Contributions to Zoology, 85 (2) 173-200 (2016) Experimental taxonomy exposes ontogenetic variability and elucidates the taxonomic value of claw configuration inMilnesium Doyère, 1840 (Tardigrada: Eutardigrada: Apochela) Witold Morek1, Piotr Gąsiorek1, Daniel Stec1, Brian Blagden2, Łukasz Michalczyk1, 3 1 Department of Entomology, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland 2 Scottish Environment Protection Agency, Inverdee House, Baxter Street, Aberdeen, AB11 9QA, United Kingdom 3 E-mail: [email protected] Key words: barcoding, diagnostic key, development tracking, in vitro culture, M. berladnicorum, M. variefidum sp. nov. Abstract Discussion ...................................................................................... 183 Claw configuration variability in the genus In this paper we describe a new apochelan species, Milnesium Milnesium ................................................................................ 183 variefidum sp. nov. from Scotland and provide novel morpho- Diagnostic traits in Milnesium ........................................... 185 logical and molecular data for Milnesium berladnicorum Composition of the genus Milnesium and quality Ciobanu et al., 2014. The new species differs from the most of species descriptions .......................................................... 189 similar M. berladnicorum by the presence of developmental di- Acknowledgements ...................................................................... 190 morphism in claw configuration, absent or weakly developed References ...................................................................................... 191 cuticular bars under claws I-III, a different arrangement of cu- Appendix ........................................................................................ 194 ticular pseudoplates, and by differences in the sequences of three nuclear DNA fragments: 18S rRNA (p-distance: 0.6%), 28S rRNA (2.0%), ITS-2 (9.3%), and on mitochondrial gene COI (12.4%). Although ontogenetic claw configuration change was Introduction suspected to occur in some Milnesium species, we are the first to document it through the combined use of traditional, molecular Claw morphology in the class Eutardigrada is thought and experimental methodologies. We discuss the implications of to be conservative and therefore it has been used to re- the observed phenomenon for the taxonomy of the genus and organise eutardigrade taxonomy at the family level (Pi- propose a new diagnostic key to all Milnesium species described up to the end of 2015. We also review other traits used for spe- lato, 1969; Bertolani et al., 2014). In the family Milne- cies differentiation in the genus and offer recommendations to siidae, the only family in the order Apochela, the gen- improve the quality of future descriptions as well as suggest a eral claw morphology is also conserved and remains need for integrative redescriptions of the known species. Finally, unique. In contrast to the parachelan taxa all four apo- we propose to suppress M. dujiangensis and M. tardigradum trispinosum and suggest that M. alpigenum and M. quadrifidum chelan genera, Milnesium Doyère, 1840, Limmenius are valid species that require thorough redescriptions. Horning et al., 1978, Milnesioides Claxton, 1999, and Bergtrollus Dastych, 2011, exhibit the same claw anato- my: the primary branch is completely separated from Contents the secondary branch. The only observed variability of claw morphology within the Milnesiidae concerns the Introduction ................................................................................... 173 presence or absence of accessory points on the single- Material and methods .................................................................. 175 branched primary branches and the number of points on Sample processing ................................................................. 175 the secondary branches, which in the currently known Microscopy and imaging ...................................................... 175 Morphometrics ....................................................................... 175 species varies from two to four. The only exception is Genotyping .............................................................................. 176 Milnesium dujiangensis Yang, 2003, a species that al- In vitro culture ........................................................................ 177 legedly lacks the primary branches and in which the Literature review and comparative material .................. 178 secondary branch of anterior claws IV is equipped only Results ............................................................................................. 180 with a single point. However, given such exceptional Taxonomic account of the new species ............................. 180 Phenotypic differential diagnosis ...................................... 182 characters and the very poor quality of the original de- Genotypic differential diagnosis ........................................ 183 scription, this species is commonly considered dubious. Downloaded from Brill.com09/23/2021 12:21:23PM via free access 174 Morek et al. – Claw configuration inMilnesium In the 19th and early 20th century, when not only Mil- species with different claw configurations, Michalczyk nesium taxonomy, but tardigrade taxonomy in general et al. (2012a, b) introduced a denotation system in was in its infancy, researchers recognised variability which the number of points on the secondary branch- in the number of points on the secondary branches in es on all legs is given as a short string of numbers, e.g. Milnesium and considered it to be a diagnostic trait at [2-3]-[3-2] for M. tardigradum (see Material and the species level. As a result, following the description methods for details). of the nominal Milnesium tardigradum Doyère, 1840, The great majority of the thirty Milnesium descrip- two further species were described solely on the basis tions published until the end of 2015 report single claw of different claw configurations:Milnesium alpigenum configurations within type populations. However, Ehrenberg, 1853 (with three points on all claws) and there are two intriguing exceptions, M. tetralamella- Milnesium quadrifidum Nederström, 1919 (with four tum Pilato and Binda, 1991 and M. barbadosense points on all claws). However, soon after, these species Meyer and Hinton, 2012, in which more than one claw were suppressed by Marcus (1928) who reconsidered configuration was described. In the type population of the taxonomic value of the number of points on the the first species Pilato and Binda (1991) identified two secondary branches and concluded that these varia- groups of specimens, one with a [2-2]-[2-2] and the tions only represent different ‘geographic races’ of M. other with a [2-3]-[2-3] claw configuration. Similarly, tardigradum. This view was supported by Ramazzotti Meyer and Hinton (2012) reported a [2-3]-[2-3] and a (1962) and by Ramazzotti and Maucci (1983) and only [3-3]-[3-3] configuration for the type population of M. very recently, at the XII International Symposium on barbadosense. Meyer and Hinton (2012) also noted Tardigrada (Portugal, July 2012), was the validity of that the specimens with the lower number of spurs the two species brought back to the debate by Marley were generally smaller than those with three spurs on (2012). However, given the poor original descriptions, all claws. These two exceptions provoke the question they are still awaiting a proper restoration of their tax- as to whether they are examples of intra- or inter-spe- onomic status. The opinion that claw configuration is cific variability. On one hand, claw morphology change unstable and therefore not useful as a taxonomic trait during development is known to occur in Milnesium in Milnesium was also supported by Dastych (1984) males, in which immature individuals have unmodi- who found various developmental aberrations in Mil- fied claws whereas in sexually mature males the sec- nesium specimens from the Antarctic. This view, com- ondary claws on the first pair of legs develop into ro- bined with the concept that tardigrades are cosmo- bust hooks (Marcus, 1928; Rebecchi and Nelson, 1998; politan, led to a severe underestimation of the number Ciobanu et al., 2015). On the other hand, it is not unu- of Milnesium species worldwide (Michalczyk et al., sual for multiple congeneric taxa to inhabit a single 2012a, b). microhabitat (e.g. Kaczmarek et al., 2011), therefore in Nevertheless, variation in other traits, such as cu- such a case different claw configurations could simply ticular sculpture and feeding apparatus morphology, represent different species, erroneously classified as a was appreciated by some researches and the genus single species (according to a personal communication ceased to be monospecific again as new Milnesium from Giovanni Pilato, in the case of M. tetralamella- species started to appear in the literature in the early tum specimens with a [2-2]-[2-2] configuration may 1990s (Binda and Pilato, 1990; Maucci, 1991). These represent a different species as they exhibit cuticular species, as well as further new species described in sculpturing compared to smooth [2-3]-[2-3] speci- the following decade, seemed to exhibit stable and mens). In principle, this conundrum could be solved by distinct claw configurations. Michalczyket al. (2012a, following the development of individuals animals and/ b) attempted to revise the taxonomic