are excessive (27 supplementary Tables), making it difficult for readers to navigate. I recommend consolidating tables and utilizing public depositories for supplementary data. The manuscript currently exceeds Nature Communication's guidelines, containing 11 main figures (exceeding the 10-figure limit) and 118 references (surpassing the 70-reference maximum), with methodology citations comprising more than half of the references.\nThe main text is overly lengthy at 30 pages and exceeds 5,000 words, with the Results & Discussion section being particularly verbose. The authors should condense the content while maintaining the scientific integrity of their findings. Despite these structural issues, the study presents interesting findings and valuable results. However, addressing these formatting and organization concerns would significantly improve the manuscript's clarity and adherence to journal guidelines.\nThe authors should address the following points for clarification:\n- L.3: Clarify the meaning and reference of the # symbol\n- L.49: Add missing \"Introduction\" header\n- L.59-60: Rephrase sentence to specify which \"other eukaryotes\" are being discussed\n- L.74-75: Content needs clarification/completion\n- L.88-97: Include methodology introduction for addressing stated hypotheses\nGeneral comments: Introduction is too brief compared to Results & Discussion and Discussion sections. Please add justification for microalga species selection and expand methodology overview for addressing research hypotheses\nResults and discussion\n- L.110: Delete \"s\" from \"genomes\".\n- L.113: Specify databases used.\n- L.116-117: The transcriptome has not been presented before, nor mentioned in the first paragraph of the results. How was the transcriptome done?\n- L.122: Define T2T acronym.\n- L.140-142: Add references for known species genomes mentioned in the text.\n- L.151-152: How have the authors performed the whole chromosome interactions, and should they briefly mention the methods used here, e.g. correlation using which metrics or algorithm?\n- L.152: Remove or justify \"As expected\" statement.\n- L.154-156: Include literature citations.\n- L.165-168: Add citations for species selection.\n- L.169: Specify and cite Cyanobacteria species used in the analysis.\n- L.175: Fix spacing between \"outgroups\" and \"(Table S10)\". Concerning Tables S10: The authors should consider transforming their Table S10 into a main table so that the reader can directly see which species they have chosen for comparison, as the authors have not included all the names of the species used in the main text.\n- L.183: Reference table/figure for 13 ortholog genes.\n- L.186: Add missing Streptophyta color in Fig.3a.\n- L.197: Replace \"exceptional position\" with more appropriate wording.\n- L.227-228: Include method reference.\n- L.261-262: Specify relevant table/figure.\n- L.273: Introduce neutrophilic freshwater FACHB-9 and cite the literature.\n- L.277: Specify C. reinhardtii strain name.\n- L.318-320: Where can we see that these genes are salt-sensitive and not associated with cell damage? This is not clear.\n- L.327-328: Same question as above, provide data location.\n- L.435: Correct table citation order (S24 before S25).\n- L.548: Reorganize Figure 11 panels for better readability.\nThe Results and Discussion section's first part needs more thorough analysis to effectively convey the findings' implications.\nDiscussion\nThere is inconsistency in the manuscript's structure regarding Results and Discussion sections. At L.98, the section is labeled as \"Results & Discussion,\" yet at L.549, a new \"Discussion\" section appears. Authors should choose either a combined Results & Discussion format or separate Results and Discussion sections. Given the limited discussion within the Results section, separating these sections would be more effective. This would allow for a focused presentation of findings in the Results section, followed by a comprehensive Discussion section that fully develops the interpretation and implications of all results. Throughout the Discussion section, the authors do not mention any figures or tables to remind the reader where to find the information discussed! Please correct this.\n- L.553: Remove \"exceptional\" unless providing comparative literature support for this characterization.\n- L.553-555: Add specific table references for coverage information.\n- L.558: Define LAI acronym at first use.\n- L.608: Move FPKM definition to its first appearance, which should be in the Results section.\n- L.621-623: Move methodological information to Methods and/or Results sections.\n- L.661-662: Define acronyms GPHGs and Ens at first mention.\n- L.722: Authors have mentioned this gene before, but have not written it in italics, except in L.722, 725, but then in L.727 the gene name is no longer in italics. Please correct this.\nConclusion\nThe conclusion section requires substantial revision as it fails to adequately summarize the study's key findings and does not address the two main hypotheses presented in the introduction. A more comprehensive conclusion should be developed that explicitly connects the research outcomes to the initial hypotheses and synthesizes the major discoveries. This expanded conclusion would provide readers with a clearer understanding of how the study's findings contribute to the field and fulfill its original objectives.\nReference\nThe manuscript exhibits significant citation imbalances, with only 49 out of 118 references supporting the core content across introduction, results, and discussion sections. The discussion section is particularly under referenced, utilizing just over 10 citations (references 38-49), indicating insufficient engagement with existing literature and inadequate result analysis. While the methodology section is extensively referenced, it needs condensing.\nReference formatting requires standardization throughout, as there are inconsistencies in DOI presentation, journal name abbreviations, and please check Nature Communication citation styles.\nData availability\nData accessibility needs improvement through the provision of complete accession numbers and relevant links. If custom bioinformatics code was used, it should be deposited in a public repository.\nRegarding figures, the caption for Figure 11 exceeds Nature Communications' 350-word limit and requires condensing.\nThe legend at L.1658 incorrectly uses identical symbols for filled circles, necessitating correction.", "reviewer_2": "This Manuscript describes the mechanisms and acquired process of the euryhaline in an undescribed species of Chrollela that can grow in a wide range of salinity environments. The manuscript presents an impressive volume and variety of data, with extensive analyses that are truly remarkable. I noticed that several expressions are uncommon in the context of scientific writing and molecular evolution, which affects the clarity of the text. Below, I outline my specific points of interest.\nMajor concerns\n1.\nThe T2T genome of MEM25 looks substantially accurate and complete. I have some concerns about its statistics. Although they are minor mistakes, they might not be as reliable as they should be.\nThere are inconsistent descriptions about N50 and contig length.\n- scaffold N50 as 4.3 Mb in MS line 107\n- the longest as 4.1 Mb in MS line 107\n- contig N50 as 3.76 Mb in Table S1\n- contig N50 as 4.1 Mb in table S2\nThere is no information on the longest contigs in Table S2.\nN50 is never longer than the longest contig (or scaffold) length because N50 is defined as the sequence length of the shortest contig at 50% of the total assembly length.\nPlease check the inconsistency of these statistics and reanalyze related calculations if necessary.\nI can find the BUSCO score with genome mode on line 115 and Table S5.\nDid you calculate the BUSCO score with protein (or transcriptome) mode?\nAlthough I will not see a problem since the BUSCO score with the genome is high, I would like to confirm it for the following analyses based on the gene prediction.\nThere is no mention of gap regions despite scaffolding with Hi-C. Is the T2T genome assembled completely gapless? If so, please appeal the completeness with a sense of pride.\n2.\nYou found that gene families were expanded in MEM25. Would MEM25’s assembly completeness and high-quality gene prediction overestimate the number of genes?\nI agree with the analysis based on the presence-absence matrix of gene families. However, the technological limitations of previous studies and the difficulty of assembling the algae genome might influence the results of the gene family expansion analysis.\nIf there are serious differences in the quality of gene prediction among species, differences in FPKM caused by the mapping accuracy of RNA-seq may be observed. Couldn’t the false correlation that larger gene families have a higher expression level affect the co-expression analysis in this study?\n3.\nThe centromere in land plants is defined by CENH3 binding. It is confirmed with ChIP-seq because the centromere is located at multiple positions and in different repeat patterns and positions among taxons.\nI’m unsure about centromere systems in Chlorophyta. Is it truly centromere? It looks like kinds of “centromere-like tandem repeat.”\n4.\nI understand that MEM25 has habitat-related genes in both saltwater and freshwater environments. I’m not sure that “MEM25 represents an early-diverging saltwater species“.\nI agree with the MEM25's earlier divergence of the MEM25 in the genus Chlorella. However, I wonder if it is early in this study’s huge variety of algae. It remains unclear whether this phylogenetic relationship indicates that the MEM25’s saltwater habitat is derived from ancestral traits or was reacquired. Based on your scope, I would like you to clarify the point of “early-diverged”, focused on the timing comparing what and the directory.\nMinor concerns\nLine 139-149\nThe absence of WGD and sequence conservation of centromeres and telomeres is unclear from Fig 2c. Almost all Angiosperm, including A. thariana have experienced WGD in the late Cretaceous. If the correspondence with A. thariana are found, MEM25 (and other chlorella species) could have been duplicated as well.\nAnd the telomeres of MEM25 seem to have the synteny to only one region in FACHB.\nLine 384-388\nI could not find these five families in Table S21. And OG0012359 was mentioned twice as a different term. Please confirm the gene family classification.\nLine 392\nYou mention “orphan genes” while you mention the gene function. Which is a true orphan genes or orthologous genes?\nLine 466\nWhat does “DW” mean?\nLine 556\nA N50 value depends on the genome and chromosome size. A relative comparison is valuable, but an absolute value makes no sense.\nLine 560\nThe chromosome-level assembly lacks several telomere repeats, right? I guess that it is difficult to find all telomere repeats in this case, especially telocentric chromosomes like chr15.\nLine 760-765\nI agree with the idea that MEM25 has a mechanism different from D. salina. As a possibility, euryhalinity (not halophilism) might be explained by sodium excretion rather than salt tolerance.