Phylogenetic Position of Crenubiotus Within Macrobiotoidea (Eutardigrada) with Description of Crenubiotus Ruhesteini Sp

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

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 homonym 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://zoobank.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 (equipped with a micrometer) according to Kaczmarek and (Katoh et al., 2002) as implemented in the MAFFT online service Michalczyk (2017); the claws were measured only if they were in (Katoh et al., 2019) and checked by visual inspection. For cox1 se- perfect lateral view. Only few specimens were suitable to be mea- quences, chromatograms were checked for presence of ambiguous sured due to their orientation on the slide. Figures were prepared bases, as sequences were translated to amino acids by using the using Adobe Photoshop 6.0 and the Picolay free software (www. invertebrate mitochondrial code implemented in MEGA7 (Kumar picolay.de) to merge pictures of different focuses. et al., 2016) to check for the presence of stop codons and therefore of pseudogenes. Sequences of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada Richters, 1926, Parachela Schuster et al., 2.2 | Phylogenetic analyses and genotyping 1980, Hypsibioidea Pilato, 1969; GenBank A.N.: MG800327 for 18S; MH079506 for 28S; MG800336 for ITS2; MG818724 for cox1) were Before molecular analysis, individuals were observed and identified used as outgroup, while other tardigrade sequences from GenBank with LM using the method in Cesari et al. (2011) to obtain pictures were also included in the analysis for comparisons (a total of 121 of the specimen (voucher specimens). Genomic DNA was extracted individuals and 232 sequences [121 for 18S; 23 for 28S; 32 for ITS2; from six single animals, four of the sample C4330(111), and two of 56 for cox1]; Table S2). Pairwise nucleotide sequence divergences the sample C4367(58) (Table 1). The extractions were performed between sequences were calculated using p-distance with MEGA7 with QuickExtract™ DNA Extraction Solution (Lucigen), following for each gene. Two different phylogenetic molecular analyses were the manufacturer's protocol. The investigations of molecular genetic inferred: one on a concatenated 1,778 bp dataset, comprising only markers were carried out using fragments of three nuclear genes 18S and 28S genes (849 bp for 18S; 929 bp for 28S) (Alignment S1), (small subunit 18S rRNA: 18S, large subunit 28S rRNA: 28S, internal and another using a concatenated 3,177 bp dataset (849 bp for 18S; transcribed spacer 2: ITS2) and a mitochondrial gene (cytochrome c 929 bp for 28S; 602 for ITS2; 797 for cox1). A Bayesian inference oxidase subunit 1: cox1). The 18S and 28S genes were amplified using (BI) dendrogram for both datasets were computed with the program the primers and PCR protocols described in Bertolani et al. (2014) MrBayes (Ronquist et al., 2012) version 3.2.7 on the CIPRES Science (18S, Forward: SSUF 5′-GCT TGT CTC AAA GAT TAA GCC-3′, Gateway Portal (http://www.phylo.org/sub_sections/portal/). Reverse: SSUR 5′-CAT TCT TGG CAA ATG CTT TCG-3′; amplicon Best fitting model evaluations were performed considering Akaike length: 873 bp) and Guidetti et al., (2014) (28S, Forward: 28S 4.8aF Information Criterion and Bayes Information Criterion (jModelt- 5′-ACC TAT TCT CAA ACT TTA AAT GG-3′, Reverse: 28S 7bR 5′-GAC est 2.1.7; Darriba et al., 2012), which identified the GTR + Gamma TTC CCT TAC CTA CAT-3′; amplicon length: 919 bp), respectively, model as the most suitable. Two independent runs, each of four while the ITS2 was amplified utilizing primers and PCR protocols de- Metropolis-coupled Markov chains Monte Carlo method, were scribed in Wełnicz et al. (2011) (ITS2, Forward: ITS3 5′-GCA TCG launched for 5 × 107 generations, and trees were sampled every ATG AAG AAC GCA G-3′, Reverse: ITS4 5′-AGT TTY TTT TCC TCC 1,000 generations. Convergence of runs was assessed by tracking GCT TA-3′; amplicon length; 526 bp). The cox1 gene was amplified the average standard deviation of split frequencies between runs using primers and PCR protocols described in Cesari et al. (2009) and by plotting the log likelihood of sampled trees in Tracer v1.5 (cox1, Forward: LCO 5′-GGT CAA CAA ATC ATA AAG ATA TTG (Rambaut & Drummond, 2007), and the first 5 × 106 sampled gener- G-3′, Reverse: HCOoutout 5′-CCT GGT AAA ATR AGA ATA TAR-3′; ations were discarded as burn-in. A maximum likelihood (ML) analy- amplicon length: 830 bp). The amplified products were gel purified sis was performed for both datasets with the program RAxML v7.2.4 using the Wizard Gel and PCR cleaning (Promega) kit. Sequencing (Stamatakis, 2006) on the CIPRES Science Gateway Portal using the reactions were performed using the ABI Prism Big Dye Terminator GTR + Gamma model. Bootstrap resampling with 1,000 replicates v. 1.1 Sequencing Kit (Applied Biosystems™) on purified amplicons. was undertaken via the rapid bootstrap procedure of Stamatakis Each sequencing reaction contained 0.2 μM of a single PCR primer et al. (2008) to assign support to branches in the ML tree. to initiate the sequencing reaction, 2 μl of BigDye, 70 ng of purified products, 4 μl of 5× BigDye Terminator v.1.1 Sequencing Buffer and

