Deceptive conservatism of claws: distinct phyletic lineages concealed within Isohypsibioidea (Eutardigrada) revealed by molecular and morphological evidence

Isohypsibioidea are most likely the most basally branching evolutionary lineage of eutardigrades. Despite being second largest eutardigrade order, phylogenetic relationships and systematics within this group remain largely unresolved. Broad taxon sampling, especially within one of the most speciose tardigrade genera, Isohypsibius
Thulin, 1928, and application of both comparative morphological methods (light contrast and scanning electron microscopy imaging of external morphology and buccal apparatuses) and phylogenetic framework (18S + 28S rRNA sequences) resulted in the most comprehensive study devoted to this order so far. Two new families are erected from the currently recognised family Isohypsibiidae: Doryphoribiidae fam. nov., comprising all aquatic isohypsibioids and some terrestrial isohypsibioid taxa equipped with the ventral lamina; and Halobiotidae fam. nov., secondarily marine eutardigrades with unique adaptations to sea environment. We also split Isohypsibius into four genera to accommodate phylogenetic, morphological and ecological variation within the genus: terrestrial Isohypsibius s.s. (Isohypsibiidae), with smooth or sculptured cuticle but without gibbosities; terrestrial Dianea gen. nov. (Isohypsibiidae), with small and pointy gibbosities; terrestrial Ursulinius gen. nov. (Isohypsibiidae), with large and rounded gibbosities; and aquatic Grevenius gen. nov. (Doryphoribiidae fam. nov.), typically with rough cuticle and claws with branches of very similar heigths. Claw morphology is reviewed and, for the first time, shown to encompass a number of morphotypes that correlate with clades recovered in the molecular analysis. The anatomy of pharynx and cuticle are also shown to be of high value in distinguishing supraspecific taxa in Isohypsibioidea. Taxonomy of all isohypsibioid families and genera is discussed, with special emphasis on the newly erected entities. Finally, a dychotomous diagnostic key to all currently recognised isohypsibioid families and genera is provided.


Introduction
Tardigrada are a phylum of microinvertebrates inhabiting almost all environments across the globe (Nelson et al., 2015). Despite the undeniable progress in disentangling tardigrade phylogeny, both tardigrade relationships with other metazoans (e.g., Campbell et al., 2011;Gross & Mayer, 2015) and many within-group affinities remain unclear (e.g., Sands et al., 2008;Bertolani et al., 2014a). One of major tardigrade groups with unresolved phylogeny and taxonomy is Isohypsibioidea Guil et al., 2019. This recently established eutardigrade order is considered problematic because it is based on traits that show high levels of morphological stasis (Marley et al., 2011). In fact, this group was erected relatively late mainly because for decades various taxa with Isohypsibius and Hypsibius type claws were traditionally grouped into a single order Hypsibioidea Guil et al., 2019. Although the erection of Isohypsibioidea clarified the taxonomy of the Eutardigrada, the few efforts to resolve phylogenetic relationships within the order suffered from insufficient sampling and resulted in prevailing polytomies Gąsiorek et al., 2019). So far, only a single study  identified a monophyletic lineage within Isohypsibioidea. They found morphological and molecular support to erect the family Hexapodibiidae Cesari et al., 2016, the only other isohypsibioid family apart from Isohypsibiidae Sands et al., 2008. Thus, in order to elucidate the taxonomy and phylogenetic relationships within the order Isohypsibioidea, here we employ comparative analyses of over fifty species representing eleven isohypsibioid genera. Our study embraces a range of analytical methods that included light and scanning electron microscopy observations of external and internal morphology as well as DNA sequencing of two nuclear markers. We uncovered four distinct phylogenetic lineages, corresponding to two previously identified and two new families. The largest tardigrade genus, Isohypsibius Thulin, 1928, as indicated earlier Cesari et al., 2016;Gąsiorek et al., 2019), is artificial and comprises at least five distinct evolutionary lineages. For three of these, we propose new formal taxonomic ranks and we demonstrate, for the first time, that even slight qualitative differences in claw anatomy, the number of macroplacoids in the pharynx, and the presence of cuticular gibbosities can be of high taxonomic importance in delineating isohypsibioid genera. This work is, therefore, another step towards making all isohypsibioid families and genera monophyletic.

Sample processing and comparative material
Tardigrades were isolated from moss, lichen, mixed moss and lichen, or water sediment samples, collected by various persons (see table 1), and processed following the protocol described by Stec et al. (2015). In addition to newly collected material, slides with type specimens of taxa described by Ramazzotti, Maucci, Pilato and Biserov, deposited in the Natural History Museum in Verona, were examined using phase contrast microscope (PCM; see table 1 for details), courtesy of Dr. Roberto Guidetti of the University of Modena and Reggio Emilia, Italy.

Microscopy and imaging
Specimens for light microscopy and morphometry were mounted on microscope slides in Hoyer's medium according to Morek et al. (2016) and examined under a Nikon Eclipse 50i phase contrast microscope (PCM) fitted with a Nikon Digital Sight DS-L2 digital camera. Specimens for imaging in the scanning electron microscope (SEM) were prepared according to Stec et al. (2015). Buccal apparatuses were extracted following the protocol provided by Eibye-Jacobsen (2001) with modifications described in Gąsiorek et al. (2016). Both animals and apparatuses were examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of Jagiellonian University, Kraków, Poland. For deep structures that could not be fully focused under PCM in a single photograph, a series of 2-6 images were taken every ca. 0.2 μm and then assembled with Corel into a single deep-focus image.

