A big tree of a tiny bear: first extensive multilocus phylogeny of the genus Minibiotus (Eutardigrada: Macrobiotidae)

Abstract Tardigrade taxonomy has undergone substantial revisions over recent decades, largely driven by advances in molecular techniques. Integrative species descriptions and redescriptions have frequently led to reclassifications at higher taxonomic levels. However, phylogenetic relationships remain unresolved for many taxa, and several higher-level groups have been shown to be polyphyletic. One such case is the genus Minibiotus Schuster, 1980, currently comprising 56 species, whose internal relationships are unclear owing to incomplete morphological datasets and the pronounced lack of molecular data. To address these issues, we analysed a 100 populations of Minibiotus from across the globe, including 12 from South America, 5 from North America, 1 from Greenland, 5 from Europe, 13 from India, 1 from New Guinea, 30 from Australia, 16 from New Zealand, and 17 from South Africa. Three nuclear markers (18S rRNA, 28S rRNA, and ITS-2) and one mitochondrial marker (COI) were sequenced for selected representatives. A phylogenetic analysis based on the three nuclear markers was conducted using Bayesian inference, resulting in the first comprehensive phylogeny of the genus Minibiotus. In addition, a species delimitation analysis based on two markers, ITS-2 and COI, revealed extensive genetic divergence within the genus. These analyses uncovered previously unknown relationships among species and identified potential evolutionary lineages corresponding to distinct taxonomic units. Our results demonstrate that Minibiotus is paraphyletic, comprising two clearly distinct clades. This study highlights both the hidden and evident diversity within Minibiotus, providing new and significant insights into its evolutionary relationships and global genetic structure.