H2O for a final volume of 20 μl. Cycling conditions for sequencing 3 | RESULTS reactions consisted of 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Both strands were sequenced with ABI Prism 3100 3.1 | Taxonomic account (Applied Biosystems™). Nucleotide sequences of the newly analyzed specimens were submitted to GenBank, accession numbers: Table 1. Crenubiotus ruhesteini sp. nov Before including all genes in the phylogenetic analyses, a sequence saturation test was assessed on all markers by using the in- ZooBank registration formation entropy-based index of substitution saturation (Xia et al., LSID: urn:lsid:zoobank.org:act:2AF3E506-C649-433F-A790- 2003), as implemented in DAMBE 7.2.138 (Xia, 2018). All genes C0E31FCFF23B. GUIDETTI et al. | 579

(a) Holotype Slide 4330s1a; five paratypes: slides 4330s1, s2, s3 (four animals), slide 4330s4 (an egg), one egg and one animal on stub for SEM analysis.

Other material Table S1.

Etymology The species name refers to the region Ruhestein, the place where (b) the species was first discovered. The Ruhestein region is a pass be- (c) tween the Murg Valley and the Acher Valley in the northern Black Forest, Germany.

Description (morphometric data in Tables S3 and S4) (d) The body is whitish. The eye spots are present in live animals (Figure 1a) and in several specimens mounted on slides, but they are not visible (probably dissolved) in most mounted animals. The entire cuticle surface is scattered with small elliptical (about 0.6–0.8 µm in diameter) and large quadrangular (about 1.0–2.5 µm in minor side) pores (Figures 2c–g, 3a–c,e and 4a–d); the larger pores have swollen (e) edges visible with SEM (Figure 2g). Small cuticular tubercles (0.5– 0.9 µm in diameter), covered with very small cones (visible only with SEM; Figure 3c), are present on a narrow posterior band positioned before the hind legs, on a wide dorsal area of the hind legs (Figures 2e, 3e and 4a,b), and on the external and internal distal portions of (f) the first three pairs of legs (Figures 3a,b and 4c,d). A large quadrangular pore lies within the tubercular area of the external surface of each leg on the first three pairs (Figures 3a,b and 4c,d). There are also very small single dots (visible only with SEM) distributed almost regularly on the entire cuticle surface (Figures 2f,g and 3c). (g) The mouth is sub-ventral and surrounded by 10 peribuccal lamel- lae. The rigid buccal tube is relatively narrow and ends within the pharynx with a thick circular margin. The anterior part of the tube is obviously bent in concurrence with the ventral lamina, which has a longitudinal apophysis at its anterior portion (Figure 1). The anterior dorsal apophysis of the buccal tube is reduced to a small thickening (Figure 1e). The stylet supports are long and inserted on the buccal tube at 71.1%–76.5% of its length. The stylet furcae have thickened, swollen condyles, which are developed and elongated laterally. With LM, the buccal armature is without bands of teeth, but presents only two long dorsal and ventral transverse crests (Figure 1c,d). The dor- FIGURE 1 Crenubiotus ruhesteini. sp. nov. (a–f) and Crenubiotus sal crests have a thickening medially (forming a mucrone) and are crenulatus (g). Feeding apparatus (Light Microscopy). (a) Buccal- pharyngeal apparatus in vivo (arrow = eye spots). (b) Macroplacoids larger than the ventral ones (Figure 1d). Within the pharynx, there in the pharynx. (c) Ventral transversal crests of