Morphometrics, terminology and classification
All measurements are given in micrometres (μm). Structures were measured only if they were intact and orientated in a flat plane. Terminology for the structures within the buccal apparatus and for the claws follows that of  and Gąsiorek et al. (2017). Additionally, in order to avoid misunderstandings and aid comparisons between isohypsibioid taxa, we propose new terminology describing the details of the oral cavity armature (OCA); see also fig. 1. All sclerified elements in the oral cavity are referred to as 'teeth' as their location and shape strongly suggest their function as teeth (see also Katholm, 2002, and, for similar proposals in eohypsibiids and macrobiotids, respectively). At the same time, we suggest to abandon the use of unspecific terms such as intrabuccal/infrabuccal 'baffles' , 'mucrones' or 'ridges' as they refer to solely shape/ appearance rather than to function and therefore may be enigmatic. In all isohypsibioids analysed with SEM, either one or two bands of teeth were observed Nelson et al., 1999;Jørgensen, 2001, the present study). The first band of teeth was present in all individuals and it was always located on the ring fold (a circular and soft portion of the oral cavity wall; Michalczyk & Kaczmarek, 2003) in the middle of the oral cavity. The second band, if present, was always located behind the first band, at the rear of the oral cavity, just before the buccal tube opening ( fig. 1). Therefore, we refer to these bands as 'the first band of teeth' and 'the second band of teeth' , respectively. The numbering of bands of teeth is introduced solely for practical reasons, to allow a concise description of the OCA, and are not for the formulation of hypotheses on homologies. Stylet support insertion point is abbreviated as SSIP, whereas apophyses for the insertion of the stylet muscles -as AISM. Claws were measured following Beasley et al. (2008). In order to quantify the relative difference in height between the secondary and the Apodibius confusus Dastych, 1983 Welzow-South,  (Ramazzotti, 1945) comb. nov.  (Thulin, 1928) comb. nov. Grevenius longiunguis (Pilato, 1974) comb. nov. Randazzo, Sicily, Italy ca. 37°52'N, 14°56'E 710-750 sediment G. Pilato + - Downloaded  Grevenius monoicus (Bertolani, 1981) comb. nov.

+ -
Grevenius pushkini (Tumanov, 2003) comb. nov. primary claw branches, we introduce the br ratio, i.e., ratio of the height of the secondary claw branch to the height of the primary claw branch (the more the branches are similar in height, the closer to 1.0 the br ratio is and the shorter the secondary branch relative to the primary branch, the lower the br ratio). During the review process of this manuscript, the tardigrade phylogeny by Guil et al. (2019) was published. Consequently, we adjusted the taxonomy of the high ranks in our work to the system proposed in Guil et al. (2019), however, in our opinion the new taxonomy is controversial and requires further work: possibly rank shift and taxon membership rearrangements, as the majority of high rank taxa (orders, families) remained in polytomies, compared to the relatively well-resolved relationships from the previous phylogeny by Bertolani et al. (2014). Given that superfamily Isohypsibioidea was elevated to the order level by Guil et al. (2019), according to the article 36.1 of International Code of Zoological Nomenclature (1999), the authorship of the superfamily (Sands et al., 2008) is now superseded by the latest authority. Schematic presentation of the oral cavity armature (OCA) in Isohypsibioidea, the first and/or the second band of teeth are marked by Roman numerals: A -a continuous peribuccal lamina, two bands of teeth (Apodibius, Grevenius gen. nov., Halobiotus, Hexapodibius); B -a continuous peribuccal lamina, the first band of teeth (Fractonotus, Isohypsibius, Ursulinius gen. nov.); C -six convex peribuccal papulae, the first band of teeth (Eremobiotus); D -rectangular peribuccal lamellae, the first band of teeth with lateral toothless intervals (Pseudobiotus); E -rectangular peribuccal lamellae, two bands of teeth (Thulinius); F -six large peribuccal lamellae, two bands of teeth (Haplomacrobiotus). Both bands of teeth contain variable number of rows, depending on the genus Genotyping DNA was extracted from individual animals using Chelex® 100 resin (Casquet et al., 2012;Stec et al., 2015). Paragenophores of all sequenced species were mounted on permanent slides and are deposited in the collection of Institute of Zoology and Biomedical Research (Pleijel et al., 2008). We sequenced two DNA fragments: a small ribosome subunit (18S rRNA) and a large ribosome subunit (28S rRNA). All fragments were amplified and sequenced according to the protocols described by Stec et al. (2015), using the primers and specific PCR programmes from: Sands et al. (2008) and Zeller (2010) (Hall 1999).

Phylogenetics
We aligned all available isohypsibioid and hypsibioid (outgroup taxa) 18S + 28S rRNA sequences from GenBank together with our new sequences (see table 2) using the Q-INS-I strategy, which considers the secondary structure of RNA, in MAFFT version 7 (Katoh et al., 2002;Katoh & Toh, 2008). Currently available partial 28S rRNA sequences for Halobiotus crispae Kristensen, 1982, Hexapodibius micronyx Pilato, 1969 and Pseudobiotus megalonyx (Thulin, 1928) represent a different region of this marker than the ones sequenced by us, thus they were not included in the dataset. The aligned fragments were edited and checked manually in BioEdit. The best substitution model and partitioning scheme for posterior phylogenetic analysis was chosen under the Akaike Information Criterion (AIC), using PartitionFinder version 2.1.1 (Lanfear et al., 2016). As best-fit partitioning scheme, PartitionFinder suggested to retain two predefined partitions separately and for each of them the best fit model was GTR+I+G. Maximum-likelihood (ML) topologies were constructed using RAxML v8.0.19 (Stamatakis, 2014). Strength of support for internal nodes of ML construction was measured using 1000 rapid bootstrap replicates. Bootstrap (BS) support values ≥70% on the final tree were regarded as significant statistical support. Bayesian inference (BI) marginal posterior probabilities were calculated using MrBayes v3.2 (Ronquist & Huelsenbeck, 2003). Random starting trees were used and the analysis was run for ten million generations, sampling the Markov chain every 1000 generations. An average standard deviation of split frequencies of <0.01 was used as a guide to ensure the two independent analyses had converged. The program Tracer v1.3 (Rambaut et al., 2014) was then used to ensure Markov chains had reached stationarity and to determine the correct 'burn-in' for the analysis which was the first 10% of generations. The ESS values were greater than 200 and consensus tree was obtained after summarising the resulting topologies and discarding the 'burn-in' . The BI consensus tree, clades recovered with posterior probability (PP) between 0.95 and 1.00 were considered well supported, those with PP between 0.90 and 0.94 were considered moderately supported and those with lower PP were considered unsupported. All final consensus tree were viewed and visualized by FigTree v.1.4.3 available from http://tree.bio.ed.ac.uk/software/figtree. The sequence HQ604951, representing E. alicatai (Binda, 1969), was characterised by highly unstable position in the trees calculated in both methods, and it never clustered with newly sequenced Eremobiotus sp. nov., suggesting it could be a misidentification or a mislabelling. Therefore, we excluded this taxon from the final dataset. Additionally, the aligned fragments were trimmed to the size of the shortest available alignment (i.e., 745 bp for  Nelson et al., 1999HQ604957 -Bertolani et al. (2014a Pseudobiotus megalonyx (Thulin, 1928) MK675931, HQ604959 MK675920 present study, Bertolani et al. (2014a) Thulinius augusti (Murray, 1907) KF360230 - Bertolani et al. (2014b) Thulinius ruffoi (Bertolani, 1982) MK675932 MK675921 present study Thulinius stephaniae (Pilato, 1974) GQ925701 -unpublished Ursulinius lunulatus (Iharos, 1966) MK675933 MK675922 present study Ursulinius pappi (Iharos, 1966) MK675934 MK675923 present study Ursulinius silvicola (Iharos, 1966) MK675935 MK675924 present study Hypsibioidea (outgroup) Acutuncus antarcticus (Richters, 1904) EU266943 - Sands et al. (2008) Adropion belgicae (Richters, 1911) HQ604925 - Bertolani et al. (2014a) Adropion scoticum (Murray, 1905 (Murray, 1907) HQ604924 - Bertolani et al. (2014a) 18S rRNA and 756 bp for 28S rRNA), and uncorrected pairwise distances were calculated using MEGA7 (Kumar et al., 2016).

