The Chlorella variabilis NC64A genome reveals adaptation to photosymbiosis, coevolution with viruses, and cryptic sex

Abstract

Chlorella variabilis NC64A, a unicellular photosynthetic green alga (Trebouxiophyceae), is an intracellular photobiont of Paramecium bursaria and a model system for studying virus/algal interactions. We sequenced its 46-Mb nuclear genome, revealing an expansion of protein families that could have participated in adaptation to symbiosis. NC64A exhibits variations in GC content across its genome that correlate with global expression level, average intron size, and codon usage bias. Although Chlorella species have been assumed to be asexual and nonmotile, the NC64A genome encodes all the known meiosis-specific proteins and a subset of proteins found in flagella. We hypothesize that Chlorella might have retained a flagella-derived structure that could be involved in sexual reproduction. Furthermore, a survey of phytohormone pathways in chlorophyte algae identified algal orthologs of Arabidopsis thaliana genes involved in hormone biosynthesis and signaling, suggesting that these functions were established prior to the evolution of land plants. We show that the ability of Chlorella to produce chitinous cell walls likely resulted from the capture of metabolic genes by horizontal gene transfer from algal viruses, prokaryotes, or fungi. Analysis of the NC64A genome substantially advances our understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.

Figure 1.

General Characteristics of the Chlorella sp NC64A Genome Assembly. This figure represents the 30 major scaffolds, which contain 89% of the total genome. GC percentage, exon density, EST density, and repeat density were calculated in 40-kb sliding windows with a step of 5 kb. Density was calculated as the percentage of nucleotide in the window covered by the relevant feature (i.e., exon, EST, or repeat sequence). Blue triangles represent telomeric repeat arrays.

Figure 2.

Features of Low-GC Regions in Chlorella sp NC64A. Nonoverlapping 40-kb segments of the NC64A genome assembly were classified into four GC content classes. The distributions of genomic segments in each of the GC content classes are depicted by box plots for the following features: EST density (as defined in the Figure 1 legend) (A), average size of introns supported by EST data (B), mean effective number of codons (ENc) per gene (C), exon density (D), and repeat density (E). (F) shows the distribution of genomic segments as a function of their GC content. The bottom and top of boxes represent the 1st and 3rd quartiles, Q1 and Q3, respectively, and the band near the middle of boxes represents the median. The extremities of the lines appearing below and above the boxes represent the lowest value still within 1.5 IQR (interquartile range = Q3 to Q1) of the lower quartile Q1, and the highest value still within 1.5 IQR of the upper quartile Q3. We applied the Kruskal-Wallis statistical test to each genomic feature to test the null hypothesis of equivalence between the distributions of values in the four GC bins. Distributions of EST density, intron size, and Enc were found to be significantly different between the four GC bins (P < 0.0001), whereas for repeat density, the difference was only marginally significant (P = 0.024). The null hypothesis of equivalence of distributions could not be rejected at α = 0.05 for exon density (P = 0.468).

Figure 3.

Heat Map of PFAM Protein Families with Significantly Biased Distribution among Chlorophyte Algae. PFAM protein families that have either significantly expanded or shrunk in one or more sequenced chlorophytes (χ² test, α = 0.05 after Bonferroni correction). Full red and black indicate 100 and 0%, respectively, of the total number of proteins in the PFAM family for the six algae. Real counts and description of PFAM protein families are given in Supplemental Table 5 online. The leftmost graph represents the hierarchical clustering of the PFAM domains by the average linkage methods using correlation coefficients between profiles.

Figure 4.

Distribution of Selected Flagellar Proteins across Chlorophytes. (A) Cladogram showing the likely evolutionary relationships of sequenced chlorophytes and T. pseudonana based on the 18S phylogenetic tree shown in Supplemental Figure 1 online. The ƒ mark shows organisms known to build motile flagella. Crei, C. reinhardtii; NC64A, Chlorella sp NC64A; Otau, O. tauri; Oluc, O. lucimarinus; M. CCMP, M. pusilla CCMP1545; M. RCC, Micromonas sp RC299. (B) Presence (dot) or absence (circle) of putative (Reciprocal Best Hit) orthologs to Chlamydomonas outer dynein proteins, inner dynein proteins, radial spoke proteins, central pair proteins, and IFT proteins.

Figure 5.

Maximum Likelihood Phylogenetic Tree of the Chitin Deacetylase and Chitinase Proteins. For both protein families, we used the WAG+I+G model of substitutions. Approximate likelihood ratio test values >50% are indicated beside branches. Phylogenetic trees are midpoint rooted. Alignments used to generate these trees are available as Supplemental Data Sets 4 and 5 online. (A) Phylogenetic tree of chitin deacetylases. The multiple sequence alignment contained 134 gap-free sites. (B) Phylogenetic tree of chitinases. The multiple sequence alignment contained 228 gap-free sites. [See online article for color version of this figure.]