Evolution of reduced and compact chloroplast genomes (cpDNAs) in gnetophytes: selection toward a lower-cost strategy

The cpDNA of Welwitschia mirabilis (the only species of Welwitschiales) was recently reported to be the most reduced and compact among photosynthetic land plants. However, cpDNAs of the other two gnetophyte lineages (viz. Ephedrales and Gnetales) have not yet been studied. It remains unclear what underlining mechanisms have downsized the cpDNA. To pin down major factors for cpDNA reduction and compaction in gnetophytes, we have determined 4 complete cpDNAs, including one from each of the 3 gnetophyte orders, Ephedra equisetina, Gnetum parvifolium, and W. mirabilis, and one from the non-Pinus Pinaceae, Keteleeria davidiana. We report that the cpDNAs of E. equisetina (109,518bp) and G.parvifolium (114,914bp) are not only smaller but more compact than that of W. mirabilis (118,919bp). The gnetophyte cpDNAs have commonly lost at least 18 genes that are retained in other seed plants. Furthermore, they have significantly biased usages of AT-rich codons and shorter introns and intergenic spaces, which are largely due to more deletions at inter-operon than intra-operon spaces and removal of segment sequences rather than single-nucleotides. We show that the reduced gnetophyte cpDNAs clearly resulted from selection for economy by deletions of genes and non-coding sequences, which then led to the compactness and the accelerated substitution rates. The smallest C-values in gnetophyte nuclear DNAs and the competitive or resource-poor situations encountered by gnetophytes further suggest a critical need for an economic strategy.