\nFor example, I know a case of adaptation to a marine beach in Vigna marina. I don’t think the same mechanism from the same origin because it is embryophytes, but this mechanism could protect from salt and grow a wide range of salt concentrations.\nDiurnal Regulation of SOS Pathway and Sodium Excretion Underlying Salinity Tolerance of Vigna marina\nNoda et al. (2025)\nhttps://doi.org/10.1111/pce.15402\nSeccion Genome-wide association in line 930\nDoes the mutant pool share SNPs? Do multiple individuals obtain mutations generated by EMS mutagenesis in the same positions?\nPlease let me know if this is a sample set that can be used for GWAS.\nline 946\nDoes BWA-MEME mean BWA-MEM?\nline 982-983\nYou show the thresholds of P-adj and FDR. What does P-adj mean, not FDR?\nline 945\nThe parameters of fastp “-t 10 -f 10 -T 10 -F 10” appear to be over-trimmed. Is there any reason?\nline 958\nDid you use these QC options? or filter out them?\nFigure 3a\nDo these legend colors indicate the background of the species’ names? Please use more distinctive colors.\nFigure 3b\nThe positions of the purple circles are unclear, and some seem to locate no branch points.\nFigure 6b\nGreen indicates bacteria. However, other colors also indicate the kind of bacteria.\nFigure 9\nPlease add the details. For example, what are the orange points in (d)? Where is the gene in (e)?\nFigure S4a shows the polar tree rather than the unrooted tree. Poler, the center of the figure, generally indicates the root.\nLegend Fig. S5\nline 1658\nWhat do the “filled circles ● and ●, respectively” mean? It looks like the same filled circles to me.", "reviewer_3": "Overall, this is a comprehensive and well written manuscript. A high-quality chlorella genome will be useful to the community. Despite extensive data assembly and analysis, the manuscript could be improved. Specifically, when increased biomass is reported this should be further quantified in terms of lipids, carbohydrates or proteins (or more cells) to better understand how more biomass is being made. Additionally, more information regarding RMI1 is encouraged. A thorough protein map with putative domains is necessary, as is alignments with homologs. Lastly, a more thorough hypothesis regarding RMI1 mechanism is encouraged."}, "author_response": "Dear Reviewers,\nThank you for the constructive and extremely valuable comments to help improving\nthe manuscript. Here we are submitting a revised version, with item-by-item responses\n(bold fonts) to the comments below.\nWe have thoroughly proofread and edited the text following the requirements. We\nbelieve that our revised version supports the main conclusion of our initial submission\nand also reveal additional insights, which were detailed below.\nComments from Reviewer 1:\n1. The manuscript titled “Cross-species dissection of saline-genes by genetically\ndeciphering a euryhaline microalga” demonstrates promising research but requires\nseveral major revisions. First, the genus name should be incorporated into the title.\nWhile the study effectively compares multiple species, it lacks detailed analysis of\nsaline-genes and their evolutionary patterns across species. The introduction needs\nstrengthening, particularly regarding the rationale for selecting Chlorella genome and\nits ecological significance in freshwater/seawater environments.\nThe manuscript’s structure and organization require substantial modification. The\nsupplementary materials are excessive (27 supplementary Tables), making it difficult\nfor readers to navigate. I recommend consolidating tables and utilizing public\ndepositories for supplementary data. The manuscript currently exceeds Nature\nCommunication's guidelines, containing 11 main figures (exceeding the 10-figure limit)\nand 118 references (surpassing the 70-reference maximum), with methodology\ncitations comprising more than half of the references. The main text is overly lengthy\nat 30 pages and exceeds 5,000 words, with the Results & Discussion section being\nparticularly verbose.\nThe authors should condense the content while maintaining the scientific integrity of\ntheir findings. Despite these structural issues, the study presents interesting findings\nand valuable results. However, addressing these formatting and organization concerns\nwould significantly improve the manuscript's clarity and adherence to journal\nguidelines.\nResponse: We greatly appreciate the reviewer’s inputs and we are sure that these\ninputs greatly help us improve the manuscript. We have carefully revised as\nadvised to improve the manuscript's clarity and adherence to journal guidelines.\nWe have removed or r eworded the Results, Discussion and Methods sections when\nnecessary.\n Title:\nAs advised, we have incorporated the genus name:\nPrevious title: Cross-species dissection of saline-related genes by genetically\ndeciphering a euryhaline microalga\nCurrent title: Cross-species dissection of saline-related genes by genetically\ndeciphering a euryhaline microalga Chlorella sp.\n Introduction:\nWe have strengthened the content regarding the rationale for selecting Chlorella\ngenome and its ecological significance in freshwater/seawater environments. Now\nthe text reads (Line 67 to line 83 in current version):\n“To shed light on the genetic and cellular adaptations that have allowed crossing\nover the salinity barrier, the generation of genomic data from closely related\nmarine and freshwater lineages is crucial 1. An appropriate choice of organism\ngroup is essential for such studies. Marine-type sister groups of Streptophyta have\nnot been confirmed 2. Therefore, it is essential to find a speciose group with ample\ngenome data available, occurring in both freshwater and saline water.\nChlorophyta microalgae of the genus Chlorella seem to satisfy these criteria. Their\nminute cell size and resistance to environmental stress facilitate long-distance\ndispersal. The genus is omnipresent in terrestrial and aquatic habitats 3. Although\nthe majority inhabit freshwater 4, a large number of species occur in the oceans,\nfrom the Antarctic, to temperate regions and the tropics 5. However, the genomes\nof saltwater Chlorella spp. have not previously been sequenced. The euryhaline\nChlorella sp. MEM25 (hereafter MEM25) was originally isolated from the inland\nsaline water of Hainan Island, China, in August 2016 6. The water is highly saline\n(> 65‰), with a year-round high temperature (from 30 to 41 °C). MEM25 grows\nvigorously across a broad range of environmental conditions, including in\nsalinities ranging from 0 to 105‰. With increasing salinity, maximum biomass\nproduction was observed at 70‰ 6.”\nMoreover, we also acknowledge the limitations of the study in the last section of\nthe main text. Now the text reads (Line 497 to line 506 in current version):\n“We acknowledge the intricate and multifaceted nature of habitat transitions in\nViridiplantae (including green algae), which encompass not only shifts from\nmarine to freshwater environments but also independent movements from\nfreshwater or terrestrial habitats to marine settings. While numerous lineages of\nViridiplantae encompass both marine and freshwater species, our choice of\nChlorella as a model for examining transition mechanisms stems from its\nremarkable ecological diversity and phylogenetic significance. However, we are\nunable to definitively establish a direct correlation between these adaptive\ncharacteristics and the transition from saltwater to freshwater environments.\nNonetheless, it is hig hly probable that these specific adaptations have played a\nfacilitating role in the transitions between marine and freshwater conditions.”\n Results:\n “Highly contiguous genome assembly” and “T2T assembled and\ncentromere annotated chromosome sequences” have been merged as “Highly\ncontiguous genome assembly”. Meanwhile, we provided SUPPLEMENTARY\nTEXT 1 to show the details of the genome quality.\n Portions of sections “MEM25 appears closest to one of the branch points\nseparating saltwater and freshwater Chlorophyta”, “MEM25 possesses general saline-\nrelated modules shared with freshwater species and exclusive ones that have not yet\nbeen explored”, and “Gains and expansions of saline-related genes are either exclusive\nto MEM25 or shared across Viridiplantae” have been removed.\nSpecifically, for the transcriptomic comparison and their metabolomic\ncounterparts, we have focus on the genes and metabolites associated with saline\nadaptation of MEM25 and removed the contents relating to the purple4 (association\nwith salinity (P = 10-6, Pearson = 0.82) and damage state (P = 10-7, Pearson = 0.85), but does not\ncorrelate with stress duration or the MEM25-specific mechanism) and yellow modules\n(associated with salinity (P = 2×10-7, Pearson = 0.85), but not with stress duration, damage state,\nor the MEM25-specific mechanism). Additionally, the contents relating to expanded\nfamilies in Chlorella spp. and expansion orthogroups in MEM25 in comparison to\nfreshwater Chlorella spp.. have been removed too. The corresponding figures and\ntables were deleted accordingly.\n Section “MEM25 shares both saltwater- and freshwater-featured genes” has been\nreworded.\nWe believe the current version is more readable while maintaining the scientific\nintegrity of the main findings.\n Discussion\n Discussion has been rephrased and the words have been decreased from 2596\nto 702.\n Methods:\nWe have moved the main content of Methods into SUPPLEMENTARY TEXT 2\nwhere detailed methods are included.\nIn general, we have performed the revision as suggested and the content of current\nversion is substantially condensed. The words of the main text (not including\nAbstract, Methods, References and Figure legends) have been decreased from\n8,332 to 5077 while the number of figures, supplementary Tables, and references\nhave been decreased from 11, 27, and 118 to 7, 17, and 35, respectively.\nWe believe our revis ed version has met all the journal requirements while the\nclarity and readability have been improved.\n2. The authors should address the following points for clarification:\n- L.3: Clarify the meaning and reference of the # symbol\nResponse: We apologize for such confusion and clarify the meaning of the # symbol.\nNow the text reads (Line 13 in current version):\n“#These authors contribute equally”\n3. L.49: Add missing \"Introduction\" header\nResponse: We greatly appreciate the reviewer’s input and have added the header\nas advised (Line 47 in current version).\n4. L.59-60: Rephrase sentence to specify which \"other eukaryotes\" are being discussed\nResponse: We greatly appreciate the reviewer’s input and have reworded the\nsentence. Now the text reads (Line 58 to line 59 in current version):\n“Higher marine-freshwater transition rates than anticipated have, however, been\nreported for both prokaryotes 7 and eukaryotes (e.g., microbial eukaryotes 1).”\n5. L.74-75: Content needs clarification/completion\nResponse: We apologize for such confusion and have clarified the content. Now\nthe text reads (Line 67 to line 72 in current version):\n“To shed light on the genetic and cellular adaptations that have allowed crossing\nover the salinity barrier, the generation of genomic data from closely related\nmarine and freshwater lineages is crucial 10. An appropriate choice of organism\ngroup is essential for such studies. Marine-type sister groups of Streptophyta have\nnot been confirmed 14. Therefore, it is essential to find a speciose group with ample\ngenome data available, occurring in both freshwater and saline water.”