the buccal are large apophyses (triangular in lateral view, bilobed in frontal armature (arrow). (d) Buccal-pharyngeal apparatus (arrow = dorsal view), two macroplacoids (the first is the longest) and a small micro- transverse crests of the buccal armature). (e) Anterior portion placoid (Figure 1). In lateral view, the macroplacoids are rod-shaped; of the buccal tube in lateral view (arrowhead = longitudinal in frontal view, the first has a central constriction, which divides apophysis of the ventral lamina; arrow = dorsal apophysis of the buccal tube). (f) Buccal-pharyngeal apparatus in lateral view the anterior heart-shaped portion from the oval posterior portion, (arrowhead = longitudinal apophysis of the ventral lamina). (g) and the second is quadrangular with a sub-terminal constriction Buccal-pharyngeal apparatus in lateral view of C. crenulatus (Figure 1b). (arrowhead = longitudinal apophysis of the ventral lamina). (a, b) The claws are Y-shaped and slender with a long basal tract DIC; (c–g), PhC. (a) sample 4330; (b) sample Probe 11; (c, d) sample and long common portion (see Lisi et al., 2020 for a more detailed 19; (e, f) sample Probe 14. Scale bars: (a–g) = 10 µm 580 | GUIDETTI et al.

(a) (a)

(b) (b)

(f) (e)

(e) (g)

FIGURE 2 Crenubiotus ruhesteini. sp. nov. Cuticle (Scanning Electron Microscopy). (a) In toto (latero-ventral view). (b) In toto FIGURE 3 Crenubiotus ruhesteini. sp. nov. Claws and cuticle (dorsal view). (c) Posterior dorsal portion of the cuticle. (d) Dorsal (Scanning Electron Microscopy). (a) Second and third pairs of legs cuticle (arrow = round pore; arrowhead = quadrangular pore). (e) (arrowhead = large pore on leg). (b) Third leg (arrowhead = large Most posterior dorsal portion of the cuticle (arrow = posterior band pore on leg). (c) Tubercles of the cuticle covered with very small of tubercles; arrowhead = band of tubercles on the hind legs). (f, g) cone-like structures (arrow). (d) Claws of the second pair of leg. Quadrangular cuticular pores (arrow = edge of the quadrangular (e) Claws of the hind leg (arrow = posterior band of tubercles; pore; arrowhead = small dot). (a–g) sample 4367. Scale bars: a, arrowhead = tubercles on hind legs). (a–e) sample 4367. Scale bars: b = 25 µm; c–e = 5 µm; f, g = 1.5 µm a = 10 µm; b, d, e = 4 µm; c = 2 µm description), the main branches have evident accessory points, and is smooth (Figure 5c), while the internal has circular irregular holes the lunula is very wide with 10–14 long thin teeth (Figures 3d,e (Figure 5d), and the tips of the processes show small dots on their and 4d). surface (Figure 5c). The surface of the processes appears reticulated The eggs are laid freely (Figure 5) and have processes in shape with very fine meshes with LM. The surface between the processes of an inverted funnel with thin distal tips that can be bifurcated seems smooth with LM (Figure 5e,f). However, it is not clearly visible (Figure 5b–d,h). The process is from 8.1 to 8.9 µm in height and from with SEM (Figure 5c,d). There are 16–18 processes on the circum- 6.2 to 9.2 µm in diameter (Table S4). The proximal margin of the ference and about 46–49 per hemisphere. Four eggs were found, process is regular with very small indentations mainly visible with one with an embryo at the last developmental stage in which the SEM (Figure 5c,d). With SEM, the external surface of the processes claws and buccal-pharyngeal apparatus were visible (Figure 5g). GUIDETTI et al. | 581

