Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca

Abstract

Autotrophic eukaryotes have evolved by the endosymbiotic uptake of photosynthetic organisms. Interestingly, many algae and plants have secondarily lost the photosynthetic activity despite its great advantages. Prototheca and Helicosporidium are non-photosynthetic green algae possessing colourless plastids. The plastid genomes of Prototheca wickerhamii and Helicosporidium sp. are highly reduced owing to the elimination of genes related to photosynthesis. To gain further insight into the reductive genome evolution during the shift from a photosynthetic to a heterotrophic lifestyle, we sequenced the plastid and nuclear genomes of two Prototheca species, P. cutis JCM 15793 and P. stagnora JCM 9641, and performed comparative genome analyses among trebouxiophytes. Our phylogenetic analyses using plastid- and nucleus-encoded proteins strongly suggest that independent losses of photosynthesis have occurred at least three times in the clade of Prototheca and Helicosporidium. Conserved gene content among these non-photosynthetic lineages suggests that the plastid and nuclear genomes have convergently eliminated a similar set of photosynthesis-related genes. Other than the photosynthetic genes, significant gene loss and gain were not observed in Prototheca compared to its closest photosynthetic relative Auxenochlorella. Although it remains unclear why loss of photosynthesis occurred in Prototheca, the mixotrophic capability of trebouxiophytes likely made it possible to eliminate photosynthesis.

Figure 1

Structure of the plastid genomes of P. stagnora and P. cutis. (a,b) Gene maps of the plastid genomes of P. stagnora and P. cutis, respectively. Genes are shown in different coloured boxes according to their putative functions. Genes on the outside of the maps are transcribed in the clockwise direction, and inner genes are transcribed in the counterclockwise direction. (c) Comparison of the gene order of the plastid genomes of C. variabilis, A. protothecoides, P. wickerhamii, P. cutis, P. stagnora, and Helicosporidium sp. Homologous genes are connected by straight lines as shown in the figure. Most of the photosynthesis-related genes (green) are absent in the non-photosynthetic lineages.

Figure 2

Phylogenetic tree and the evolutionary scenario of the plastid gene losses in Chlorellales. (a) Maximum Likelihood (ML) tree constructed using 38 plastid-encoded proteins. Bootstrap support (BP) is indicated above the lines, and Bayesian posterior probability (BPP) is indicated below the lines. BP <50 or BPP <0.95 are not shown. Bold lines represent 100% BP and 1.00 BPP. The dotted branches are shown in half-length. (b) ML tree of 58 nucleus-encoded proteins. All the nodes were supported with 100% BP. (c) Evolutionary scenario of gene losses in P. wickerhamii, P. cutis, P. stagnora, and Helicosporidium sp. Eliminated plastid genes are indicated on the tree. Genes shown in green and blue have independently disappeared two and three times in the AHP lineage. Loss of group-I intron is presented by intron [trnL(UAA)].

Figure 3

Comparison of nucleus-encoded proteins among Prototheca, Auxenochlorella, and Helicosporidium. (a) Venn diagram of shared gene families among P. cutis, P. stagnora, A. protothecoides, and Helicosporidium sp. (b) The number and size of gene families. Gene families consisting of multiple genes are shown in red, green, and purple according to their family size (two, three, and more than four). (c) The number of genes according to KEGG classification.

Figure 4

Gene contents related to plastid biosynthesis in Prototheca, Auxenochlorella, and Helicosporidium. Genes for plastid-related proteins were categorized into eight groups according to their functions: carbon fixation, and biosynthesis of starch, carotenoid, tetrapyrrole, fatty acids, terpenoid, Phe/Tyr/Trp, and Val/Leu/Ile. Green coloured boxes indicate the presence of genes for the plastid-related proteins as shown by EC numbers. The colour gradient represents the copy number of genes.

Figure 5

Synteny analysis of the nuclear genomes between P. cutis and A. protothecoides. (a) Syntenic regions between P. cutis and A. protothecoides nuclear genomes are indicated by coloured lines. Numbers beside the scheme represent a scaffold number. (b–d) Syntenic regions including photosynthesis-related genes (green) in A. protothecoides. Homologous genes (orange) between P. cutis and A. protothecoides are connected by dotted lines.