\n6. L.88-97: Include methodology introduction for addressing stated hypotheses\nGeneral comments: Introduction is too brief compared to Results & Discussion and\nDiscussion sections. Please add justification for microalga species selection and expand\nmethodology overview for addressing research hypotheses\nResponse: We greatly appreciate the reviewer’s input and have provided more\ndescription to justify microalga species selection. Please refer to our response to\nComment 5 and 6 for details. Moreover, we have expanded methodology overview\nand provided more results in Introduction. Now the text reads (Line 84 to line 102\nin current version):\n“Here, we present genome sequences of MEM25 that represents one of the early\ndivergences in the genus. The genome of MEM25, likely positioning at one of the\nbifurcation points separating saltwater and freshwater species, may reveal the\nsuite of traits that faci litated saline adaptation. The high-quality genome assembly\nof MEM25 approaches the gold standard telomere-to-telomere (T2T) assembly\nthrough a meticulous blend of sequencing technologies, ensuring exceptional\ncoverage and precision. By deciphering the multi-omics data via integrated\nstrategies, we propose that the genetic products of green plant heritage genes\n(GPHGs) and evolutionary novelties (ENs) collaboratively sculpt the adaptation\nof MEM25 to salinity changes. Comparison of MEM25 with freshwater green\nalgae and halophilic Dunaliella salina underscores the intricate evolutionary\ndynamics entailed in transitioning between saltwater and freshwater ecosystems\nand allow us outlined a list of fundamental genes related to salinity that offer\npotential for enhancing tolerance to saline conditions.\nThese findings allowed us to address two questions important when exploring the\nevolutionary history of habitat transitions. (1) How did freshwater species evolve\nfrom their saltwater ancestors, or vice versa? (2) What gene repertoires contribute\nto the euryhaline characteristics of MEM25? Moreover, the de novo discovery of\nthe “lost” or “acquired” saline-related genes during these transitions could\npotentially enable the improvement of saline resistance of crops.”\nResults and discussion\n7. L.110: Delete \"s\" from \"genomes\".\nResponse: We apologize for the typo and have deleted “s” as advised. Moreover,\nwe have moved the details of the genome quality into SUPPLEMENTARY TEXT\n1. Please also see our response to Comment 1.\n8. L.113: Specify databases used.\nResponse: We have removed the sentence from current version.\n9. L.116-117: The transcriptome has not been presented before, nor mentioned in the\nfirst paragraph of the results. How was the transcriptome done?\nResponse: We have deleted the sentence from current version.\n10. L.122: Define T2T acronym.\nResponse: We greatly appreciate the reviewer’s input. As advised, we have\nspecified T2T in the Introduction section. Now the text reads (Line 87 to line 90 in\ncurrent version):\n“The high-quality genome assembly of MEM25 approaches the gold standard\ntelomere-to-telomere (T2T) assembly through a meticulous blend of sequencing\ntechnologies, ensuring exceptional coverage and precision.”\n11. L.140-142: Add references for known species genomes mentioned in the text.\nResponse: As advised, the relevant references have been added. Now the text reads\n(Line 116 to line 120 in current version):\n“An investigation int o intragenomic synteny among the sixteen chromosomes of\nMEM25 (Fig. S4) and among three Chlorella genomes (MEM25, Chlorella\npyrenoidosa FACHB-9 20, and Chlorella sorokiniana 1230 21), along with the\ngenomes of Chlamydomonas reinhardtii 22 and Arabidopsis thaliana 23 (Fig. 1c),\nrevealed no whole-genome duplications (WGDs) in the MEM25 genome.”\n12. L.151-152: How have the authors performed the whole chromosome interactions,\nand should they briefly mention the methods used here, e.g. correlation using which\nmetrics or algorithm?\nResponse: We greatly appreciate the reviewer’s input and provided the method\nused. Now the text reads (Line 125 to line 130 in current version):\n“A 100 kb-resolution 3D map of the MEM25 genome (Fig. S4) and whole-\nchromosomal interactions (Fig. 1d) were created using HiC-GNN and visualized\nusing UCSF Chimera. Altogether, the quality, accuracy, and completeness of the\nMEM25 genome assembly is higher than the available reference sequences 8, 9,\nensuring the reliability of subsequent genomic analyses (See Supplementary text\n1 for the details of genome assembly).”\nMoreover, we provided more details in the Methods section (current\nSupplementary text 2). Now the text reads (Line 35 to line 43 in Supplementary\ntext 2):\n“3D-DNA (version 180922) 6 and ALLHiC (version 0.9.8) 7 were used for draft\ngenome correction and genome assembly, respectively. Finally, Juicebox (version\n1.11.08) 8 and Pilon (version 1.24) 9 were used to correct genome fragments (based\non the chromosome contact frequency matrix) and base errors of the genome with\nIllumina and RNA-seq data, respectively. The Hi-C interaction frequency matrix\nwas analyzed by Homer, FitHic (version 2.0.8) 10, HiCExplorer (version 3.7.2) 11,\nand HiCCUPS (version 5.14) 12. The 3-D chromosome coordinate location was\ncalculated using HiC-GNN 13 and was visualized using UCSF Chimera (version\n1.15) 14. ”\nResults and discussion\n13. L.152: Remove or justify \"As expected\" statement.\nResponse: As advised, we have deleted “As expected”. Now the text reads (Line 48\nto line 52 in Supplementary text 1):\n“Although two sets of chromosomes, Chr09 through Chr12 and Chr14 through\nChr15 (Fig. S4), were associated more closely with each other than the remaining\nones, more intense intra-chromosomal interactions (within the same chromosome)\nthan inter-chromosomal interactions (between different chromosomes) were\ndetected (Student’s t-test, P < 0.05, Fig. 1d).”\n14. L.154-156: Include literature citations.\nResponse: The sentence has been removed from current version.\n15. L.165-168: Add citations for species selection.\nResponse: We greatly appreciate the reviewer’s input.\nConsidering the limit of the number of references and the number of species\nselected in this study (more than 30), we have provided citations of all the selected\nspecies in Table S4 (Species used for genome comparison analysis). Please refer to\nthe supplementary tables for details. Now the text reads (Line 144 to line 146 in\ncurrent version):\n“For the selected species, habitat information and genomic sources were\nthoroughly verified (See Table S4 for details).”\n16. L.169: Specify and cite Cyanobacteria species used in the analysis.\nResponse: As advised, we have specified the cyanobacteria. Considering the limit\nof the number of references, we have provided citations in Table S4 Now the text\nreads (Line 142 to line 144 in current version):\n“Cyanobacteria (e.g., Synechococcus moorigangaii and\nSynechocystis sp. PCC 6803) were also included to provide a holistic view of\ngenetic diversity in microalgal evolution.”\nPlease also refer to our response to Comment 15.\n17. L.175: Fix spacing between \"outgroups\" and \"(Table S10)\". Concerning Tables S10:\nThe authors should consider transforming their Table S10 into a main table so that the\nreader can directly see which species they have chosen for comparison, as the authors\nhave not included all the names of the species used in the main text.\nResponse: We greatly appreciate the reviewer’s input. As advised, we have fixed\nspacing between “outgroups” and “(Table S10)”. As for Table S10, we think twice\non whether to move it as a main table or not. Considering the numbers of the words\nand the references have already exceeded the limit of the journal’s requirements,\nwe finally decide to keep it as supplementary tables (Table S4 in current version).\nResponse: We greatly appreciate the reviewer’s input. As advised, we have\nprovided an additional table for the 13 ortholog genes as Supplementary Table 5\n(Supplementary Table 5. Chlorophyta-conserved single-copy orthogroups\nidentified across 36 species of chlorophyta). Please refer to Supplementary Tables\nfor details.\n19. L.186: Add missing Streptophyta color in Fig.3a.\nResponse: We greatly appreciate the reviewer’s input. As advised, we have added\nthe missing Streptophyta color in the figure (Fig. 2 in current version). Moreover,\nthe figure has been re-organized where the background has been changed into\nmore distinctive color s to indicate the species from different phyla. Now the text\nreads (Line 682 to line 690 in current version):\nFig. 2. Phylogenetic position of Chlorella sp. MEM25. Rooted phylogenetic tree\nfeatured 38 Chlorophyta species and representative species from cyanobacteria,\nRhodophyta, Glaucophyta, Streptophyta, and Embryophyta. The species tree was\ndeduced using the most closely related genes within single-copy or multi-copy\northogroups (n=199), based on the Species Tree from All Genes (STAG) algorithm.\nSpecies from different phyla are indicated in different background colors. Freshwater\nand seawater species are shown in green and orange dots. Bootstrap with values of 100%\nor above 70% are indicated in smaller or bigger dots at the branching points,\nrespectively.\n20. L.197: Replace \"exceptional position\" with more appropriate wording.\nResponse: As advised, we have reworded the sentence. Now the text reads (Line\n156 to line 158 in current version):\n“The highly credible species trees (bootstrap support exceeding 60%) align with\nthe chloroplast phylogenetic tree (Fig. S6b), positioning MEM25 closest to the\nbranch point separating freshwater Chlorella spp. from the halotolerant marine\nspecies Picochlorum spp (Fig. 2).”\n21. L.227-228: Include method reference.\nResponse: We have removed the description on the cutoff method in previous\nversion and substantially condensed the section with method reference included.\nNow the text reads (Line 181 to line 190 in current version):\n“To identify the distinctive gene families of freshwater and saltwater Chlorophyta\nspecies (Table S10), we initiated with a two-stage machine learning approach\ninvolving 86 models and model combinations. Specifically, we generated a\npresence-absence matrix for gene families and applied various machine learning\nalgorithms to differen tiate between freshwater and saltwater algae (see Methods\nfor details). The Random Forest + Elastic Net [0.6], identified as the most effective\ncombination among the 86 models, achieved a classification accuracy of 97% and\npinpointed 138 critical gene families with distinct functions in freshwater versus\nsaltwater microalgae (Table S12). These genes were further categorized into\nsaltwater (n=44) and freshwater-specific groups (n=94) based on their weight\ncoefficients.”\n22. L.261-262: Specify relevant table/figure.\nResponse: We apologize for such confusion and as provided the relevant table as\nadvised. Now the text reads (Line 195 to line 197 in current version):\n“In comparison, the genome of Chloropicon primus (a prevalent species in marine\nphytoplankton communities 10) contains 23 saltwater-featured genes and 34\nfreshwater-featured genes, respectively (Table S7).”