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Highlights •LSU ribosomal RNA is the ribozyme that catalyzes translation.•Variation in its GC content was measured across regions of the ribosome, in many Bacteria, Eukaryota, Archaea.•GC variation across taxa increases, smoothly, from the deep to the superficial regions.•This reflects the in-out pattern of increasing variation of all the nucleotides (A, C, G, T).•This GC mapping will help to improve models used for phylogenetic-tree reconstruction.

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Calcareous sponges are taxonomically difficult, and their morpho-systematic classification often conflicts with molecular phylogenies. Consequently, species descriptions that rely solely on morphological characters,and taxonomic revisions appear to provide little to no information about phylogenetic affiliations and integrative approaches, combining DNA and morphological data, are applied more frequently. However, a standardized database that combines DNA sequence and morphological specimen information is still missing for calcareous sponges. The mitochondrial cytochrome oxidase subunit 1 gene (COI) is the marker of choice for rapid species identification in many other animal taxa, including demosponges, for which COI sequences and morphological information have been compiled in the sponge barcoding database (). But due to the peculiarities of calcarean mitochondrial genomes, sequencing COI in Calcarea is methodologically challenging. We here propose the use of one more commonly used DNA marker, the C-region of the 28S gene (LSU), as standard barcoding marker for Calcarea, after also considering the internal transcribed spacer (ITS) region for such proposes. Especially in the subclass Calcaronea, we observed severe problems of high intra- and intergenomic variation that impedes pan-calcarean ITS alignments. In contrast, the C-region of LSU provides a short but phylogenetically informative DNA sequence, alignable across both subclasses with the help of a newly developed secondary structure and which also can be used to address exemplary taxonomic questions. With our work, we start to close the gap of Calcarea in the sponge barcoding project () and provide a resource for biodiversity studies and potentially for DNA-guided species identification.

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