(a) FIGURE 4 Crenubiotus ruhesteini. sp. nov. Claws and cuticle (Light Microscopy). (a, b) Posterior cuticle in lateral view (asterisk = posterior band of tubercles; circle = band of tubercles on the hind legs; arrows = small and large pores). (c) Second and third pair of legs (arrowhead = large pore on leg; arrow = tubercles on the internal side of the leg). (d) Second leg (arrowhead = external tubercles; arrow = internal tubercles). (a) DIC; (b–d) PhC. (a) sample Probe 6; (b) sample 4330; (c) sample Probe 14; (d) sample 6. Scale bars: a–d = 10 µm

Type locality Sample C4330(111) moss growing on an exposed tree root on the soil surface, Schliffkopf, Black Forest (Germany), N 48° 32' 20.34, E 8° 13' 9.84. The species was found in many other localities of the Black Forest (Table S1). (b)

Type repositories The holotype (slide 4330s1a), 90 paratypes, and three eggs are deposited in the Bertolani collection at the Department of Life Sciences, University of Modena and Reggio Emilia, Italy, 25 paratypes and one egg are deposited in the Museum of Natural History of Verona, Italy.

Differential diagnosis The new species Crenubiotus ruhesteini sp. nov. differs from the only two species of the genus by the following features:

(c) • From C. crenulatus (Figure 1g) by: the presence of large quadrangular pores, the smaller size of the larger elliptical pores (<2.5 µm in C. ruhesteini sp. nov., >2.9 µm in C. crenulatus), the narrower buccal tube (pt 13.2–14.4 in C. crenulatus, 9.2–12.1 in C. ruhesteini sp. nov.), the shorter first macroplacoids (pt 25.6– 26.5 in C. crenulatus, 17.7–23.0 in C. ruhesteini sp. nov.), the shorter claws of the fourth pair of legs (pt 25.3–26.1 in C. crenulatus, 22.6–25.9 in C. ruhesteini sp. nov.), the more evident cuticular tubercles on the external and internal sides of the first three pairs of legs. • From C. revelator by: the presence of large quadrangular pores, the larger size of the larger elliptical pores (<1.9 µm in C. revelator, >2.0 µm in C. ruhesteini sp. nov.), the lack of a medioventral mucrone in the buccal armature, the more evident cuticular tubercles on the external and internal sides of the first three pairs of legs. (d)

3.2 | Molecular characterization and phylogenetic analyses

The analyses of the DNA sequences of C. ruhesteini sp. nov. were based on sequences from six specimens for the 18S, 28S, and ITS2 genes, and on sequences from three specimens for the cox1 gene. All analyzed specimens had the same haplotype for each analyzed gene, except the cox1 gene, for which two haplotypes were found. The p-distance on cox1 sequences analysis showed low values 582 | GUIDETTI et al.

(a) (e)

(b) (f)

(c)

(g)

(d) (h)

FIGURE 5 Crenubiotus ruhesteini. sp. nov. Eggs (Scanning Electron Microscopy [SEM], Light Microscopy). (a) In toto. (b–d) Egg processes (arrow = internal side of a process; arrowhead = bifurcation of a process). (e, f) In toto (two sides of the same egg). (g) Egg with an embryo in the last developmental stage (arrow = claws; arrowhead = cuticular pore). (h) Egg processes. (a–d), SEM; (e-h), PhC. (a, c, g) sample 4330; (b, d) sample 4368; (e, f, h) sample 4327. Scale bars: a = 20 µm; b, e–h = 10 µm; c, d = 5 µm

(0.0%–0.3%; Table S5) among the individuals of C. ruhesteini sp. nov. Table S5). While, the p-distances of 18S and 28S sequences were The comparison with sequences belonging to other macrobiotoid low compared with the other Richtersiidae (0.9%–3.3%, 1.0%–2.0%, species shows high values of genetic p-distance for the ITS2 (27.8%– respectively; Tables S7 and S8). 44.9%; Table S6). High values of genetic distance were also found Phylogenetic molecular analyses of the Macrobiotoidea spe- in the comparison with Richtersiidae cox1 sequences (18.6%–22.1%; cies were computed by both Bayesian and ML inferences on two GUIDETTI et al. | 583