\n23. L.273: Introduce neutrophilic freshwater FACHB-9 and cite the literature.\nResponse: We have reworded the sentence and provided a brief introduction of\nFACHB-9 and the relevant reference. Now the text reads (Line 213 to line 217 in\ncurrent version):\n“To investigate species similarities and differences in salinity adaptation\nmechanisms between saltwater and freshwater species, we conducted a\ncomparative analysis of the global trancriptomic and metabolic dynamics of the\neuryhaline MEM25 and the freshwater FACHB-9 8 (See Supplementary text 2 for\ngrowth conditions of FACHB-9).”\n24. L.277: Specify C. reinhardtii strain name.\nResponse: We have removed the sentences relating to C. reinhardtii.\n25. L.318-320: Where can we see that these genes are salt-sensitive and not associated\nwith cell damage? This is not clear.\nResponse: We greatly appreciate the reviewer’s input and have removed the\ncontent relating to the yellow module. Please also refer to our response to\nComment 1 for details.\n26. L.327-328: Same question as above, provide data location.\nResponse: We greatly appreciate the reviewer’s input and have reorganized the\nsection and provided relevant data as Table S11. Now the text reads (Line 242 to\nline 244 in current version):\n“Noteworthy is the distinct association of the RecQ-mediated genome instability\nprotein 1 (RMI1; CP5g5156) with the saline adaptation of MEM25 (Table S11).”\n27. L.435: Correct table citation order (S24 before S25).\nResponse: We apolog ize for such confusion and have double checked all citations\nfor figures and tables throughout the main text.\n28. L.548: Reorganize Figure 11 panels for better readability.\nResponse: We greatly appreciate the reviewer’s input and have removed Figure\n11. Please refer to our response to Comment 1.\nResults and discussion\n29. The Results and Discussion section’s first part needs more thorough analysis to\neffectively convey the findings’ implications.\nResponse: We greatly appreciate the reviewer’s input. As advised, we have merged\nthe 1st and 2nd section of Results and removed the description that not so relevant.\nMeanwhile we provided Supplementary text 1 to give a full picture of the genome\nassembly. The figures and tables have been revised accordingly. Please also refer\nto our response to Comment 1.\n30. Discussion\nThere is inconsistency in the manuscript's structure regarding Results and Discussion\nsections. At L.98, the section is labeled as \"Results & Discussion,\" yet at L.549, a new\n\"Discussion\" section appears. Authors should choose either a combined Results &\nDiscussion format or separate Results and Discussion sections. Given the limited\ndiscussion within the Results section, separating these sections would be more effective.\nThis would allow for a focused presentation of findings in the Results section, followed\nby a comprehensive Discussion section that fully develops the interpretation and\nimplications of all results. Throughout the Discussion section, the authors do not\nmention any figures or tables to remind the reader where to find the information\ndiscussed! Please correct this.\nResponse: We apologize for such confusion and have revised as advised.\nSpecifically, the sections are now labeled as “Results” and “Discussion”, separately.\nMoreover, we have substantially condensed “Discussion” section and provided\nrelevant references when necessary. Please see our response to Comment 1 and the\nDiscussion section of main text in the revised version.\n31. L.553: Remove \"exceptional\" unless providing comparative literature support for\nthis characterization.\nResponse: We greatly appreciate the reviewer’s input and have removed the\nphrase from the main text.\n32. L.553-555: Add specific table references for coverage information.\nResponse: We greatly appreciate the reviewer’s input and have removed this\nsection from the main text. Please also refer to our response to Comment 1 and 29.\n33. L.558: Define LAI acronym at first use.\nResponse: We have removed this section from the main text. Please also refer to\nour response above. Moreover, we have defined LAI acronym at first use. Now the\ntext reads (Line 108 to line 112 in current version):\n“A high level of genome scaffold continuity was revealed by the long scaffold N50\n(longest, 4.3 Mb; Table S1) and a high value of Long Terminal Repeat Assembly\nIndex (LAI) (15.33; Fig. S2), meeting the quality benchmark for reference\ngenomes (i.e., between 10 and 20) (Table S2) 19.”\n34. L.608: Move FPKM definition to its first appearance, which should be in the Results\nsection.\nResponse: We have removed this section from the main text. We also double\nchecked all acronyms to make sure they are defined at first appearance.\n35. L.621-623: Move methodological information to Methods and/or Results sections.\nResponse: We have removed this section from the main text. Please also refer to\nour response to Comment 1.\n36. L.661-662: Define acronyms GPHGs and ENs at first mention.\nResponse: We greatly appreciate the reviewer’s input. The acronyms have been\ndefined in the Introduction section where they are first mentioned. Now the text\nreads (from Line 90 to line 92 in current version):\n“By deciphering the multi-omics data via integrated strategies, we propose that\nthe genetic products of green plant heritage genes (GPHGs) and evolutionary\nnovelties (ENs) collaboratively sculpt the adaptation of MEM25 to salinity\nchanges.”\n37. L.722: Authors have mentioned this gene before, but have not written it in italics,\nexcept in L.722, 725, but then in L.727 the gene name is no longer in italics. Please\ncorrect this.\nResponse: We apologize for such typo and have corrected and double checked\nthroughout the main text.\nConclusion\n38. The conclusion section requires substantial revision as it fails to adequately\nsummarize the study's key findings and does not address the two main hypotheses\npresented in the introduction. A more comprehensive conclusion should be developed\nthat explicitly connects the research outcomes to the initial hypotheses and synthesizes\nthe major discoveries. This expanded conclusion would provide readers with a clearer\nunderstanding of how the study's findings contribute to the field and fulfill its original\nobjectives.\nResponse: We greatly appreciate the reviewer’s input and have merged the\n“Conclusion” into “Discussion” section. The current version has been\nsubstantially revised. We focus the line on the two proposed hypotheses. Now the\ntext reads (from Line 432 to Line 495 in current version):\n(1) How did freshwater species evolve from their saltwater ancestors, or vice versa?\n“The dynamic adaptation of green algae from saltwater to freshwater is a complex\nevolutionary process that showcases the diversity of selective pressures from\nmarine and freshwater environments. Both saltwater and freshwater species have\nindependently evolved or inherited various adaptive mechanisms from their\npredecessors. MEM25 acts as a transitional point crossing over the salinity barrier,\ndemonstrating dual environmental adaptation mechanisms. The genetic products\nof GPHGs and ENs collaboratively sculpt the adaptation of MEM25 to salinity\nchanges. MEM25 possesses general modules related to salinity that are shared\namong freshwater Chlorophyta species (potentially extending to all green plants),\nalongside distinctive modules acquired through HGTs (e.g., OG0011211) or other\nyet undiscovered mechanisms (e.g., orphan genes).\nIn the hypothetical scenario of transitioning from saline to freshwater habitats, the\nshared modules may encompass a suite of genes (referred to as GPHGs) inherited\nfrom saltwater forebears, equipping freshwater species with analogous, though\nlargely mitigated, mechanisms to confront salinity fluctuations. These genes’\nfunctionalities revolve around mitigating or intensifying cellular harm in saline\nsettings, representing a ubiquitous mechanism present in both saltwater and\nfreshwater species. An example is that MEM25 employs proline as an\nosmoprotectant, commonly found in embryophytes. This indicates that the\nmechanism of employing proline as an osmoprotectant in higher plants may\noriginate from their shared ancestral lineage with MEM25. Furthermore, as an\nearly-diverging marine alga, MEM25 has commenced the traversal of the salinity\nthreshold. While boasting numerous genes specific to saltwater environments,\nMEM25 is in the process of assimilating genes characteristic of freshwater milieus.\nMEM25 harbors distinctive modules (referred to as ENs) that likely evolved\nautonomously, enhancing its ability to thrive in its unique saline habitat.\nAlternatively, in a scenario where MEM25’s ancestors originated in freshwater,\nthe acquisition of ENs transpired during the specialization phase towards an\nenvironment with higher salinity levels. For instance, the gene family OG0011211\n(belonging to the NAD-dependent epimerase/dehydratase family) could have been\nassimilated from bacteria through HGTs (Fig. S13). These ENs embody genes\nassociated with salinity that may have been “lost” during the transition from\nmarine to freshwater ecosystems. The gradual disappearance of ENs may have\nconferred a competitive edge for saltwater ancestors adapting to freshwater\nenvironments. In both conjectures, these genes likely facilitated MEM25’s\nacclimatization to challenging habitats and warrant further investigation for\nenhancing resilience to salinity.”\n(2) What gene repertoires contribute to the euryhaline characteristics of MEM25?\n“MEM25 and D. salina represent two typical adaptive strategies among\neuryhaline and halop hilic green algae, respectively. What sets MEM25 apart is its\nability to adapt to both freshwater and saltwater environments, maintaining its\nstatus as a dominant species in both ecological settings. The euryhaline nature of\nMEM25 enhances its competitiveness across a wide range of ecological\nenvironments, whereas the extreme salt tolerance specialization of D. salina limits\nits ecological adaptability. As a possibility, euryhalinity might be explained by\nsodium excretion rather than salt tolerance as seen in some euryhaline\nembryophytes 12. It is apparently the case for halophilic D. salina. D. salina’s salt\ntolerance primarily relies on accumulating high concentrations of glycerol as an\nosmoprotectant to maintain cellular osmotic pressure, relying on efficient ion\nexclusion systems, and employing antioxidant enzymes to reduce reactive oxygen\nspecies 13. However, many of these mechanisms are specialized for high-salinity\nenvironments, resulting in significantly reduced metabolic flexibility under low\nsalinity or even freshwater conditions. In contrast, MEM25 exhibits a more\ndiversified adaptability, with its salt tolerance mechanisms encompassing not just\nthe accumulation of osmoprotectants but also long-term evolutionary mechanisms\nat the genomic level, mid-term adaptation mechanisms at the metabolomic level,\nand rapid response mechanisms at the transcriptomic level. This allows it to thrive\necologically in both high-salinity and low-salinity environments.”