Received: 3 August 2020 | Revised: 2 December 2020 | Accepted: 4 December 2020 DOI: 10.1111/jzs.12449 ORIGINAL ARTICLE When DNA sequence data and morphological results fit together: Phylogenetic position of Crenubiotus within Macrobiotoidea (Eutardigrada) with description of Crenubiotus ruhesteini sp. nov Roberto Guidetti1 | Ralph O. Schill2 | Ilaria Giovannini1 | Edoardo Massa1 | Sara Elena Goldoni1 | Charly Ebel3 | Marc I. Förschler3 | Lorena Rebecchi1 | Michele Cesari1 1Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Abstract Italy The integration of morphological data and data from molecular genetic markers is 2Institute of Biomaterials and Biomolecular Systems, University of important for examining the taxonomy of meiofaunal animals, especially for eutardi- Stuttgart, Stuttgart, Germany grades, which have a reduced number of morphological characters. This integrative 3 Department of Ecosystem Monitoring, approach has been used more frequently, but several tardigrade taxa lack molecular Research and Conservation, Black Forest National Park, Freudenstadt, Germany confirmation. Here, we describe Crenubiotus ruhesteini sp. nov. from the Black Forest (Germany) integratively, with light and electron microscopy and with sequences of Correspondence Lorena Rebecchi, Department of Life four molecular markers (18S, 28S, ITS2, cox1 genes). Molecular genetic markers were Sciences, University of Modena and also used to confirm the recently described Crenubiotus genus and to establish its Reggio Emilia, via G. Campi 213/D, 41125 Modena, Italy. phylogenetic position within the Macrobiotoidea (Eutardigrada). The erection of Email: [email protected] Crenubiotus and its place in the family Richtersiidae are confirmed. Richtersiidae is redescribed as Richtersiusidae fam. nov. because its former name was a junior homo- nym of a nematode family. KEYWORDS integrative taxonomy, phylogeny, Richtersiidae, Richtersiusidae, Tardigrada 1 | INTRODUCTION Sarascon Guil et al., 2014), while most of them were found by acquir- ing new information from molecular and/or morphological data of In the last 10 years, 39 new genera of tardigrades have been de- species or taxa previously described. An integrated approach based scribed (Degma et al., 2020). Twenty-two of them belong to the on multiple sources of information (e.g., morphological, ultrastruc- class Eutardigrada, which comprises species with a reduced num- tural, molecular, karyological; Bertolani et al., 2014; Gąsiorek, Morek, ber of characters useful for taxonomy and systematics (Guidetti & et al., 2019; Gąsiorek, Stec, et al., 2019; Guidetti et al., 2005; Guidetti Bertolani, 2005). Some of these eutardigrade genera were discov- et al., 2009; Guidetti et al., 2016; Sands et al., 2008; Vicente et al., ered in entirely new phylogenetic lines (i.e., Austeruseus Trygvadóttir 2013; Zawierucha et al., 2018) is more useful for investigating the tax- & Kristensen, 2011, Bergtrollus Dastych, 2011, Bindius Pilato, 2009, onomy and phylogeny of tardigrades, mainly due to the presence of Cryoconicus Zawierucha et al., 2018, Meplitumen Lisi et al., 2019, and cryptic species (e.g., Guidetti et al., 2009; Guidetti et al., 2016; Faurby Contributing author: Roberto Guidetti ([email protected]), Ralph O. Schill ([email protected]), Ilaria Giovannini ([email protected]), Edoardo Massa ([email protected]), Sara Elena Goldoni ([email protected]), Charly Ebel ([email protected]), Marc I. Förschler ([email protected]), Michele Cesari ([email protected]) ZooBank Link: LSID: http://zooba nk.org/urn:lsid:zoobank.org:pub:99D2731E-A6D5-40FC-9180-CBA6B5D8A810 576 | © 2021 Wiley-VCH GmbH wileyonlinelibrary.com/journal/jzs J Zool Syst Evol Res. 2021;59:576–587. GUIDETTI et AL. | 577 et al., 2011; Morek et al., 2019) and homoplasies (Guil et al., 2013). of C. crenulatus is based on the description of M. dentatus and on the Nevertheless, there are several eutardigrade genera described with remarks reported by Binda (1988) and Lisi et al. (2020). only morphological support (e.g., Austeruseus, Bergtrollus, Bindius, The discovery of a new species of the genus Crenubiotus in the Crenubiotus Lisi et al., 2020, Dastychius Pilato, 2013, Meplitumen, Black Forest (Germany) allowed us to describe this new taxon and Sarascon, Tenuibiotus Pilato & Lisi, 2011, Thalerius Dastych, 2009, to obtain DNA sequences to confirm the validity and systematic Vladimirobius Kaczmarek et al., 2020, and Weglarskobius Kaczmarek position of the genus and to establish its phylogenetic relationships et al., 2020) and other genera that do not find a molecular phylo- within the Macrobiotoidea superfamily. genetic support, resulting para- or polyphyletic (e.g., Adropion Pilato, 1987 (see Gąsiorek & Michalczyk, 2020), Borealibius Pilato et al. 2006, Doryphoribius Pilato, 1969, Minibiotus Schuster, 1980, Murrayon 2 | MATERIAL AND METHODS Bertolani & Pilato, 1988, and Xerobiotus Bertolani & Biserov, 1996 (see Bertolani et al., 2014)). Therefore, the acquisition of information 2.1 | Tardigrade sampling and morphological from DNA sequences about these taxa is important to support their analyses validity and phylogenetic position. The genus Crenubiotus has been recently erected (Lisi et al., 2020) A total of 133 animals (and four eggs) of the new species were ex- based on the morphology of the claws and feeding apparatus of the tracted from 39 samples of mosses collected in October 2016 by R. species Crenubiotus crenulatus (Richters, 1904) and Crenubiotus reve- O. Schill in the Black Forest (Schwarzwald, Germany; Table S1). These lator Lisi et al., 2020. The erection of Crenubiotus and its insertion specimens were morphologically analyzed with Light Microscopy in the Richtersiidae family (Macrobiotoidea Thulin, 1928) lacked (LM; 117 animals + two eggs) and Scanning Electron Microscopy confirmation from the analyses of molecular markers. Further issues (SEM; 10 animals + 2 eggs) and studied with molecular genetic meth- with this new genus are related to the troubled taxonomic history ods (six animals). of its type species: C. crenulatus. Crenubiotus crenulatus was de- To extract tardigrades from their substrates, fragments of all scribed in 1904 by Richters as Macrobiotus crenulatus from material samples were placed in distilled water for about half an hour. After collected in the Svalbard Islands, then it was synonymized in 1911 soaking, the samples were sieved (sieves meshes: 500 and 38 μm) to with Macrobiotus echinogenitus Richters, 1903 by the same author separate tardigrades and eggs from the substrate; animals and eggs (Richters, 1911). After 77 years, it was still considered a valid species were then isolated using a needle and a glass pipette under a stereo- by Binda (1988), and then transferred to the new genus Crenubiotus microscope. Specimens were mounted on slides in Hoyer's medium by Lisi et al. (2020) (for more details see Binda, 1988; Lisi et al., 2020; for observations with LM. Additional specimens from sample C4367 Richters, 1911). In the meantime, Macrobiotus dentatus Binda, 1974 were prepared for SEM observations by fixing them in boiling abso- was described from specimens in a moss collected in the Italian Alps lute ethanol for a few minutes. Then, they were rinsed three times in (Binda, 1974). Subsequently, Binda (1988) synonymized M. dentatus absolute ethanol, desiccated by evaporation, mounted on stubs, and with C. crenulatus, identifying the former holotype of M. dentatus as sputter coated with a thin layer of gold (Guidetti, Massa, et al., 2019). the neotype for C. crenulatus. This synonymy (C. crenulatus–M. denta- Observations with SEM were carried out with a Nova Nano SEM tus) was based not on the observation of the type material of C. cren- 450 (FEI company), available at the “Centro Interdipartimentale ulatus (from Svalbard Islands, which has been lost, according to Lisi Grandi Strumenti” at the University of Modena and Reggio Emilia et al., 2020), but of specimens identified as M. crenulatus and depos- (UNIMORE). ited at the National Museum of Scotland (Binda, 1988). According to Observations with LM and measurements were carried out Morgan (1977), these specimens were collected by James Murray in under both phase contrast (PhC) and differential interference con- the Shetland Islands. Moreover, the description of C. crenulatus by trast (DIC) up to the maximum magnification (100× oil objective) Richters (1904) is quite concise and lacks several characters used in with a Leica DM RB microscope equipped with a Nikon DS-Fi 1 the current tardigrade taxonomy. Therefore, the current description digital camera, at the Department of Life Sciences, UNIMORE. The TABLE 1 Analyzed specimens, GenBank accession numbers (GenBank#), and type of voucher specimens GenBank# Specimen 18S 28S cox1 ITS2 Voucher C4330(111) T1 MW074384 MW074390 MW074336 MW074367 paragenophore C4330(111) T2 MW074385 MW074391 MW074337 MW074368 paragenophore C4330(111) T3 MW074386 MW074392 MW074369 paragenophore C4330(111) V6 MW074387 MW074393 MW074338 MW074370 hologenophore C4367(58) V1 MW074388 MW074394 MW074371 hologenophore C4367(58) V3 MW074389 MW074395 MW074372 hologenophore 578 | GUIDETTI et AL. measurements of the lengths of the animals and their cuticular parts were found with little saturation. Thus, the 18S, 28S, ITS2, and (i.e., claws, feeding apparatus) were done with a Leitz dialux 20 mi- cox1 nucleotide sequences were aligned with the MAFFT algorithm croscope