Head morphology and peribuccal structures in Isohypsibioidea
The head in all Isohypsibioidea is terminated bluntly, with anteroventral mouth opening (figs. 3-4). The frontal part of the head is smooth or equipped with either of two types of structures: frontal lobes or cephalic papillae   Møbjerg et al., 2007), are less clearly delimited from the surrounding cuticle than lobes. Frontal lobes, on the other hand, are present in some remaining isohypsibioids, although they can vary in size and shape, for example they are smaller and slightly more roundish in Ursulinius pappi ( fig. 4B) than in Apodibius confusus Dastych, 1983 (fig. 4E). Paradiphascon Dastych, 1992 has large, domeshaped frontal lobes (Dastych, 1992). Given that Halobiotidae fam. nov., in contrast to all remaining Isohypsibioidea, secondarily adapted to marine environment, cephalic papillae are most likely a halobiotid autapomorphy. If, as hypothesised by Dastych (1992), frontal lobes are homologous remnants of heterotardigrade cephalic papillae, they should be considered an isohypsibioid plesiomorphy. Moreover, in some genera, additional regular circular cuticular wrinkles can be present around the mouth opening (Apodibius and Hexapodibius   Schuster et al., 1980 for the complete fusion of lamellae into a continuous lamina in Thulinius saltursus ). The number of peribuccal lamellae is considered a generic trait (12 in Thulinius, 30 in Pseudobiotus and an undermined number in Paradiphascon; Schuster et al., 1980;Bertolani, 1982;Nelson et al., 1999;Dastych, 1992). Finally, in Eremobiotus, and likely in Dastychius Pilato, 2013, six peribuccal papulae are present Pilato, 2013). The continuous peribuccal lamina is definitely the most widespread morphotype, and likely the ancestral one, which independently evolved into divided or semi-divided peribuccal lamellae in two doryphoribiid genera. Nonetheless, our SEM observations question the validity of peribuccal lamellae as the main trait distinguishing Thulinius and Pseudobiotus (figs. 5G-H), since these structures have variable morphology.

Oral cavity armature in Isohypsibioidea and other Eutardigrada
In the great majority of isohypsibioid species, OCA is visible only under SEM and all our observations are based on this technique. In all analysed taxa, the oral cavity was equipped with at least one band of conical teeth located on the ring fold, in the central part of the oral cavity ( fig. 1). However, in the majority of isohypsibioid genera a second band of teeth was also detected (Apodibius, Grevenius gen. nov., Halobiotus, Hexapodibius, Thulinius; the second band could be present also in Pseudobiotus, see below for details). There are no SEM observations of the oral cavity for Dastychius.
The OCA system in Paradiphascon is obscure (Dastych, 1992) Nelson et al., 1999). The second band of teeth, composed of 1-4 rows of teeth, comprises conical teeth that are typically larger than those in the first band, and are located immediately behind the first band and before the buccal tube opening (figs. 5D, F-G, I, 13B-C, 14B). In all examined species, the second band was continuous, except for Hexapodibius micronyx, in which the band was divided into a short dorsal and ventral row of irregular teeth ( fig. 5J).
In a wider context, so far, greatest attention was paid to OCA in Macrobiotoidea Guil et al., 2019(Thulin, 1911Pilato, 1975;Michalczyk & Kaczmarek, 2003;Guidetti et al., 2012) and Eohypsibioidea Guil et al., 2019(e.g., Hansen et al., 2017, but very little is known about OCA in Hypsibioidea Stec et al., 2017Stec et al., , 2018 and the topic has not been addressed systematically in Isohypsibioidea (OCA was only mentioned occasionally in several species, e.g., in Pilato, 1975;Jørgensen, 2001;Lisi, 2011). The lack of data for Hypsibioidea prevents the formulation of sound hypotheses about the evolution of OCA both within isohypsibioids and in all eutardigrades.
Nevertheless, OCA in isohypsibioids seems to have supra-generic significance. Our observations showed that Isohypsibiidae have only one band of teeth, whereas the three other families exhibit two bands ( fig. 1). The only exception in Doryphoribiidae fam. nov. -Pseudobiotus with only the first band of teeth visible in the oral cavity -has to be treated with caution as the first band of teeth in this genus is very large and it obscures the view of the posterior part of the oral cavity, therefore it is not possible to say whether the second band of teeth is lacking or it is simply not visible when looking through the mouth opening. However, based on phylogeny, we hypothesise that Pseudobiotus exhibits two bands of teeth. Given that the two sister clades constituting Isohypsibioidea (i.e., Isohypsibiidae vs Halobiotidae fam. nov. + Hexapodibiidae + Doryphoribiidae fam. nov.) exhibit one vs two bands of teeth in the oral cavity, it is not possible to state whether the last common ancestor for all isohypsibioids had one or two bands of teeth.
The differences in the location of teeth in the OCA between Isohypsibioidea and both Eohypsibioidea and Macrobiotoidea may suggest that bands of teeth in Isohypsibioidea and in the two latter orders are not homologous. OCA in Eohypsibioidea and Macrobiotoidea consists of three bands of teeth: first (minute cones located in the very anterior of the oral cavity), second (larger cones or ridges parallel to the main axis of the buccal apparatus, located in the rear of the oral cavity just behind the ring fold), and third (a system of ventral and dorsal transverse crest/ridgeshaped teeth, located in the rear of the oral cavity just behind the second band of teeth and before the buccal tube opening in Macrobiotidae Thulin, 1928, Murrayidae Guidetti et al., 2000and some Richtersiidae Guidetti et al., 2016 or a band of conical teeth in some Richtersiidae). Thus, Isohypsibioidea do not exhibit the most anterior band of teeth, termed as the first (or anterior) band, that is present in both Eohypsibioidea and Macrobiotoidea in the very anterior of the oral cavity. Moreover, except for Richtersius Pilato & Binda, 1989 (which exhibits a highly modified OCA), neither in Eohypsibioidea nor in Macrobiotoidea were the teeth observed on the ring fold. The only congruence in the location of teeth in the oral cavity concerns the most posterior teeth: in Eohypsibioidea and Macrobiotoidea the third band of teeth is located immediately behind the ring fold, i.e., in the same place as the second band of teeth in Isohypsibioidea. Nevertheless, as already mentioned above, the current state of knowledge on the OCA in eutardigrades does not allow to conclude whether the third band in eohypsibiids and macrobiotids is homologous with the second band in isohypsibioids.
Interestingly, regardless of phylogenetic relationships and location of teeth in the oral cavity, larger teeth (e.g., in the first band in Pseudobiotus and in the third band in Eohypsibiidae Bertolani & Kristensen, 1987 and Macrobiotidae) tend to be arranged in two rows, ventral and dorsal. We hypothesise that lateral toothless intervals are necessary to allow stylet extrusion through the oral cavity and mouth opening (stylets are positioned laterally, parallel to the buccal tube, and they are extruded in a scissor-like manner; Guidetti et al., 2013).