\nThe de novo discovery of the “lost” or “acquired” saline-related genes during these\ntransitions could potentially enable the improvement of saline resistance of crops.\n“Last but not least, we have outlined a list of fundamental genes related to salinity\nthat offer potential for enhancing tolerance to saline conditions. The array of genes\nunveiled in this study has shown significant effectiveness in enhancing plant\ntolerance to salt stress by manipulating a single gene, offering hope in addressing\nconcerns related to genetically modified organisms by creating organisms through\ngenome editing that are indistinguishable from conventionally bred varieties.\nUnfortunately, only two genes have been confirmed in both microalgae and higher\nplants, indicating the need for further exploration. Despite this limitation, this\nresearch provides essential genetic reservoirs crucial for the advancement of\nhalotolerant plants.”\nReference\n39. The manuscript exhibits significant citation imbalances, with only 49 out of 118\nreferences supporting the core content across introduction, results, and discussion\nsections. The discussion section is particularly under referenced, utilizing just over 10\ncitations (references 38-49), indicating insufficient engagement with existing literature\nand inadequate result analysis. While the methodology section is extensively referenced,\nit needs condensing.\nResponse: As advised, we have reorganized the references. The number of\nreferences has been decreased from 118 to 37. Moreover, due to word limit, we\nhave substantially revised the Methods section and move most of the contents into\nSupplementary text 2.\nState Key Lab of Marine Resource\nUtilization in South China Sea, Hainan\nU niversity\nMailing: 58 Renmin AveCnuoel leg e o f O c e a n o l o g y , H a Tienl: a86n-8 98-66257612\nHaikou, Hainan, China, 570228 Fax: 86-898-66251258\nUniversity (HN U)\n40. Reference forma tting requires standardization throughout, as there are\ninconsistencies in DOI presentation, journal name abbreviations, and please check\nNature Communication citation styles.\nResponse: We greatly appreciate the reviewer’s input and have thoroughly\nchecked all references. We believe our revised version has met all the journal\nrequirements described in the submission guidelines.\n41. Data accessibility needs improvement through the provision of complete accession\nnumbers and relevant links. If custom bioinformatics code was used, it should be\ndeposited in a public repository.\nResponse: We greatly appreciate the reviewer’s input and have provided all\nrelevant data and code in public repositories. The genome sequence and\nannotation of MEM25, along with detailed studies on gene families and essential\nfindings from HiC analysis, transcriptomics, metabolomics, and GWAS analysis\nare accessed on the public data platform Figshare\n(https://figshare.com/articles/journal_contribution/Data_of_MEM25/29062721).\nFor the bioinformatics code used for cleaning raw sequencing data, mutation\ndetection, GWAS analysis, and result display can be obtained at\nhttps://github.com/aoqiwang/MEM25. Now the text reads (from Line 623 to line\n632 in current version):\n“Data availability\nData is deposited in National Microbiology Data Center (NMDC) with accession\nnumbers NMDC10019441. The genome sequence and annotation of MEM25,\nalong with detailed studies on gene families and essential findings from HiC\nanalysis, transcriptomics, metabolomics, and GWAS analysis are accessed on the\npublic data platform Figshare\n(https://figshare.com/articles/journal_contribution/Data_of_MEM25/29062721).\nFor the bioinformatics code used for cleaning raw sequencing data, mutation\ndetection, GWAS analysis, and result display can be obtained at\nhttps://github.com/aoqiwang/MEM25.”\n42. Regarding figures, the caption for Figure 11 exceeds Nature Communications' 350-\nword limit and requires condensing.\nResponse: We fully agree with the reviewer and have removed Figure 11.\n43. The legend at L.1658 incorrectly uses identical symbols for filled circles,\nnecessitating correction.\nResponse: We apologize for such confusion. We have differentiated the symbols\nusing different colors and specified in the legend. Now the text reads (from Line\n801 to line 803 in current version):\n“Significantly expanded or contracted orthologs are denoted by filled red circles\n● and filled blue cir cles●, respectively.”\nComments from Reviewer 2:\nThis Manuscript describes the mechanisms and acquired process of the euryhaline in\nan undescribed species of Chlorella that can grow in a wide range of salinity\nenvironments. The manuscript presents an impressive volume and variety of data, with\nextensive analyses that are truly remarkable. I noticed that several expressions are\nuncommon in the context of scientific writing and molecular evolution, which affects\nthe clarity of the text. Below, I outline my specific points of interest.\nMajor concerns\n1. The T2T genome of MEM25 looks substantially accurate and complete. I have some\nconcerns about its statistics. Although they are minor mistakes, they might not be as\nreliable as they should be.\nThere are inconsistent descriptions about N50 and contig length.\n- scaffold N50 as 4.3 Mb in MS line 107\n- the longest as 4.1 Mb in MS line 107\n- contig N50 as 3.76 Mb in Table S1\n- contig N50 as 4.1 Mb in table S2\nThere is no information on the longest contigs in Table S2.\nN50 is never longer than the longest contig (or scaffold) length because N50 is defined\nas the sequence length of the shortest contig at 50% of the total assembly length.\nPlease check the inconsistency of these statistics and reanalyze related calculations if\nnecessary.\nResponse: We apologize for the confusion and greatly appreciate the inputs of the\nreviewer. We have thoroughly checked the manuscript and clarified all confusion\npoints caused by different calculation method. Specifically, the value “4.1 Mb”\nwas calculated based on improper binary conversion (1024²), which is invalid in\ngenomics (standard: 1 Mbp = 10⁶ bp). Employing samtools-faidx, all chromosome\nlengths have been re-calculated. The total genome length is 53,506,137 bp (50% =\n26,753,068.5 bp) while N50 is 4.3 Mb (chr05), instead of 3.76 Mb (chr06) or 4.1 Mb\n(calculated based on improper binary conversion). Accordingly, we have\nthoroughly checked the main text and supplementary materials. Moreover, to\nmake the manuscript more concise (based on the comments from Reviewer 1),\nwe have moved portion of results relating to genome quality to SUPPLEMENTARY\nTEXT 1.\n2. I can find the BUSCO score with genome mode on line 115 and Table S5.\nDid you calculate the BUSCO score with protein (or transcriptome) mode?\nAlthough I will not see a problem since the BUSCO score with the genome is high,\nI would like to confirm it for the following analyses based on the gene prediction.\nResponse: We apolog ize for the confusion and have clarified the mode used for\nBUSCO calculation. Now the text reads (from Line 15 to Line 18 in Supplementary\ntext 1):\n“The genome assembly successfully captured 97.2% (with transcriptome mode;\n93.7% with protein mode) of the Chlorophyta Benchmarking Universal Single\nCopy Orthologs (BUSCO) dataset, indicating a high level of gene region\ncompleteness in the genome assembly (Table 4).”\n3. There is no mention of gap regions despite scaffolding with Hi-C. Is the T2T genome\nassembled completely gapless? If so, please appeal the completeness with a sense of\npride.\nResponse: We appreciate the input here and are quite proud of the completeness\nof the genome. There is a single gap in the assembled genome at the end of chr15\n(1,311,674 bp) with a length of 100 bp from 1,286,591 bp to 1,286,690 bp. We have\nclarified this point in Supplementary text 1. Now the text reads (from Line 8 to\nline 10 in Supplementary text 1):\n“A single gap was present in the assembled genome at the end of chr15 (1,311,674\nbp) with a length of 100 bp from 1,286,591 bp to 1,286,690 bp.”\n4. You found that gene families were expanded in MEM25. Would MEM25’s assembly\ncompleteness and high-quality gene prediction overestimate the number of genes?\nI agree with the analysis based on the presence-absence matrix of gene families.\nHowever, the technological limitations of previous studies and the difficulty of\nassembling the algae genome might influence the results of the gene family expansion\nanalysis.\nIf there are serious differences in the quality of gene prediction among species,\ndifferences in FPKM caused by the mapping accuracy of RNA-seq may be observed.\nCouldn’t the false correlation that larger gene families have a higher expression level\naffect the co-expression analysis in this study?\nResponse: We greatly appreciatethe inputs here. As a matter of fact, we have a\nsame concern when we conducted transcriptome comparison among different\nspecies. We understand unified standardized annotation for all species by setting\na reference gene set could eliminate potential “pseudo-amplification”. However,\nour attempt to achieve such a reference gene set failed due to dramatic difference\nin evolution or genome quality of species of green algae. Therefore, we employed\nseveral strategies to avoid potential bias caused by differences in the quality of\ngene prediction among species:\n Admitting that most green algae have poor genomic assembly and annotation\nquality, we included species with high-quality genomes, such as Arabidopsis\nthaliana and rice.\n Both RNA-seq mapping rates and conserved domain annotations were\nemployed to ensu re the correctness of gene prediction of MEM25. It means\npredicated genes of MEM25 are bona fide, rather than false genes with no real\nexpression.\n To mitigate biases stemming from differences in gene annotation quality\nbetween the two species, we employed a Weighted Correlation Network\nAnalysis (WGCNA). We chose to include both single-copy and multi-copy\northogroups to encompass a wider array of gene family expressions that could\nbe crucial for salinity adaptation, rather than focusing solely on single-copy\northogroups. While single-copy orthologous gene families offer a simpler\napproach for comparison, restricting the dataset to single-copy orthogroups\nsolely would exclude many functionally significant gene families potentially\ninvolved in the salinity adaptation. By aggregating the expression of genes\nwithin each orthogroup to create a “metagene” expression level, we aimed to\ncapture co-expression patterns reflective of shared functional roles,\nirrespective of individual gene copy numbers within each gene family.\nMoreover, this method emphasizes coordinated expression trends (e.g.,\nsynchronized up/downregulation) rather than expression levels in different\nspecies. Although gene family size correlates positively with cumulative\nexpression, this correlation did not confound module-trait associations. This\nstrategy aligned with WGCNA’s purpose of identifying biologically relevant\nmodules, enabling a more comprehensive exploration of traits associated with\nfreshwater/saltwater adaptation across species.\n5. The centromere in land plants is defined by CENH3 binding. It is confirmed with\nChIP-seq because the centromere is located at multiple positions and in different repeat\npatterns and positions among taxons.