FIGURE 6 Phylogenetic molecular tree of the Macrobiotoidea superfamily (Bayesian and Maximum likelihood analyses) based on combined dataset (18S + 28S + ITS2 + cox1 sequences) under the FIGURE 7 Phylogenetic tree of the families Murrayidae and GTR + Gamma model. Numbers above the branches show posterior Richtersiusidae (Bayesian and Maximum likelihood analyses) based probability values, while those in italics under the branches show on combined dataset (18S + 28S + ITS2 + cox1 sequences) under the bootstrap values. Asterisks denote highly supported nodes (both GTR + Gamma model. Numbers above the branches show posterior posterior probability value = 1 and bootstrap value = 100). In probability values, while those in italics under the branches Table S2, the Gen Bank acc. n. of all the Macrobiotoidea species show bootstrap values. Asterisks denote highly supported nodes used in the phylogenetic analyses are reported. Scale bar = number (both posterior probability value = 1 and bootstrap value = 100). of substitutions per site. The area of tree delimited by the dotted Individuals newly analyzed for this paper are in bold. In Table S2, line is showed in detail in Figure 7 the GenBank acc. n. of all the Macrobiotoidea species used in the phylogenetic analyses are reported. Scale bar = number of substitutions per site different datasets: one comprising sequences of 18S, 28S, ITS2, and cox1 molecular markers and the other including 18S and 28S (((Minibiotus spp.)((Paramacrobiotus)(Minibiotus furcatus Ehrenberg, genes only (Figures 6 and 7, Figure S1). The phylogenetic tree ob- 1859, Tenuibiotus)))). The phylogenetic tree obtained with only the tained with all four molecular markers shows two main clades 18S + 28S genes shows a different topology with an unresolved node (Figures 6 and 7): the first groups the genera Mesobiotus, Xerobiotus, at the base of the tree, with a polytomy showing three clades, not and Macrobiotus Schultze, 1834; the second is not well supported well supported (Figure S1). The three clades show the following rela- and groups two clusters. One cluster is well supported and groups tionships: (a) ((Minibiotus intermedius (Minibiotus furcatus, Tenuibiotus the genera Diaforobiotus Guidetti et al. 2016, Richtersius Pilato & voronkovi (Tumanov, 2007), Paramacrobiotus spp.))(Mesobiotus spp. Binda, 1989, Crenubiotus, Adorybiotus Maucci & Ramazzotti, 1981, (Xerobiotus pseudhufelandi (Iharos, 1966), Macrobiotus polonicus Murrayon, and Dactylobiotus Schuster, 1980, with the following re- Pilato et al., 2003, Macrobiotus spp.))); (b) ((Murrayon, Dactylobiotus) lationships ((Diaforobiotus, Richtersius)((Crenubiotus, Adorybiotus) (Adorybiotus, Crenubiotus)); (c) (Diaforobiotus, Richtersius). With both (Murrayon Dactylobiotus))); the other is less supported and groups datasets (all four markers and 18S + 28S), the genera Minibiotus and Minibiotus, Tenuibiotus, and Paramacrobiotus Guidetti et al., 2009: Murrayon are not monophyletic, and the genus Xerobiotus is included 584 | GUIDETTI et al. within Macrobiotus. The family Macrobiotidae results supported, al- armature, two macroplacoids), and cuticle (i.e., with pores), and though with low support, only in the 18S + 28S dataset. on well-supported phylogenetic relationships based on molecular markers for Richtersius and Diaforobiotus. The phylogenetic position of Adorybiotus is unclear and, according to the present 4 | DISCUSSION study and Guidetti et al. (2016), needs to be clarified with further DNA sequences. This unclear phylogenetic relationship is The discovery of the new species C. ruhesteini sp. nov. and of C. rev- also evident for the family Macrobiotidae. In fact, the support for elator strongly indicate that within the genus Crenubiotus there Macrobiotidae and the position of Adorybiotus change consider- are several species not yet discovered (as pointed out also by Lisi ably according to the dataset utilized, that is, the more conserved et al., 2020), and that the widespread distribution of C. crenulatus dataset (18S + 28S; Figure S1) versus the more variable dataset (all (Kaczmarek et al., 2014, 2015, 2016; McInnes, 1994) represents four markers; Figures 6 and 7). the distribution of the genus instead of the species, as previously Lisi et al. (2020) placed the genus Crenubiotus within the fam- recorded for several other eutardigrade species: for example, ily Richtersiidae (based on the characters of the claws and feeding Macrobiotus hufelandi Schultze, 1834 (Bertolani & Rebecchi, 1993), apparatus) while waiting for confirmation based on the analyses of Hypsibius dujardini (Doyère, 1840) (Gąsiorek et al., 2018), molecular markers. In our analyses, Crenubiotus