Buccal apparatus morphology in the Doryphoribiidae fam. nov. + Hexapodibiidae clade
Two evolutionary pathways can be recognised in the anatomy of the buccal apparatus in Doryphoribiidae fam. nov.: buccal tube without ventral lamina and with unmodified AISM (Grevenius gen. nov., Pseudobiotus, Thulinius), and the other with the buccal tube enforced with ventral lamina, which is associated with modifications of AISM (Apodibius, Doryphoribius) . The buccal apparatus of Grevenius pushkini (Tumanov, 2003) comb. nov. and other aquatic "Isohypsibius" spp. is generally more similar to that in Thulinius spp. than to terrestrial Isohypsibius spp. (figs. 13, 14), which is not surprising given the close phylogenetic relationship between the two taxa ( fig. 2) and same, aquatic, habitat. Specifically, aquatic "Isohypsibius" spp. and Thulinius spp. have two rows of teeth in the oral cavity (figs. 13B-C, 14B) and narrow apophyses for the insertion of the stylet muscles (AISM) (figs. 13D, 14C) whereas terrestrial Isohypsibius spp. exhibit one row of buccal teeth ( fig. 5A) and proportionally broader AISM. The anatomy of buccal apparatus in Hexapodibiidae is more conserved, since only the morphotype with ventral lamina exists . Buccal apparatus of Hexapodibius, similarly to that of Haplomacrobio-tus, has reduced AISM due to the developed ventral lamina (figs. 15A-C, 16D). Isohypsibioid taxa equipped with ventral lamina, i.e., Hexapodibiidae and some Doryphoribiidae fam. nov.: Doryphoribius and Apodibius, share extreme resemblance of the buccal apparatus anatomy. For example, they all exhibit unmodified Hypsibius type furcae (figs. 15D, 16) and two or three short, often almost granular macroplacoids in the pharynx (figs. 15E, 16;Hohberg & Lang, 2016). This is in contrast to taxa without the ventral lamina, Thulinius, Pseudobiotus, and Grevenius gen. nov., which all have elongated macroplacoids. Interestingly, ventral lamina is present in terrestrial but not in freshwater representatives of the Halobiotidae fam. nov. + Hexapodibiidae + Doryphoribiidae fam. nov. clade (single exceptions can be found in polyphyletic Doryphoribius). Ventral lamina in both Doryphoribius and Hexapodibiidae has two different morphotypes: a short, delicate lamina reaching no farther than to the half of the buccal tube length in Apodibius, Hexapodibius, or some Parhexapodibius , and Doryphoribius (figs. 15A-C, 16A, C-D, F); or a long, robust lamina reaching almost the level of the stylet support insertion point in some Doryphoribius and Parhexapodibius (figs. 16B, E). The presence of ventral lamina in all hexapodibiids but only in some doryphoribiids suggests that either the common ancestor of Hexapodibiidae + Doryphoribiidae fam. nov. exhibited the lamina, which was later independently lost in Thulinius, Pseudobiotus, and Grevenius gen. nov., or lamina evolved independently two or three times: in (1) Hexapodibiidae, (2) ancestor of Apodibius and some Doryphoribius spp., and (3) in remaining Doryphoribius spp. (see fig. 2). The lack of ventral lamina in Heterotardigrada Marcus, 1927, Apotardigrada, Hypsibioidea and many Isohypsibioidea suggests that it is a derived trait that evolved independently in Isohypsibioidea and in the ancestor of Macrobiotoidea. In other words, the presence of the ventral lamina should be treated as an example of parallel evolution within Eutardigrada, being at the same time the autapomorphy of Macrobiotoidea as well as of Hexapodibiidae and some genera of Doryphoribiidae fam. nov. (Marley et al., 2011).

