![]() Only three of these share positions, and they cannot be traced back to their common ancestor. However, we identified five additional ITSs in the 28S rRNA of all analysed metakinetoplastids, and up to twelve in euglenids. Similarly, no additional ITSs were detected in basal prokinetoplastids. In all nine diplonemids, 28S rRNA seems to be contiguous, with no additional ITSs detected. The rDNA sequences acquired for several euglenids and kinetoplastids were used to provide the background for the analysis. We used available genomic (culture and single-cell) sequencing data to assemble complete or almost complete rRNA operons for three classical and six deep-sea diplonemids. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. Currently, the rDNA organization has not been researched for any diplonemid. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. In Euglenida rDNA is located on extrachromosomal circular DNA. Members of Euglenozoa (Discoba) are known for unorthodox rDNA organization. ![]() Immediate actions need to be taken if we want to prevent this unique species from going extinct. Isoetes wormaldii is critically endangered, known only from one (to a few) minor populations. Moreover, it shortens the branch length to its living sister genus Selaginella, and may enhance node age estimation in future studies. The here demonstrated sister-relationship between the phylogenetically, morphologically and genetically distinct Isoetes wormaldii and the remaining family appears to bridge the morphological gap between Isoetes and its extinct relatives, although further studies are needed. Previously shown difficulties with node age estimation increase the problem. The evolutionary and biogeographical history of Isoetes is not easily explained, and may conceivably include ample extinction and a mixture of ancient and more recent processes. For example, tropical-southern African species occur in at least five clades, and Indian, Australian and Mediterranean species in at least three clades each. While the phylogeny shows geographic structure, the patterns are complex. The remaining species of Isoetes are resolved in five major clades, also indicated in previous work. Its leaves are flattened with a rounded point, which sharply contrasts with the awl-shaped leaves of most other species of Isoetes. The species, Isoetes wormaldii, is a rare endemic to the Eastern Cape of South Africa. Our results reveal an unexpected discovery of an “ Amborella syndrome” in Isoetaceae: a single poorly known species is sister to the remaining family. We investigate evolutionary relationships in Isoetes, using molecular data and an extended sample of species compared to previous work, adding species that have never before been included in a phylogenetic study. Genetic variation has also proven limited, which has hampered phylogenetic inference. They display little morphological variation the lobed corm has helically arranged leaves with internal air channels and basal sporangia. Modern day species are aquatic, semi-aquatic or terrestrial and occur almost worldwide. Merlin’s grass ( Isoetes, Isoetaceae, Lycopsida), is the extant remnant of the isoetalean wood-producing lycopsids that originated during the Paleozoic, possibly in aquatic or boggy habitats.
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