is the sister taxon Ramazzottius oberhaeuseri (Doyère, 1840) (Stec, Morek, et al., 2018), of Adorybiotus, supporting the hypothesis of Lisi et al. (2020) that and Paramacrobiotus richtersi (Murray, 1911) (Guidetti, Cesari, Crenubiotus belongs to Richtersiidae based on morphological data. et al., 2019). The redesciption of C. crenulatus based on specimens However, our study shows that Crenubiotus and Adorybiotus are more from the type locality is an important goal to resolve this situation. related to the Murrayidae genera (Murrayon and Dactylobiotus) than Furthermore, the phylogenetic results highlight that more than one to the other genera of Richtersiidae (Richtersius and Diaforobiotus). species is also present in the genera Diaforobiotus and Richtersius (as Although the statistical support for this relationship is quite high, reported also by Guidetti et al. (2016) and Stec et al. (2020)), which no evident morphological synapomorphies link the four genera currently are considered monospecific (Degma et al., 2020). Crenubiotus, Adorybiotus, Murrayon, and Dactylobiotus. Richtersius The surface of the cuticle of C. ruhesteini sp. nov. is characterized and Crenubiotus have the same claw structure according to Lisi et al. by three types of structures: the very small dots distributed quite (2020), further investigations of Adorybiotus and Diaforobiotus are uniformly on the surface (Figure 2g), the cuticular pores (Figure 2c), needed to confirm if they share a similar claw structure. Therefore, and the tubercles covered by very small conical structures (Figure 3c). before reconsidering the two families Richtersiidae and Murrayidae, The very small dots on the cuticle surface have previously been noted it is necessary to acquire more morphological and molecular informa- in Macrobiotus species (e.g., Stec, Krzywański, et al., 2018). They are tion from DNA sequences (e.g., analyzing more species and/or genes). probably present in several other species but overlooked, being only At present, the Richtersiidae comprises the genera Adorybiotus, visible with SEM. Their nature and function remain unknown, but so Crenubiotus, Diaforobiotus, and Richtersius. Due to the inclusion of far, they have been found only in Macrobiotoidea species. Crenubiotus in Richtersiidae and the synonymy of this family with Evident cuticular pores-like structures (“pearls”) are exclusively the family of nematodes Richtersiidae, we establish the new family present in Macrobiotoidea genera. The different sizes and shapes of Richtersiusidae fam. nov. (see section 4.1). the pores have been already used as characters for species discrimi- nation within Richtersiidae (Guidetti et al., 2016). The biological sig- nificance of cuticular pores is also unclear, but Guidetti et al. (2016) 4.1 | Taxonomic account hypothesized that they can play a role in cuticle permeability; the holes found within the pores (Figure 2f) support this idea. Eutardigrada Richters, 1926 In Crenubiotus species, the cuticular tubercles covered by small Parachela Schuster, Nelson, Grigarick & Christenberry, 1980 conical structures are present in positions that suggest they can in- Macrobiotoidea Thulin, 1928 crease the grip of the legs (and the animal) on the substrate, enhanc- Richtersiusidae fam. nov. ing locomotion. In fact, similar tubercles and/or small cones are also ZooBank registration LSID: urn:lsid:zoobank.org:act:4BC5EC33- present in very similar positions on other Macrobiotoidea species: 1705-4100-9947-CF85113F7566 for example, in the genera Minibiotus and Macrobiotus (Michalczyk Junior homonym: Richtersiidae Guidetti et al., 2016 is invalid because & Kaczmarek, 2003, 2006), Calcarobiotus Dastych, 1993 (Kaczmarek the junior homonym of Richtersiidae Kreis, 1929, a family of nematodes. et al., 2006), Paramacrobiotus (Kaczmarek & Michalczyk, 2009), and Richtersi- is the stem of the type genus of this family of tardigrades, Mesobiotus (Guidetti, Gneuß, et al., 2019). Richtersius Pilato & Binda, 1989, and Richtersia Steiner, 1916 is the type The family Richtersiidae (Macrobiotoidea) was erected by genus of the nematode family Richtersiidae, which has priority. Guidetti et al. (2016) for the genera Adorybiotus, Diaforobiotus, Etymology: In order to avoid the homonymy in fami- and Richtersius based on morphological characters related to ly-group names (Art. 29.6 of the International Code of Zoological claws (i.e., lunula with large teeth), feeding apparatus (i.e., thick- Nomenclature (ICZN, 1999)), the name Richtersiusidae should ness on the anterior buccal tube, no transverse crests in the buccal be used for the new family of tardigrades, in accordance with the GUIDETTI et al. | 585