Cuticle morphology in Isohypsibioidea
In contrast to the majority of eutardigrades, isohypsibioids frequently exhibit distinct cuticular sculpturing (figs. 3, 6). Five major kinds of sculpturing can be distinguished within the order: (I) reticulum, (II) circular tubercles of various size, (III) pointy gibbosities, (IV) round gibbosities, and (V) plaques. The most unique type of cuticle morphology characterises Fractonotus, which has symmetrically arranged dorsal plaques (figs. 3A, 6B) as well as densely arranged smooth tubercles that cover the entire dorsum and limbs (figs. 3A, 6A; Gąsiorek et al., 2019). Isohypsibioid gibbosities can be generally divided into two types: small, weakly demarcated (almost flat in LM) and pointy gibbosities present in Dianea gen. nov. (figs. 3B, 6C), or large, mamillose and round gibbosities with developed reticulum or complex ornamentation in Ursulinius gen. nov. and many Doryphoribius spp. (figs. 3C, 6D-F; Ramazzotti & Maucci, 1983). Gibbosities of Dianea gen. nov. are less regular and clearly narrow towards the apex in contrast to hemispherically convex gibbosities in the two latter genera. The usage of dorsal gibbosities as a generic trait was a subject of criticism (Pilato, 1982), as, according to some descriptions, in one species there could be a considerable variation in gibbosity development (e.g., Binda & Pilato, 1971). The same variability was ascribed to cuticular sculpturing in general (Kristensen & Hallas, 1980). However, recent data show that in a single sample, numerous, potentially closely related or pseudocryptic species can be found (e.g., see Faurby et al., 2011;Morek et al., 2019). Therefore, the reports of such profound variability in the development of gibbosities given without genetic data should be taken with caution. In species devoid of Fractonotus type tubercles or gibbosities, e.g., in Grevenius gen. nov., Thulinius or Pseudobiotus (figs. 3D, 6G), quite often the entire dorsal cuticle is covered with homogenous, rough sculpturing that forms wrinkly epicuticular reticulum or processes ( fig. 6H; Bertolani, 1982;Chang et al., 2007;Bertolani et al., 2014b). The richness and variability of cuticular sculpturing within Isohypsibioidea indicate independent, autapomorphic origin and prevent hypothesising whether the ancestral cuticle state was smooth or sculptured.

Claw morphology in Isohypsibioidea
Isohypsibioid claws can be divided into six general morphotypes: (I) Isohypsibius type, as defined by Ramazzotti & Maucci (1983), the most widespread morphotype, with external and internal claws on the same limb of similar size and with branches forking at a ca. right (90°) angle, (figs. 7A-D, H, K, 8, 9B, 10); (II) Eremobiotus type, with all claws with branches forming an obtuse, approaching a straight (ca. 180°) angle, and external and internal claws on the same limb of similar size, but dissimilar branch heights (br < 70%), which is a highly modified Isohypsibius type (figs. 7E-F, 9C-D; ; (III) Fractonotus type, with all claws with V-shaped branches and with secondary branches forming a continuous curve with the basal tract and significantly shorter than the primary branches (br < 70%), which could be seen as an intermediate morphotype between the Isohypsibius and the Hypsibius type claw (figs.  >70%, see table 2), elongated basal tracts, and typically prominent humps on primary branches of internal and anterior claws (figs. 7I-J, L-P, 11). The Pseudobiotus type claws are common for the genera Pseudobiotus, Halobiotus, Thulinius and Grevenius gen. nov. The peculiar morphology of OCA (see above), together with anatomical modifications related to copulation and parental care in Pseudobiotus (hooklike claws on the first pair of legs in males ( fig. 7N) and reduced hind claws in females who carry shed exuviae with eggs), seem to be more suitable taxonomic criteria to differentiate Pseudobiotus and Thulinius rather than the number of peribuccal lamellae, as Thulinius is parthenogenetic (or at least does not exhibit sexual dimorphism, however thelytoky was confirmed in T. augusti (Murray, 1907) -see Bertolani, 1976, and T. ruffoi (Bertolani, 1981)see Kosztyła et al., 2016) and lacks parental care and associated morphological modifications (Rebecchi & Nelson, 1998)).
Morphotypes II-VI are internally homogenous, however Isohypsibius morphotype can be further divided into three distinct subgroups: (Ia) I. prosostomus type, with secondary branches clearly shorter than primary branches (br ranges from around 40% to 70%, see table 3), claw bases without pseudolunulae, and with single bars under claws (figs. 7A-B, 8A-D); (Ib); I. dastychi type, with branches forking at an obtuse, approaching a straight (ca. 180°) angle, with developed pseudolunulae, br ≈ 70% and double bars under claws ( fig. 8E; according to Tumanov (2005), bars are absent only in I. panovi Tumanov, 2005); (Ic) U. pappi type, with evident pseudolunulae, and double bars under claws (br ≈ 50%-70%; figs. 7C-D, 10). The ancestral state of claw morphology remains unknown, as relationships within Isohypsibiidae s.s. are unclear ( fig. 2). Types II-VI have been already used in erections of supraspecific entities, and we hypothesise that all subtypes of type I could also be suitable for differentiating higher taxonomic levels.

Taxonomy of Isohypsibiidae sensu stricto
Isohypsibius Thulin, 1928 and related genera For a considerable time, Isohypsibius was the second largest tardigrade genus (Degma & Guidetti, 2007;Degma et al., 2009-18). Despite the erections of new genera from Isohypsibius, including those erected in the present study, the genus still remains relatively speciose (42 spp. vs 16, 36 and 35 spp. in the newly erected Dianea, Ursulinius and Grevenius gen. nov., respectively; see Appendix). However, as recently suggested by Gąsiorek et al. (2019), some Isohypsibius spp. appear more closely related to Fractonotus than to Isohypsibius s.s., which could explain the current paraphyletic character of Isohypsibius with respect to Fractonotus ( fig. 2). Moreover, there are at least two more claw morphotypes that are divergent from the I. prosostomus (i.e., Isohypsibius s.s.) type defined in this work (figs. 8A-D). The distinctiveness of the first group, I. dastychi group, has been already noticed by Tumanov (2005). The I. dastychi group exhibits claws with branches forking at a very wide, approaching a 180° angle, present also in Eremobiotus ( fig. 8E). Interestingly, the topology of the tree indicates the affinity of these two groups as I. dastychi and Eremobiotus sp. nov. are in a single polytomous clade (that includes also Ursulinius gen. nov.). The second morphotype is currently represented only by a single species, Isohypsibius chiarae Maucci, 1987. Secondary branches in this species are reduced, being short and acute ( fig. 8F). Taking into consideration that in the present study, morphological peculiarities of a similar magnitude induced the erections of three new genera, including one representing a different family (Grevenius gen. nov., in Doryphoribiidae fam. nov.), it should be noted that I. chiarae does not belong to Isohypsibius s.s.   . 10) (see also Lisi et al., 2016). However, the monophyly of the genus should be treated with caution since claws in E. ovezovae , unlike claws of the remaining two described Eremobiotus spp., are significantly reduced (compare figs. 9C-D). Thus, the possibility that E. ovezovae represents an independent evolutionary line that has convergently evolved claws with widely angled branches must be considered. In fact, it would not be surprising if this claw morphotype evolved more than once in Isohypsibiidae, especially that, for example, claw reduction has been shown to evolve independently in several eutardigrade lineages (Bertolani & Biserov, 1996).

