Sequencing and Phylogenetic Analysis of Chloroplast Genes in Freshwater Raphidophytes

Abstract

The complex evolution of chloroplasts in microalgae has resulted in highly diverse pigment profiles. Freshwater raphidophytes, for example, display a very different pigment composition to marine raphidophytes. To investigate potential differences in the evolutionary origin of chloroplasts in these two groups of raphidophytes, the plastid genomes of the freshwater species Gonyostomumsemen and Vacuolariavirescens were sequenced. To exclusively sequence the organelle genomes, chloroplasts were manually isolated and amplified using single-cell whole-genome-amplification. Assembled and annotated chloroplast genes of the two species were phylogenetically compared to the marine raphidophyte Heterosigmaakashiwo and other evolutionarily more diverse microalgae. These phylogenetic comparisons confirmed the high relatedness of all investigated raphidophyte species despite their large differences in pigment composition. Notable differences regarding the presence of light-independent protochlorophyllide oxidoreductase (LIPOR) genes among raphidophyte algae were also revealed in this study. The whole-genome amplification approach proved to be useful for isolation of chloroplast DNA from nuclear DNA. Although only approximately 50% of the genomes were covered, this was sufficient for a multiple gene phylogeny representing large parts of the chloroplast genes.

Keywords: LIPOR; chloroplast genome; phylogeny; raphidophytes; whole-genome-amplification.

Figure 1

Sequence similarities based on blastn between to the circular chloroplast genome of H. akashiwo (~160 kbp) and chloroplast contigs of G. semen and V. virescens visualized with CGView. Coloured regions on the G. semen and V. virescens rings correspond to fragments matching the reference genome. The GC content of the H. akashiwo chloroplast genome averages at 30.5%.

Figure 2

Maximum likelihood phylogeny of 53 concatenated chloroplast protein sequences, 14,896 amino acids in length, built with RAxML from an alignment of 49 taxa with bootstrap values <100 displayed on the internal branches. Raphidophyceae are highlighted in green, while curved lines indicate the position of other taxonomic groups. Circles on the branches indicate presence/absence of light-independent protochlorophyllide oxidoreductase (LIPOR) genes based on [31] and NCBI: green = presence, black = absence, grey = pseudogenes, empty = unknown.