Bertolani, R., & Rebecchi, L. (1993). A revision of the Macrobiotus Article 29.3.3 of the ICZN (1999), which states that it is possible to hufelandi group (Tardigrada, Macrobiotidae), with some form a family-group name by adding the suffix –idae to the entire observations on the taxonomic characters of eutardi- name of the type genus. grades. Zoologica Scripta, 22(2), 127–152. https://doi. Diagnosis: Double claws Y-shaped, with the two branches form- org/10.1111/j.1463-6409.1993.tb00347.x Binda, M. G. (1974). Tardigradi della Valtellina. Animalia, 1(113), 201–216. ing an evident common tract of variable length. Large teeth on all Binda, M. G. (1988). Ridescrizione di Macrobiotus echinogenitus Richters, lunules. Buccal tube with ventral lamina and a cuticular thickness on 1904 e sul valore di buona specie di Macrobiotus crenulatus Richters, the anterior portion of the dorsal wall of the buccal tube (which can 1904 (Eutardigrada). Animalia, 15, 201–210. form a large apophysis). Two macroplacoids in the pharynx. Cuticular Cesari, M., Bertolani, R., Rebecchi, L., & Guidetti, R. (2009). DNA barcoding in Tardigrada: The first case study on Macrobiotus macro- pores (at least in one phase of the life cycle). Eggs laid freely with calix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). processes on their surface. Molecular Ecology Resources, 9, 699–706. https://doi. Type genus: Richtersius. org/10.1111/j.1755-0998.2009.02538.x Composition: Richtersius, Diaforobiotus, Crenubiotus, Adorybiotus. Cesari, M., Giovannini, I., Bertolani, R., & Rebecchi, L. (2011). An example of problems associated with DNA barcoding in tardigrades: A novel Remarks: The phylogenetic relationship of Crenubiotus and method for obtaining voucher specimens. Zootaxa, 3104, 42–51. Adorybiotus with the other genera of the family needs to be con- https://doi.org/10.11646/zoota xa.3104.1.3 firmed by further studies (see also Guidetti et al., 2016). Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: More models, new heuristics and parallel computing. Nature Methods, 9, 772. https://doi.org/10.1038/nmeth.2109 ACKNOWLEDGEMENTS Degma, P., Bertolani, R., & Guidetti, R. (2020). Actual checklist of The authors thank all persons who supported the inventories and Tardigrada species. Retrived from http://www.evozoo.unimore.it/ monitoring of tardigrades in the Black Forest National Park, Prof. site/home/tardigrade -tools/ artic olo10 80044 528.html. https://doi. Oscar Lisi for providing us information about C. crenulatus and C. rev- org/10.25431/11380_1178608. Accessed July 9, 2020. Faurby, S., Jørgensen, A., Kristensen, R. M., & Funch, P. (2011). elator, Prof. Diane Nelson for the English revision of the manuscript, Phylogeography of North Atlantic intertidal tardigrades: and the two anonymous referees and the Editors for their sug- Refugia, cryptic speciation and the history of the Mid-Atlantic gestions. We would like to thank Dr. Alexey Chernyshev (National Islands. Journal of Biogeography, 38(8), 1613–1624. https://doi. Scientific Center of Marine Biology, Vladivostok, Russia), and Dr. org/10.1111/j.1365-2699.2011.02533.x Louise Allcock (National University of Ireland Galway) for their in- Gąsiorek, P., & Michalczyk, Ł. (2020). Phylogeny of Itaquasconinae in the light of the evolution of the flexible pharyngeal tube in Tardigrada. formation and advice on the synonymy of the family Richtersiidae. Zoologica Scripta, 49, 