The Arracacia clade (Apiaceae, Apioideae) is a heterogeneous assemblage of 12 genera, comprising 111 known species distributed in high montane temperate and sub-alpine habitats of meso- and South America. Previous studies have indicated that the genera Arracacia, Coulterophytum, and Prionosciadium are polyphyletic, but for the most part relationships among the members of the clade are largely unknown. Initially, cladistic analyses of nrDNA ITS sequences were carried out on 212 accessions (122 taxa), representing 92 species of the Arracacia clade and outgroups from the closely-related páramo genera Cotopaxia, Niphogeton, and Perissocoeleum and members of the Perennial Endemic North American clade and its allies. Using the ITS results to inform sampling of a small subset of taxa, a pilot study examining the phylogenetic utility of 20 noncoding chloroplast loci was subsequently performed to identify those regions most useful at resolving relationships. A cost-benefit analysis determined that five loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, psbD-trnT, ndhA intron) would maximize resolution and branch support in the clade. Cladistic analyses of four of these loci (trnQ-5'rps16, trnD-trnT, rpl32-trnL, ndhA intron) and the ITS region, separately and combined, revealed that Arracacia, Coaxana, Coulterophytum, Prionosciadium, and Rhodosciadium are each polyphyletic and that Donnellsmithia and Myrrhidendron are each monophyletic. Although most relationships in the Arracacia clade and among the closely-related genera Cotopaxia, Niphogeton, and Perissocoeleum are poorly resolved and supported, ten groups are recognized for future revisionary studies. Polyploidy and rapid species radiation have likely confounded generic circumscriptions and interpretation of relationships.
We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, methylated DNA immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.