Systematic position of Grevenius gen. nov.
Genetic distinctiveness of Grevenius gen. nov. became first apparent in Sands et al. (2008), where close affinities between I. asper (Murray, 1906), I. granulifer, and Thulinius stephaniae (Pilato, 1974) (Jørgensen, 2001) comb. nov. is described as 'intrabuccal baffles') and in claw morphology (claws elongated, with a clear hump on the primary branch and with relatively elongated secondary branches (br > 70%) in Grevenius gen. nov., figs. 11A-F vs claws of the Isohypsibius type, without the hump on the primary branch and with a considerable difference in primary and secondary branch height (br ≤ 70%) in Isohypsibius, figs. 8A-D). Moreover, Grevenius gen. nov. inhabits a different ecological niche than in Isohypsibius s.s. (freshwater vs terrestrial). Claws in Grevenius gen. nov., similarly to those in Pseudobiotus and Thulinius (e.g., see Nelson et al., 1999;Bertolani, 2003), are clearly elongated, which is most likely an adaptation to the aquatic habitat ( fig. 12). Moreover, internal claws in the new genus have a clear hump (as in Thulinius) and the cuticle is typically rough (as in Pseudobiotus; e.g., see Bertolani, 1982;Chang et al., 2007. All these similarities suggest a close affinity of the new genus with both Pseudobiotus and Thulinius, which is in agreement with the molecular phylogeny ( fig. 2). However, the exact phyletic relationships between the three genera and relationships within Doryphoribiidae fam. nov. are not fully solved. Thus, more DNA sequences, in particular for intertidal Grevenius gen. nov. spp., are needed to better understand its relationships with other doryphoribiid genera.

Morphology of Hexapodibiidae
The problematic systematic position of calohypsibiid genera and species (order Hypsibioidea) has been a subject of long debate (Pilato, 1989;Guil et al., 2013;Bertolani et al., 2014a;Gąsiorek et al., 2019) Pilato & Beasley, 1987, Haplomacrobiotus May, 1948 and instituted a new family rank for eutardigrades equipped with the ventral lamina and exhibiting various degrees of claw reduction. All four hexapodibiid genera share the same general morphology of the buccal apparatus, i.e., reduced dorsal AISM, ventral lamina and three granular macroplacoids (compare figs. 15, 16D-F herein and the buccal apparatus of Haplomacrobiotus in Cesari et al., 2016). Interestingly, a similar buccal apparatus morphotype is also present in two doryphoribiid genera: Apodibius and Doryphoribius (although with two macroplacoids in some species), but absent in the remaining doryphoribiid genera (Pseudobiotus, Thulinius and Grevenius gen. nov.). Thus, at the moment, it is not possible to establish whether a similar buccal apparatus morphotype evolved independently in Hexapodibiidae as well as in Apodibius (Hohberg & Lang, 2016) and Doryphoribius  or whether the similarity indicates the ancestral state of Hexapodibiidae + Doryphoribiidae fam. nov. Nevertheless, an independent (convergent) origin of the ventral lamina within this clade seems more likely as it is a more parsimonious explanation: given that the evolution of the ventral lamina is tightly linked with the reduction of dorsal AISM (same pattern was observed also in other eutardigrades), hypothesising that the ancestor of Hexapodibiidae + Doryphoribiidae fam. nov. had ventral lamina, which was secondarily lost, and ridge-like AISM evolved again, appears less probable. In other words, a plesiomorphic ventral lamina would require a subsequent atrophy of this structure, re-establishing of the dorsal apophysis and the restoration of the overall symmetry of AISM in Grevenius gen. nov., Pseudobiotus and Thulinius.
Despite representing different families, both Hexapodibius and Apodibius exhibit peculiar peribuccal circular wrinkles (figs. 5F, J). Interestingly, it must be noted via free access that these structures are found exclusively in soil isohypsibioids (Haplomacrobiotus being an exception ). Similarly, frontal lobes are present mainly in soil genera (Apodibius, Haplomacrobiotus, Paradiphascon) and they occur also in Ursulinius gen. nov. Thus, these organs could be another adaptation to the terrestrial habitat.
In accordance with analyses of Guil et al. (2013), claw morphology in Hexapodibiidae represents three levels of reduction: (I) shortened secondary branches and the basal tract being continuous with cuticle surface (reduced pseudolunulae in Parhexapodibius; see Manicardi & Bertolani, 1987), (II) shortened primary branches and lack of claws IV in Hexapodibius (see fig. 7Q), and (III) complete reduction of secondary branches in Haplohexapodibius and Haplomacrobiotus (see Cesari et al., 2016). The reduction is commonly viewed as an adaptation to soil habitat, preferred by hexapodibiids (Bertolani & Biserov, 1996;Hohberg et al., 2011).

Autapomorphies of Halobiotidae fam. nov.
The erection of Halobiotidae fam. nov. is firmly supported both by DNA sequences as well as unique morphology and anatomy, which are most likely the result of secondary adaptation to marine habitat. Traits exclusive to Halobiotus, such as cephalic papillae, peribuccal chemosensory organs, and gigantic Malpighian tubules, most probably serve in perception of external stimuli and osmoregulation, respectively (Kristensen, 1982;Møbjerg & Dahl, 1996;Møbjerg et al., 2007;Halberg et al., 2013). Claws of Halobiotus (figs. 7I-J) are similar to the most common morphotype of doryphoribiid claws, i.e., with elongated stalks and branches of similar heigths, present also in Grevenius gen. nov., Paradiphascon, Pseudobiotus, Thulinius, and some Doryphoribius spp. Therefore, presumably the ancestral claw type of the clade [Halobiotidae fam. nov. (Doryphoribiidae fam. nov. + Hexapodibiidae)] was close to this morphotype.