\nI’m unsure about centromere systems in Chlorophyta. Is it truly centromere? It looks\nlike kinds of “centromere-like tandem repeat.”\nResponse: We greatly appreciatethe inputs here. We acknowledge that the CENH3\nchromatin immunoprecipitation sequencing (ChIP-seq) technique is effective in\ndefining centromeres. Although we have not confirmed our predicted MEM25\ncentromeres using ChIP-seq, we ensure the accuracy of the predication via rigid\nbioinformatics analysis.\nFirst, the completeness of the MEM25 genome assembly is high with only a single\n100-bp gap present at the end of chr15. Second, we have tried to determine the\ncentromeres in MEM genome by comparing with recently reported centromeres\nof two Chlorella strains during the review of this manuscript. Specifically, 12 and\n13 centromeres have been identified in Chlorella sorokiniana NS4-2 and Chlorella\npyrenoidosa DBH, respectively where CENH3 ChIP-seq technique have been used\nto validate these centromeric regions (PMID: 38600443). The comparison revealed\nidentical results as our former predication. Although confirmation of the MEM25\ncentromeres would fu rther improve the accuracy, we believe that the featured\nsequences discovered in this study would be valuable for future researches relating\nto microalgal centromeres.\n6. I understand that MEM25 has habitat-related genes in both saltwater and freshwater\nenvironments. I’m not sure that “MEM25 represents an early-diverging saltwater species”.\nI agree with the MEM25’s earlier divergence of the MEM25 in the genus Chlorella.\nHowever, I wonder if it is early in this study’s huge variety of algae. It remains unclear\nwhether this phylogenetic relationship indicates that the MEM25’s saltwater habitat is\nderived from ancestral traits or was reacquired. Based on your scope, I would like you\nto clarify the point of “early-diverged”, focused on the timing comparing what and the\ndirectory.\nResponse: We fully agree with the reviewer. MEM25 is earlier divergence in the\ngenus Chlorella while it may not be early in this study’s huge variety of algae. Our\nmolecular clock analysis supports this hypothesis as that the emergence of MEM25\npredates all examined Chlorella spp. and ranking among the oldest Chlorophyta.\nAlthough the shared saltwater ancestors remain elusive, certain clades exhibited\nearlier divergence times than MEM25. It hints that unlike the swift and dramatic\ntransition to a terrestrial environment spurred by abrupt climate shifts, the\ntransition from saltwater to freshwater settings unfolded in localized regions, with\ndistinct lineages evolving independently within specific niches. Therefore, to\nclarify the point of “early-diverged”, focused on the timing comparing what and\nthe directory, MEM25 predates all examined Chlorella spp. and is only one of the\nmany representatives at the points separating saltwater and freshwater\nChlorophyta. These points have been intensified in the main text. Now the text\nreads:\nLine 159 to line 163 in current version:\n“The molecular clock places the emergence of MEM25 at 632 Ma, predating all\nexamined Chlorella spp. and ranking among the oldest Chlorophyta (Fig. S6).\nWhile certain clades exhibited earlier divergence times than MEM25, the shared\nsaltwater ancestors of these lineages remain elusive, hinting at a dispersed\nevolution during the shift from saltwater to freshwater habitats.”\nLine 205 to line 210 in current version:\n“This suggests that MEM25, potentially as an early-diverging saltwater species,\nhas evolved numerous genes critical for freshwater-saltwater transitions,\npositioning it at one of the many pivotal junctures separating saltwater and\nfreshwater Chlorophyta.”\nMoreover, we have acknowledged this in the section “Limitations of the study”.\nNow the text reads (from Line 496 to line 506 in current version):\n“Limitations of the study\nWe acknowledge the intricate and multifaceted nature of habitat transitions in\nViridiplantae (includ ing green algae), which encompass not only shifts from\nmarine to freshwater environments but also independent movements from\nfreshwater or terrestrial habitats to marine settings. While numerous lineages of\nViridiplantae encompass both marine and freshwater species, our choice of\nChlorella as a model for examining transition mechanisms stems from its\nremarkable ecological diversity and phylogenetic significance. However, we are\nunable to definitively establish a direct correlation between these adaptive\ncharacteristics and the transition from saltwater to freshwater environments.\nNonetheless, it is highly probable that these specific adaptations have played a\nfacilitating role in the transitions between marine and freshwater conditions.”\nMinor concerns\n7. Line 139-149\nThe absence of WGD and sequence conservation of centromeres and telomeres is\nunclear from Fig 2c. Almost all Angiosperm, including A. thaliana have experienced\nWGD in the late Cretaceous. If the correspondence with A. thaliana are found, MEM25\n(and other chlorella species) could have been duplicated as well.\nResponse: We fully understand the reviewer’s concern that MEM25 (and other\nchlorella species) could have been duplicated as well since A. thaliana have\nexperienced WGD and considerable amounts of homologous A. thaliana genes\nhave been detected in MEM25 (gray lines in Fig. 1c). The main purpose of Fig. 1c\n(Fig. 2c in previous version) is to display the quality, accuracy, and completeness\nof the MEM25 genome assembly. Collinearities in gene sequences, telomeres, and\ncentromeres are delineated by the gray, purple, and yellow lines. The collinearity\nfor telomeres and centromeres among three Chlorella genomes is higher than that\nbetween MEM25 and C. reinhardtii or A. thaliana, suggesting the sequence\nconservation of centromeres and telomeres in chlorella strains. Despite of many\nhomologous genes between A. thaliana and MEM25, their specific position and\ndistribution on the genomes is unknown. Therefore, we couldn’t come to the\nconclusion that MEM25 (and other chlorella species) could have been duplicated\nas well as A. thaliana which has undergone WGD. Therefore, we conducted an\naddition analysis of WGD events in MEM25 genome to investigate the\nintragenomic synteny among the sixteen chromosomes of MEM25. The results\nfurther support that no WGD events occur in MEM25 genome (Fig. S4). Now the\nfigure reads:\nFig. S4. Synteny analysis of the sixteen chromosomes of MEM25.\nPairwise gene alignments were performed using BLAST. Genes exhibiting the highest\nsequence similarity are indicated by red dots, while blue dots represent genes among\nthe top four homologous hits. Diagonal linear patterns reflect self-alignments, whereas\noff-diagonal linear arrangements suggest large-scale duplication events, including\npotential whole-genome duplications.\n8. And the telomeres of MEM25 seem to have the synteny to only one region in FACHB.\nResponse: We greatly appreciate the valuable feedback.\nThe apparent synteny to a single region in the FACHB-9 genome is a consequence\nof its highly fragmented assembly, which comprises 1,336 contigs. In reality, the\ntelomeric regions of MEM25 show synteny with 25 different contigs in the C.\npyrenoidosa FACHB-9 genome. The corresponding telomeric sequences depicted\nfor FACHB-9 in Fig. 1c (formerly Fig. 2c) actually represent multiple genomic\nregions. We have now clarified this point in the main text. The revised sentence\n(Lines 676–678 in current version) reads:\n“The apparent synteny of MEM25 telomeres to a single region in the FACHB-9\ngenome results from the latter’s fragmented assembly (1,336 contigs).”\n9. Line 384-388\nI could not find these five families in Table S21. And OG0012359 was mentioned twice\nas a different term. Please confirm the gene family classification.\nResponse: We apologize for the confusion and incorrect citation here. The five\ngene families have been provided in Table S13 while peroxidase is family OG0015353,\nbut not OG0012359. Now the text reads (from Line 273 to line 280 in current\nversion):\n“Out of these, four ha ve been annotated and are associated with oxidative stress\n(i.e., peroxidase, family OG0015353), osmotic adjustment (i.e., volume-regulated\nanion channel subunit LRRC8A, family OG0021391; amino acid permease 2,\nfamily OG0026817), and response to salty stimuli (E3 ubiquitin-protein ligase,\nE3L; family OG0012359) 27-29 (Table S13), suggesting these saline-related genes\nwere already present in the last common ancestor of MEM25 and green plants\nprior to their divergence into either saltwater or freshwater preferred adaptations.”\n10. Line 392\nYou mention “orphan genes” while you mention the gene function. Which is a true\norphan genes or orthologous genes?\nResponse: We apologize for such confusion and have reworded the section as\nbelow (from Line 281 to line 300 in current version):\n“On the contrary, the majority of expanded groups are unique to MEM25 (84 in\n89). We have designated these genes as ENs of MEM25. The large number of\nMEM25-specific genes may reflect the phylogenetic distance of MEM25 from\nother Chlorella species with sequenced genomes. Assuming a freshwater origin of\nthe ancestor of MEM25, portion of these genes may have been acquired during the\nspecialization of MEM25 to a salty niche. For example, we found an exclusive\npresence of gene family OG0011211 in the MEM25 genome (Fig. S10). OG0011211\nencodes a NAD-dependent epimerase/dehydratase family protein, absent in other\nChlorophyta and plant lineages but found in three copies within MEM25 genomes\n(Fig. S10). This gene family is believed to have originated from bacteria where it\nis involved in osmotic stress 30. Loss of NAD-dependent epimerase/dehydratase is\nknown to cause osmosensitivity in bacteria 30, indicating that MEM25 may have\nacquired specialized adaptive mechanisms from bacteria through horizontal gene\ntransfers (HGTs), representing an evolutionary innovation associated with\nsalinity-related genes in MEM25.\nFor the remaining of MEM25-specific genes, we would assume them as orphan\ngenes which have no apparent homology to genes in other evolutionary lineages\noccur in all genomes. They could be candidates for the de novo evolution of genes\nand may contribute to evolutionary novelties, which can become relevant for\nlineage-specific adaptations. Consequently, MEM25 has developed a collection of\nexpanded salinity-related genes, categorized as GPHGs and ENs, shaping its\neuryhaline nature.”\n11. Line 466\nWhat does “DW” mean?\nResponse: “DW” stands for “dry weight”. As a matter of fact, it has been explained\nat first mention in Line 344. Now the text reads (Line 346 to line 349 in current\nversion):\n“Subsequently, 365 mutants with stable phenotypes after ten generations were\nchosen to assess varia tions in cell number, cell size, and dry weight (DW) under\nthe high-salinity conditions (Fig. 6a).”\n12. Line 556\nA N50 value depends on the genome and chromosome size. A relative comparison is\nvaluable, but an absolute value makes no sense.\nResponse: We fully agree with the reviewer and have removed the sentence.