A thorny bush: rich, but cryptic species diversity in the Australasian <i>Minibiotus aculeatus</i> complex (Eutardigrada: Macrobiotidae)

Abstract Species defined by striking morphological traits are often identified primarily through these characters, which can obscure subtler variation and lead to underestimation of true diversity and overestimation of geographical ranges of particular species. This issue is evident in the genus Minibiotus, whose species exhibit high morphological similarity and are characterized by long-standing taxonomic uncertainties. Minibiotus aculeatus, originally described by Murray in 1910 and later redescribed by Claxton in 1998, exemplifies this problem. For decades it was identified solely by its conspicuous dorsolateral cuticular spines, regarded as its primary diagnostic characteristic. We examined 19 populations fitting the scarce description of M. aculeatus: 5 from Australia and 14 from New Zealand. Morphological and morphometric traits were analysed using phase contrast and scanning electron microscopy, and molecular analyses were performed on four markers: three nuclear (18S rRNA, 28S rRNA, and ITS-2) and one mitochondrial (COI) DNA fragment. Phylogenetic relationships were reconstructed using Bayesian inference, and species boundaries were estimated with genetic and phenotypic data (both qualitative and quantitative). Our results suggest the presence of not one but ten species, constituting the Minibiotus aculeatus complex. However, the relationships between the species were not fully resolved, resulting in multiple lineages recovered in polytomy. Such a topology indicates that most of the diversification within the complex took place in a short period of time, in comparison to the subsequent evolution of the recovered lineages. Moreover, low levels of congruency between phenotypic and genetic variation of the analysed individuals suggest morphological stasis and/or a young age of the complex, which did not provide sufficient time for the evolution of divergent traits. The stasis and/or insufficient time for morphological differentiation resulted in apparent species crypsis, a phenomenon more and more often integratively documented in various tardigrade groups, which creates serious challenges for delineation and identification of species also in the M. aculeatus complex. Owing to the lack of eggs, we were unable to redescribe M. aculeatus and designate a neotype series and neotype DNA sequences, but we reinstate Minibiotus subjulietae stat. rev. as a valid species. Nevertheless, despite problems with phylogenetic resolution and species delineation, the geographical distribution of the M. aculeatus complex across south-eastern Australasia clearly suggests stenothermic tolerance and preferences for a temperate to cold climate for all species in the complex.