Incertae sedis: Ramajendas Pilato & Binda, 1991 and Thalerius Dastych, 2009
Exhibiting a mixture of hypsibioid and isohypsibioid morphological traits, two enigmatic genera, Ramajendas and Thalerius, are a subject of an ongoing debate on their taxonomic affinity. Originally placed in Isohypsibiidae (Marley et al., 2011;Guil et al., 2013), they were later tentatively transferred to Ramazzottiidae Sands et al., 2008 and most recently, moved back, also provisionally, to Isohypsibiidae (Zawierucha et al., 2018). On one hand external and posterior claws, by having elongated and flexible primary branch, seem to resemble those in the family Ramazzottiidae (Hypsibioidea). On the other hand, however, the shape of internal and anterior claws is similar to that found in some species representing both Hypsibioidea and Isohypsibioidea. Moreover, the two genera lack body pigmentation and paired cephalic elliptical organs (present in Ramazzottidae), which speak against the close affinity with ramazzottiids (Zawierucha et al., 2018). The body shape and the bucco-pharyngeal apparatus morphology (including AISM shape) in Ramajendas are indeed near those in aquatic doryphoribiid genera. However, this genus comprises both terrestrial taxa and a marine species (R. renaudi (Ramazzotti, 1972)) which strongly indicates that Ramajendas may be polyphyletic, as it was shown above that distinct evolutionary lineages often correspond with the type of environment. Furthermore, Thalerius exhibits the bucco-pharyngeal apparatus similar to many isohypsibioid genera (three granular macroplacoids, widespread in Isohypsibioidea but rare in Hypsibioidea, except for the polyphyletic Mixibius Pilato, 1992 and Diphascon Plate, 1889) and claws with concave bases present in some Itaquasconinae ( Hypsibioidea).
In fact, this perplexing mix may indicate a need to create a new higher taxon for Thalerius. We are of the opinion that neither buccopharyngeal apparatus nor claw morphology should be given priority (see Schuster et al., 1980, andPilato, 1982, for opposing views on the relevance of these structures used in the formulation of eutardigrade classification on higher taxonomic levels), making clarification of the status of the two genera impossible without molecular data. To conclude, the mixture of traits exhibited by both Ramajendas and Thalerius make it difficult to ascribe them to any of the isohypsibioid families distinguished in this work. Therefore, we designate the two genera as incertae sedis within Isohypsibioidea, pending molecular verification of their taxonomic positions within this or a different eutardigrade order.

Taxonomic account of the families and genera of Isohypsibioidea
Type genera are underlined with a double line. Phylum: Tardigrada Doyère, 1840Class: Eutardigrada Richters, 1926Order: Isohypsibioidea Guil et al., 2019 Amended diagnosis (modified from Bertolani et al., 2014a): Double claws asymmetrical with respect to the median plane of the leg (2121), normally with a similar shape and size on each leg; double claws with the external secondary branches inserted perpendicularly on the claw basal tract, or partly reduced (very short, without the common basal tract, with a base as large as the sum of the primary and secondary branch widths, and with an evident suture between the primary and the secondary branch), or elsewhere absent. Buccal tube rigid (apart Paradiphascon) and often relatively large, without the ventral lamina (Dastychius, Dianea gen. nov., Eremobiotus, Grevenius gen. nov., Halobiotus, Isohyp-sibius, Ursulinius gen. nov., Paradiphascon, Pseudobiotus, Ramajendas, Thalerius, Thulinius) or with the ventral lamina (Apodibius, Doryphoribius, Haplomacrobiotus, Haplohexapodibius, Hexapodibius, Parhexapodibius). Pharyngeal apophyses and placoids present.
Smooth eggs laid in exuviae.
Family: Isohypsibiidae Sands et al., 2008 Amended diagnosis: Terrestrial eutardigrades with six peribuccal lobes or with a continuous peribuccal ring, and peribuccal lamina. Lacking peribuccal lamellae and ventral lamina on the buccal tube. AISM ridge-like and asymmetrical with respect to the frontal plane (only in Fractonotus) or symmetrical (remaining five genera). Stylet furcae of the Hypsibius type. Claws with secondary branches clearly shorter than primary branches (br ≤ 0.70).
Composition: Dastychius Pilato, 2013, Dianea gen. nov., Eremobiotus Biserov, 1992, Fractonotus Pilato, 1998, Isohypsibius Thulin, 1928 Remarks: Molecular data are not available for representatives of some genera of former Isohypsibiidae s.l., thus their taxonomic assignment may change when the data are obtained. Dastychius improvisus (Dastych, 1984) is kept in the family since Dastychius type AISM are modified Isohypsibius type AISM (ridged AISM, exceptionally elongated towards the SSIP). Together with peculiar cuticular cavities and typical Isohypsibius type claws, they currently prevent any taxonomic re-shuffling of this genus.  . 2), and at least one more representative of this group ought to be sequenced to confirm the monophyly of the I. dastychi group, the dastychi complex is not erected as a separate genus. Moreover, as recently indicated by Gąsiorek et al. (2019), relationships between Isohypsibius and Fractonotus need clarification as the only sequenced species, F. verrucosus (Richters, 1900), is embedded within the Isohypsibius clade ( fig. 2). Etymology: When observed in SEM en face, the first row of dorsal gibbosities look like pointy ears on the head, which results in a teddy bear-like appearance of animals of the new genus (e.g., see fig. 4B). Therefore, the name of the new genus is derived from the Latin word "ursus" (bear), being a diminutive to mean "a small bear". nov., Paradiphascon Dastych, 1992, Pseudobiotus Nelson, 1980, Thulinius Bertolani, 2003 Remarks: Paradiphascon manningi Dastych, 1992 is transferred from the family Isohypsibiidae primarily on the basis of large peribuccal lamellae. Pilato & Binda (1996) considered lamellae in this taxon as papulae, but the term "papulae" refers to rounded peribuccal structures, present e.g., in Calohypsibius . However, extremely peculiar morphological autapomorphies of the genus (highly modified AISM, annulated pharyngeal tube, dorsoposterior apodeme on the border between the buccal and pharyngeal tube, external and posterior claws with wide bases) require molecular and new morphological evidence to verify the tentative affiliation within Doryphoribiidae fam. nov.