499–515. https://doi.org/10.1111/zsc.12424 Gąsiorek, P., Morek, W., Stec, D., & Michalczyk, Ł. (2019). Untangling AUTHOR CONTRIBUTIONS the Echiniscus Gordian knot: Paraphyly of the “arctomys group” RG, ROS, and LR conceived the study, supervised the laboratory (Heterotardigrada: Echiniscidae). Cladistics, 35, 633–653, https:// doi.org/10.1111/cla.12377 work. MC performed the molecular analyses. RG, IG, and SEG per- Gąsiorek, P., Stec, D., Morek, W., & Michalczyk, Ł. (2018). An integrative formed the morphological and taxonomic analyses. EM collected redescription of Hypsibius dujardini (Doyère, 1840), the nominal the morphometric data and prepared the images. CE and MIF col- taxon for Hypsibioidea (Tardigrada: Eutardigrada). Zootaxa, 4415(1), lected the samples. ROS and LR financed the research. All authors 45–75. https://doi.org/10.11646/zoota xa.4415.1.2 Gąsiorek, P., Stec, D., Morek, W., & Michalczyk, Ł. (2019). Deceptive discussed results and contributed to a revision of the manuscript. All conservatism of claws: Distinct phyletic lineages concealed within authors read and approved the final manuscript. Isohypsibioidea (Eutardigrada) revealed by molecular and morphological evidence. Contributions to Zoology, 88(1), 78–132. https:// DATA AVAILABILITY STATEMENT doi.org/10.1163/18759866-20191350 Guidetti, R., & Bertolani, R. (2005). 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Xia, X., Xie, Z., Salemi, M., Chen, L., & Wang, Y. (2003). An index of sub- Table S4. Morphometric data for the eggs of Crenubiotus ruhesteini stitution saturation and its application. Molecular Phylogenetics and sp. nov. Evolution, 26(1), 1–7. https://doi.org/10.1016/S1055-7903(02)00326 -3 Table S5. Uncorrected p-distance of cox1 computed among Zawierucha, K., Stec, D., Lachowska-Cierlik, D., Takeuchi, N., Li, Z., & Michalczyk, Ł. (2018). High mitochondrial diversity in a new water Richtersiidae. bear species (Tardigrada: Eutardigrada) from mountain glaciers in cen- Table S6. Uncorrected p-distance of ITS2 computed among tral Asia, with the erection of a new genus Cryoconicus. Museum and Macrobiotoidea. Institute of Zoology, Polish Academy of Sciences, Annales Zoologici, 68(1), Table S7. Uncorrected p-distance of 18S computed among 179–202. https://doi.org/10.3161/00034541ANZ2018.68.1.007 Macrobiotoidea. Table S8. Uncorrected p-distance of 28S computed among SUPPORTING INFORMATION Macrobiotoidea. Additional supporting information may be found online in the Alignment S1. Alignment matrix 18S + 28S+ITS2 + cox1 of Supporting Information section. Macrobiotoidea.

Figure S1. Phylogenetic tree of the Macrobiotoidea superfamily (Bayesian and Maximum likelihood analyses) based on the combined How to cite this article: Guidetti R, Schill RO, Giovannini I, et al. When DNA sequence data and morphological results fit 18S + 28S dataset under the GTR + Gamma model. Table S1. Sampling sites within the Black Forest (Germany) in which together: Phylogenetic position of Crenubiotus within Crenubiotus ruhesteini sp. nov. was found. Macrobiotoidea (Eutardigrada) with description of Crenubiotus Table S2. GenBank accession numbers (acc. n.) of the sequences ruhesteini sp. nov. J Zool Syst Evol Res. 2021;59:576–587. used in this study and retrieved from GenBank, with the species https://doi.org/10.1111/jzs.12449 name reported in the NCBI database. Table S3. Morphometric data for the animals of Crenubiotus ruhesteini sp. nov.