\n13. Line 560\nThe chromosome-level assembly lacks several telomere repeats, right? I guess that it is\ndifficult to find all telomere repeats in this case, especially telocentric chromosomes\nlike chr15.\nResponse: Right. While all sixteen chromosomes were clearly marked with the\nplant-specific telomeric repeats (TTTAGGG, Table S3), twelve of them containing\ntelomeric repeats at both ends. A single telomeric repeat was found for the\nremaining four chromosomes, including chr15 (Fig. 1b).\n14. Line 760-765\nI agree with the idea that MEM25 has a mechanism different from D. salina. As a\npossibility, euryhalinity (not halophilism) might be explained by sodium excretion\nrather than salt tolerance.\nFor example, I know a case of adaptation to a marine beach in Vigna marina. I don’t\nthink the same mechanism from the same origin because it is embryophytes, but this\nmechanism could protect from salt and grow a wide range of salt concentrations.\nDiurnal Regulation of SOS Pathway and Sodium Excretion Underlying Salinity\nTolerance of Vigna marina\nNoda et al. (2025)\nhttps://doi.org/10.1111/pce.15402\nResponse: We greatly appreciatethe inputs here and have cite the reference to\nfurther support our argument. Now the text reads (from Line 472 to Line 476 in\ncurrent version):\n“As a possibility, euryhalinity might be explained by sodium excretion rather than\nsalt tolerance as seen in some euryhaline embryophytes 36. It is apparently the case\nfor halophilic D. salina while whether sodium excretion is involved in MEM25’s\neuryhalinity remains elusive.”\n15. Section Genome-wide association in line 930\nDoes the mutant pool share SNPs? Do multiple individuals obtain mutations generated\nby EMS mutagenesis in the same positions?\nPlease let me know if this is a sample set that can be used for GWAS.\nResponse: We greatly appreciatethe inputs here.\nA genome-wide assoc iation study (abbreviated GWAS) is a research approach\nused to identify genomic variants that are statistically associated with a risk for a\nparticular trait. The method involves surveying the genomes of many lines,\nlooking for genomic variants that occur more frequently in those with a specific\ntrait compared to those without the trait. Once such genomic variants are\nidentified, they are typically used to search for nearby variants that contribute\ndirectly to the trait. In this particular case, our interested trait is the salty feature.\nWe first generated a mutant pool for MEM25 by EMS mutagenesis. In principle,\neach mutant in the pool has more than one SNPs in difference sites in the genome.\nHowever, they might share some SNPs in a same position. Because our interested\ntrait of the alga is the salty feature. These mutants were further assessed for\ngrowth in either the low salinity (i.e., 35‰) or the high salinity (i.e., 70‰)\nconditions. Finally, we obtained 195 salinity-related mutants. For these mutants,\nthey have a high possibility to share some SNPs at same position which may\ncontribute to salinity-related feature. Therefore, genomes of the 195 salinity-\nrelated mutants and three WT samples were resequenced to identify their SNPs,\nshared SNPs in particular.\nNow to answer the questions: Does the mutant pool share SNPs? Do multiple\nindividuals obtain mutations generated by EMS mutagenesis in the same positions?\nNo. Each mutant has very diversity SNPs. But the mutants may have some SNPs\nin common. In particular, for the 195 salinity-related mutants used in this study,\nthey have high possibility to share some SNPs in a same gene(s) contributing to\nthe saline feature.\nWe hope above explanation would address your concerns.\n16. line 946\nDoes BWA-MEME mean BWA-MEM?\nResponse: We greatly appreciatethe inputs here. BWA-MEME, the first full-\nfledged short read alignment software that achieves up to 3.45× speedup in seeding\nthroughput over BWA-MEM2. We could treat BWA-MEME as an upgrade of\nBWA-MEM2.\n17. line 982-983\nYou show the thresholds of P-adj and FDR. What does P-adj mean, not FDR?\nResponse: We greatly appreciatethe inputs and have corrected the sentence as\nbelow. Now the text reads (from Line 166 to Line 169 in Supplementary text 2):\n“Differentially expressed genes were defined as those with FDR-adjusted p-value\n≤ 0.05 and |log FoldChange| ≥ 1.”\n2\n18. line 945\nThe parameters of fastp “-t 10 -f 10 -T 10 -F 10” appear to be over-trimmed. Is there\nany reason?\nResponse: We greatly appreciatethe inputs here. As matter of fact, we have\nconducted a comparison between the data before and after the trimming. The trim\nat 3’-end eliminates phasing-induced low-quality cycles that would decrease the\ncoverage and increase false positives. On the other hand, the 5’-end trim removes\na reproducible A/C and adapter remnants concentrated in the first 8 bp that would\nlead to batch-specific sequence bias. Because all libraries share a 248 bp insert\npeak, 20-bp trim for each read yields cleaner and more homogeneous data without\ncausing R1/R2 overlap or impairing variant localization.\nBased on our quantitative QC analysis, the trim removed 4.7 % of total reads and\nobtained reads with a mean length of 129 bp (above the anchoring thresholds of\nstandard aligners). The Q30 fraction, MAPQ values, and coverage uniformity all\nincreased. It would facilitate downstream variant calling and GWAS due to lower\nerror rates and more uniform coverage by improving both the sensitivity for rare\nalleles and the specificity of association signals.\nTable 1. Summary of the comparison between the data before and after the trimming.\nMetric (sample: the 19-1) Before trimming After trimming Difference\nData retention (%) 100 95 5↓\nTotal reads 9815354 9356990 4.67%↓\nQ20 proportion R1 (%) 95.8 96.94 1.14↑\nQ20 proportion R2 (%) 93.7 95.51 1.81↑\nQ30 proportion R1 (%) 90.96 92.49 1.53↑\nQ30 proportion R2 (%) 86.99 89.29 2.3↑\nAlignment rate (%) 93.3 93.19 0.11↓\nHigh-quality alignment (MAPQ ≥30) (%) 86.94 87.8 0.86↑\nCoverage uniformity (CV) 1.396 0.655 ↑\nPaired reads proportion (%) 90.47 91.95 1.48↑\nUnpaired reads proportion (%) 9.53 8.05 1.48↓\nBecause the space limit, we prefer not to include the table in either the main text\nor supplementary materials.\n19. line 958\nDid you use these QC options? or filter out them?\nResponse: Right. We used these QC options. As a new model species, MEM25 lacks\nreferences data for mutation analysis. Therefore, we employed a very strict\nfiltering approach to ensure the quality of detected mutation by using GATK.\nQuality control on SNPs and INDELs was performed separately. Details of the\nparameters were provided in Methods (Supplementary text 2) and the public\nrepository (https://git hub.com/aoqiwang/MEM25).\n20. Figure 3a\nDo these legend colors indicate the background of the species’ names? Please use more\ndistinctive colors.\nResponse: We greatly appreciatethe inputs here and, as advised, we have now used\nmore distinctive colors to indicate the background of the species from different\nphyla (Fig. 2 in current version). Moreover, we add the previously missing\nbackground color for Streptophyta. Please also refer to our response to Comment\n19 from Reviewer 1.\n21. Figure 3b\nThe positions of the purple circles are unclear, and some seem to locate no branch points.\nResponse: We apologize for the confusion. In fact, as the reviewer says, some\npurple circles seem to locate no branch points. It is because that too many species\nhave been included in this analysis and some branches are overlapped. An\nadditional reason is that only branches with bootstrap values exceeding 90% are\nhighlighted with purple dots. It has been signified in the legend of previous Fig. 3c\n(Fig. S8 in current version). Now the text reads (from Line 810 to Line 812 in\ncurrent version):\n“Purple dots denote branches with bootstrap support values exceeding 90% based\non 1,000 replicates. Apparent displacement of the dots from branch points is due\nto overlapping branches.”\nsynteny of MEM25 telomeres to a single region in the FACHB-9 genome results\nfrom the latter’s fragmented assembly\n22. Figure 6b\nGreen indicates bacteria. However, other colors also indicate the kind of bacteria.\nResponse: We apologize for the confusion. The bacteria indicated by green color\nhave been nominated as “other type of bacteria” in the revised figure.\n23. Figure 9\nPlease add the details. For example, what are the orange points in (d)? Where is the\ngene in (e)?\nResponse: We apologize for the confusion and, as advised, we have re-organized\nFig. 9d and 9e (Fig. 6d and 6e in the current version) (from Line 741 to Line 749\nin current version):\n(d) Location of the S04_3657761 region. This region is situated downstream of gene\nCP4g4326 and upstream of CP4g4327. Exons are indicated as orange boxes and introns\nare shown in gray boxes. Empty boxes are intergenic regions. Colored dots indicate the\nSingle Nucleotide Polymorphisms (SNPs) proximal to S04_3657761 in Chr04. Red\narrow indicates the position of nucleotide variation in S04_3657761. (e) Mapping of\ntopologically associating domains (TADs) and loops on chromosome 4. TADs are\nhighlighted with yellow lines, while loops are depicted by blue lines. The interaction\nbetween S04_3657761 and gene CP4g4237 is illustrated.\n24. Figure S4a shows the polar tree rather than the unrooted tree. Poler, the center of\nthe figure, generally indicates the root.\nResponse: We apologize for such typo and fully agree with the reviewer that Figure\nS4a is a rooted tree. We have provided the tree files in the repository:\nhttps://figshare.com/articles/journal_contribution/Data_of_MEM25/29062721.\n25. Legend Fig. S5\nline 1658\nWhat do the “filled circles ● and ●, respectively” mean? It looks like the same filled\ncircles to me.\nResponse: We apologize for such confusion and have specified the legend for Fig.\nS5 (current Fig. S7). Now the text reads (from Line 801 to Line 803 in current\nversion):\n“Significantly expanded or contracted orthologs are denoted by filled red circles\n● and filled blue circles●, respectively.”\nPlease also refer to ou r response to Comment 43 of Reviewer 1.\nComments from Reviewer 3:\nOverall, this is a comprehensive and well written manuscript. A high-quality chlorella\ngenome will be useful to the community.\nDespite extensive data assembly and analysis, the manuscript could be improved.\nSpecifically, when increased biomass is reported this should be further quantified in\nterms of lipids, carbohydrates or proteins (or more cells) to better understand how more\nbiomass is being made. Additionally, more information regarding RMI1 is encouraged.\nA thorough protein map with putative domains is necessary, as is alignments with\nhomologs. Lastly, a more thorough hypothesis regarding RMI1 mechanism is\nencouraged.\nResponse: We sincerely appreciate the reviewer’s valuable feedback. We believe\nthat these suggestions will significantly contribute to refining our research and\nuncovering additional interesting findings. Regarding the six algal mutant lines\ncreated in this study, we have evaluated their biomass and cell density under high\nsalinity conditions (70‰), where both metrics were consistently elevated across all\nsix lines compared to the wild-type strain. These results not only lend support to\nour hypothesis that the genes identified in this study enhance salinity tolerance,\nbut also demonstrate the reliability of the methodology employed to identify genes\nassociated with salt tolerance.