Evolution of traits within the order in relation to other lineages of Eutardigrada
Isohypsibioidea are most likely the most basal lineage in the order Eutardigrada (Sands et al., 2008;Bertolani et al., 2014a). Therefore, unravelling phyletic affinities within this group is of special importance for understanding the evolution of Eutardigrada (Kiehl et al., 2007;Sands et al., 2008). Of the four currently recognised eutardigrade orders, Isohypsibioidea, alongside Hypsibioidea and Eohypsibioidea, exhibit asymmetrical (heteronych) claws and only Macrobiotoidea are characterised by symmetrical (isonych) claws ( fig. 12). Given the phylogenetic relationships between the orders , asymmetrical claws are most likely a plesiomorphy of the Eutardigrada whereas claw symmetry should be considered as a macrobiotid autapomorphy. Nevertheless, in comparison to Hypsibioidea, in which a number of claw morphotypes were recognised (e.g., Hypsibius, Ramazzottius or Calohypsibius type), isohypsibioid claws have always been defined as of a single, general "Isohypsibius type", which suggests prevalent conservatism in their morphology Marley et al., 2011). However, our study implies that details of claw shape together with the presence or absence of other pedal structures such as pseudolunulae and cuticular bars, which were often considered as of minor taxonomic significance (e.g., they were omitted in the only comprehensive morphological phylogeny of eutardigrades by Guil et al., 2013), may hold sound phylogenetic signal. Some isohypsibioid taxa seem to have claws intermediate between the Isohypsibius and the Hypsibius types. For example, claws in Fractonotus (Hypsibius-like claw curvature) or Paradiphascon (Hypsibius-like difference in the size of external and internal claws), may signalise a closer affinity between Isohypsibioidea and Hypsibioidea than with the two remaining orders. As the relationships between the basal families of Hypsibioidea, Calohypsibiidae Microhypsibiidae Pilato, 1998, are not resolved ( fig. 12), the plesiomorphic condition for this order remains unknown. However, in the recent phylogenies, the polytomy embraced also Mixibius and Acutuncus Pilato & Binda, 1997, having either hypsibiid-isohypsibiid claws or typical hypsibiid claws, respectively Cesari et al., 2016). This suggests that hypsibiid ancestor had claws nearing to the present Hypsibiidae , and that the claws of Calohypsibiidae and Microhypsibiidae are considerably modified. Similarly to the hypothesised closer affinity between Isohypsibioidea and Hypsibioidea, the relationship between Eohypsibioidea and Macrobiotoidea is well-supported in the development of true, strongly sclerotised lunulae (in contrast to pseudolunulae present in the former), and narrowing of the basal portion of the claw, which became the peduncle ( fig. 12). Aquatic isohypsibioid species are scattered between more numerous terrestrial taxa, and the basal family, Isohypsibiidae s.s., comprises entirely land taxa ( fig. 2). Concerning the entire class Eutardigrada, limnic forms occur only in some Doryphoribiidae fam. nov., Microhypsibiidae, some Hypsibiidae and Eohypsibiidae, and Murrayidae, whereas marine -in Halobiotidae fam. nov. and in some Doryphoribiidae fam. nov. (Nelson & Marley, 2000). The current phylogeny indicates all these are examples of independent invasion of aquatic habitats (figs. 2, 12). Maucci (1973Maucci ( -1974 first formulated the hypothesis on the evolution of claw morphotypes in relation to the inhabited ecological niche for Hypsibiidae and Ramazzottiidae: he noted that aquatic species exhibit longer claws compared to terrestrial taxa. The correlation between secondarily aquatic environment and claw morphology is also expressly visible in Isohypsibioidea, in which aquatic taxa have elongated claws with branches of almost similar heigths, whereas terrestrial species exhibit robust claws with markedly shorter secondary branches (table 3; figs. 7-12; see also Bertolani, 1982 (Iharos, 1964) then], Eremobiotus alicatai [Isohypsibius alicatai then], Isohypsibius marcellinoi Binda & Pilato, 1971, I. prosostomus, U. pappi comb. nov., U. ronsisvallei Binda & Pilato, 1969.
Concerning buccal apparatus morphology, all isohypsibioid AISM shapes could be seen as derived states of the Isohypsibius type, i.e., ridged AISM (Marley et al., 2011), suggesting this shape as plesiomorphic for the Eutardigrada. Pilato (2013) also hypothesised about the ancestral state of eutardigrade (parachelan then) AISM shape within Isohypsibioidea, suggesting however the Dastychius rather than Isohypsibius type (long ridges reaching to the level of SSIP vs short ridges limited to the buccal crown) as a potential candidate. He hypothesised that in the course of evolution, the Dastychius AISM became shorter, which resulted in the Isohypsibius type AISM. Nonetheless, the current state of knowledge does not allow to determine confidently which of these types is plesiomorphic. The highly modified AISM types in Fractonotus and Halobiotus probably evolved by the division of both ventral and dorsal apophyses, and subsequent reduction of lateral AISM portions or by forming hook-like portions. On the other hand, the most modified AISM type, with reduced dorsal apophysis, is present in Hexapodibiidae and some groups within Doryphoribiidae fam. nov. (figs. 15-16). The magnitude of these changes is most likely associated with the parallel evolution of ventral lamina, which constitutes an important stylet muscle attachment and therefore changes the distribution of forces in the buccal apparatus, rendering dorsal apophyses less important for the functioning of the stylet musculature. The pattern of reduction of the dorsal AISM is consistently found in all eutardigrades exhibiting the ventral lamina (i.e., Macrobiotoidea;  Another instance of parallel evolution, next to the independent origin of the ventral lamina, is the development of dorsolateral gibbosities. Among Eutardigrada, cuticular gibbosities evolved most likely independently in two orders, Isohypsibioidea and Hypsibioidea. Mamillose, sculptured gibbosities of a very similar appearance are present in four genera representing four families: Ursulinius gen. nov. (Isohypsibiidae), some Doryphoribius spp. (Doryphoribiidae fam. nov.), the majority of Pilatobius Bertolani et al., 2014 spp. (Hypsibiidae), and in Ramazzottius szeptyckii (Dastych, 1979) (Ramazzottiidae). On the other hand, small, terminated at point and wrinkly gibbosities of Dianea gen. nov. (Isohypsibiidae) are a unique feature of this genus, therefore they should be recognised as its autapomorphy.

Taxonomic composition of isohypsibioid families
Type genera are underlined by a double line, and type species by a single line. Taxa described as species dubiae are either synonyms of other species or their descriptions are too general and do not allow confident identifications; whereas nomina inquirenda embrace most likely valid species, but insufficiently described. This distinction and assessment was done after a careful analysis of the original species descriptions.