A Systematic Revision of North American Tolypella A. Braun (Charophyceae, Charophyta)

Charophyta comprises the algal classes Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae and the land plants. However, the precise phylogenetic position of these algal classes with respect to land plants is unresolved as are the phylogenetic relationships among genera in Charophyceae (Characeae). Characeae contains two tribes with six genera: tribe Chareae (Chara, Lamprothamnium, Lychnothamnus and Nitellopsis) and tribe Nitelleae (Nitella and Tolypella). Tolypella was considered the third most species-rich genus but, in the most comprehensive taxonomic treatment of Characeae, 16 Tolypella species were consolidated into two species, T. nidifica and T. intricata in sections Rothia and Tolypella, respectively. It was further suggested that Tolypella might be a derived group within the closely related genus Nitella. Currently, there are no comprehensive molecular phylogenetic studies of Tolypella. In this investigation into North American Tolypella, sequence data from the plastid genes atpB, psbC and rbcL and the nuclear ribosomal operon 18S-ITS1-5.8S-ITS2-28S were assembled for a broad range of charophytic algae and land plants in order to address Tolypella species diversity and resolve their relationship to other genera in Characeae. In addition, mitochondrial and plastid genomes were completely sequenced for three Tolypella species for a comparative analysis of genome architecture and gene content and for inclusion into a multi-gene phylogeny of the Charophyta. Phylogenetic analyses of the plastid and nuclear gene data showed that Nitelleae is paraphyletic with Nitella sister to Chareae. Genus Tolypella and Tolypella section Tolypella were recovered as monophyletic. Tolypella section Rothia was weakly to moderately supported as monophyletic or weakly supported as paraphyletic in several analyses. Several lineages of the traditionally recognized Tolypella species in addition to a new species, T. ramosissima sp. nov., were identified, which suggests greater species diversity in Tolypella than currently recognized. Comparative analyses of mitochondrial and plastid genomes of three Tolypella species, T. canadensis, T. glomerata and T. intricata, showed that gene content, gene order and genome architecture are relatively conserved, however, T. glomerata showed a complete loss of the Ndh 1-complex genes. Phylogenetic analyses also recovered a monophyletic Tolypella and sections Rothia and Tolypella but showed conflicting results regarding the sister taxon to land plants with either Charophyceae or Zygnematophyceae as their closest living relatives