\nFurther investigations into metabolite profiling and a more comprehensive\ncharacterization of RMI1, including motif prediction, validation, interactions with\ntranscription factors, and the establishment of regulatory networks centered\naround RMI1, would significantly deepen the scope of our study. A research\nproposal incorporating these aspects could prove to be quite valuable.\nIn fact, with the six algal mutant lines and the plant mutants now established, we\nhave begun to explore potential upstream and downstream regulators interacting\nwith the knocked-out genes. We hope to provide additional evidence in future\nstudies that will further support the conclusions presented in this submission and\npossibly reveal further insights.\nAgain, thank you and the anonymous reviewers for the constructive comments that have\nimproved our manuscript greatly.\nThank you for your consideration.\nSincerely yours,\nState Key Lab of Marine Resource\nUtilization in South China Sea, Hainan\nU niversity\nMailing: 58 Renmin AveCnuoel leg e o f O c e a n o l o g y , H a Tienl: a86n-8 98-66257612\nHaikou, Hainan, China, 570228 Fax: 86-898-66251258\nUniversity (HN U)\nYandu LU, Ph.D\nEngineering & Research Center of Marine Bioactives & Bioproducts of Hainan\nProvince\nHainan University, Haikou, China\nEmail: ydlu@hainanu.edu.cn\nhttp://orcid.org/0000-0002-0136-2252\nReferences:\n1. Jamy, M. et al. Global patterns and rates of habitat transitions across the eukaryotic tree of life.\nNature Ecology & Evolution 6, 1458-1470 (2022).\n2. Dittami, S.M., Heesch, S., Olsen, J.L. & Collén, J. Transitions between marine and freshwater\nenvironments provide new clues about the origins of multicellular plants and algae. Journal of\nPhycology 53, 731-745 (2017).\n3. Hodač, L. et al. Widespread green algae Chlorella and Stichococcus exhibit polar-temperate\nand tropical-temperate biogeography. FEMS Microbiology Ecology 92 (2016).\n4. Eckardt, N.A. The Chlorella genome: big surprises from a small package. The Plant cell 22,\n2924 (2010).\n5. Barati, B., Lim, P.-E., Gan, S.-Y., Poong, S.-W. & Phang, S.-M. Gene expression profile of\nmarine Chlorella strains from different latitudes: stress and recovery under elevated\ntemperatures. Journal of Applied Phycology 30, 3121-3130 (2018).\n6. Lu, X. et al. Sustainable development of microalgal biotechnology in coastal zone for\naquaculture and food. Science of The Total Environment 780, 146369 (2021).\n7. Ramachandran, A., McLatchie, S. & Walsh, D.A. A novel freshwater to marine evolutionary\ntransition revealed within Methylophilaceae bacteria from the Arctic ocean. mBio 12, e0130621\n(2021).\n8. Fan, J. et al. Genomic foundation of starch-to-lipid switch in oleaginous Chlorella spp. Plant\nphysiology 169, 2444-2461 (2015).\n9. Blanc, G. et al. The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis,\ncoevolution with viruses, and cryptic sex The Plant cell 22, 2943-2955 (2010).\n10. Lemieux, C., Turmel, M., Otis, C. & Pombert, J.-F. A streamlined and predominantly diploid\ngenome in the tiny marine green alga Chloropicon primus. Nature Communications 10, 4061\n(2019).\n11. Ou, S., Chen, J. & Jiang, N. Assessing genome assembly quality using the LTR Assembly Index\n(LAI). Nucleic Acids Research 46, e126-e126 (2018).\n12. Noda, Y. et al. Diurnal regulation of SOS pathway and sodium excretion underlying salinity\ntolerance of Vigna marina. Plant, Cell & Environment n/a.\n13. Polle, J.E.W. et al. Genomic adaptations of the green alga Dunaliella salina to life under high\nsalinity. Algal Research 50, 101990 (2020).\n14. Deneka, D. et al. Allosteric modulation of LRRC8 channels by targeting their cytoplasmic\ndomains. Nature Communications 12, 5435 (2021).\n15. Castro, B. et al. Stress-induced reactive oxygen species compartmentalization, perception and\nsignalling. Nature Plants 7, 403-412 (2021).\n16. Wang, S. et al. Roles of E3 ubiquitin ligases in plant responses to abiotic stresses. International\nJournal of Molecular Sciences 23 (2022).\n17. Petrovova, M., Tkadlec, J., Dvoracek, L., Streitova, E. & Licha, I. NAD(P)H-hydrate\ndehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation. PLoS\nOne 9, e112590 (2014)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors of the manuscript entitled \"Cross-species dissection of saline-related genes by genetically deciphering a euryhaline microalga Chlorella sp.\" have submitted a detailed revised manuscript addressing the reviewers' comments. I appreciate the extensive effort they have invested in this revision process. However, several modifications are still required before publication:\nThe methodology section requires significant improvement. Currently, it merely lists section headings without providing adequate experimental details. Authors must develop each methodological section with sufficient information to ensure reproducibility. If the comprehensive methodology would make the main text too lengthy, detailed protocols should be provided as supplementary files. The current format, which presents only section titles without substantive content, is inappropriate for a scientific publication. Please revise this section to include complete methodological descriptions.", "reviewer_2": "I am highly impressed with the completeness of the T2T-level assembly, notably achieving a single gap.\nIs there any support for a gap length of 100 bp? The software might arbitrarily insert the round number. Although the quality of the genome with a single gap is not compromised, if a 100bp gap is an artifact, it is appropriate not to mention it.\nFig. 2\nDistinctive colors make this figure clear. However, the background colors are in the front, making the characters hard to see.\nPlease exchange the layers’ orders.", "reviewer_3": "The authors have adequately addressed my concerns."}, "author_response": "Nature Communication s\nJuly 4, 2025\nDear Reviewers,\nThank you for the constructive and extremely valuable comments to help improving\nthe manuscript. Here we are submitting a revised version, with item-by-item responses\n(bold fonts) to the comments below.\nWe have thoroughly proofread and edited the text following the requirements. We\nbelieve that our revised version supports the main conclusion of our initial submission\nand also reveal additional insights, which were detailed below.\nComments from Reviewer 1:\n1. The authors of the manuscript entitled \"Cross-species dissection of saline-related\ngenes by genetically deciphering a euryhaline microalga Chlorella sp.\" have submitted\na detailed revised manuscript addressing the reviewers’ comments. I appreciate the\nextensive effort they have invested in this revision process. However, several\nmodifications are still required before publication:\nThe methodology section requires significant improvement. Currently, it merely lists\nsection headings without providing adequate experimental details. Authors must\ndevelop each methodological section with sufficient information to ensure\nreproducibility. If the comprehensive methodology would make the main text too\nlengthy, detailed protocols should be provided as supplementary files. The current\nformat, which presents only section titles without substantive content, is inappropriate\nfor a scientific publication. Please revise this section to include complete\nmethodological descriptions.\nResponse: We greatly appreciate the reviewer’s inputs. As the journal has no word\nlimit for Methods, we have moved the Methods from Supplementary Note 2 to the\nMain Text. We believe that the methodological section is now with sufficient\ninformation to ensure reproducibility.\nComments from Reviewer 2:\nI am highly impressed with the completeness of the T2T-level assembly, notably\nachieving a single gap.\nIs there any support for a gap length of 100 bp? The software might arbitrarily insert\nthe round number. Although the quality of the genome with a single gap is not\ncompromised, if a 100bp gap is an artifact, it is appropriate not to mention it.\nState Key Lab of Marine Resource\nUtilization in South China Sea, Hainan\nU niversity\nMailing: 58 Renmin AveCnuoel leg e o f O c e a n o l o g y , H a Tienl: a86n-8 98-66257612\nHaikou, Hainan, China, 570228 Fax: 86-898-66251258\nUniversity (HN U)\nResponse: We fully u nderstand the reviewer’s concern. As a matter of fact, the\nregion upstream of the gap (5,000 bp) exhibits complex repetitive structures, such\nas tandem repeats, representing inherent assembly challenges. Therefore, the\nlength of 100 bp is not the actual size of the gap but represents a small gap where\nsequence continuity cannot be established. To avoid potential confusion, we\nrevised the previous sentencein Supplementary note 1 (A single gap was present\nin the assembled genome at the end of chr15 (1,311,674 bp) with a length of 100\nbp from 1,286,591 bp to 1,286,690 bp). Now the text reads:\n“A single gap was present in the assembled genome at the end of chr15 (1,311,674\nbp due to the difficulty in assembling repetitive fragments of the genome)”\n2. Fig. 2\nDistinctive colors make this figure clear. However, the background colors are in the\nfront, making the characters hard to see. Please exchange the layers’ orders.\nResponse: We have changed the layers’ order as advised. Now the figure reads:\nFig. 2. Phylogenetic position of Chlorella sp. MEM25. Rooted phylogenetic tree\nfeatured 38 Chlorophyta species and representative species from cyanobacteria,\nRhodophyta, Glaucophyta, streptophyte algae, and embryophytes. The species tree was\ndeduced using the most closely related genes within single-copy or multi-copy\nState Key Lab of Marine Resource\nUtilization in South China Sea, Hainan\nU niversity\nMailing: 58 Renmin AveCnuoel leg e o f O c e a n o l o g y , H a Tienl: a86n-8 98-66257612\nHaikou, Hainan, China, 570228 Fax: 86-898-66251258\nUniversity (HN U)\northogroups (n=199), based on the Species Tree from All Genes (STAG) algorithm.\nSpecies from different phyla are indicated in different background colors. Freshwater\nand seawater species are shown in green and orange dots. Bootstrap with values of 100%\nor above 70% are indicated in smaller or bigger dots at the branching points,\nrespectively.\nComments from Reviewer 3:\nThe authors have adequately addressed my concerns.\nResponse: We sincerely appreciate the reviewer’s valuable feedback. We believe\nthat these suggestions will significantly contribute to refining our research and\nuncovering additional interesting findings.\nAgain, thank you and the anonymous reviewers for the constructive comments that have\nimproved our manuscript greatly.\nThank you for your consideration.\nSincerely yours,\nYandu LU, Ph.D & Professor\nEngineering & Research Center of Marine Bioactives & Bioproducts of Hainan\nProvince\nHainan University, Haikou, China\nEmail: ydlu@hainanu.edu.cn\nhttp://orcid.org/0000-0002-0136-2252"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I would like to commend the authors for addressing all of the editor's and reviewers' comments thoroughly. The improvements to the manuscript are clear, and the authors have done an excellent job of revising it."}, "author_response": ""}]

Decision letter