Resolving the systematics of Richtersiidae by multilocus phylogeny and an integrative redescription of the nominal species for the genus Crenubiotus (Tardigrada)

The family Richtersiidae, although established recently with the use of phylogenetic methods, was considered potentially paraphyletic at the time of its erection. Until now, the family comprised four genera, Richtersius, Diaforobiotus, Adorybiotus and a newly erected genus Crenubiotus. However, the genetic characterisation for the latter two genera was very limited or absent. To address concerns about the phylogenetic affinity of these two genera, we present a multilocus phylogeny of the families Richtersiidae and Murrayidae based on four molecular markers (18S rRNA, 28S rRNA, ITS-2 and COI). Our results show a distinct evolutionary lineage composed of Adorybiotus and Crenubiotus, which is sister to Murrayidae. In order to accommodate the phylogenetic and morphological distinctiveness of this lineage, we erect a new family, Adorybiotidae fam. nov. The new taxon differs morphologically from other families in the superfamily Macrobiotoidea by a unique combination of traits: (1) the presence of tubercles/cushions with aggregations of microgranules on their surfaces present on all legs and on the dorso-caudal cuticle, (2) a system of internal septa in claws, and (3) buccal apparatus morphology. Moreover, in order to stabilise the taxonomy and nomenclature in the genus Crenubiotus, we redescribe its type species, Crenubiotus crenulatus, by means of integrative taxonomy and designate a new neotype based on a population from the original terra typica.

node supports full support in BI and ML (pp=1.00, BS=100) or ML (pp<1.00 and/or BS<100) pp=1.00 * Figure 1. Phylogenetic reconstruction of the superfamily Macrobiotoidea based on concatenated 18S rRNA + 28S rRNA + ITS-2 + COI nucleotide sequences. Topology and branch length of BI reconstruction. Nodes with BI support under 0.70 were collapsed. Values above branches indicate BI posterior probabilities (pp), values below branches indicate ML bootstrap (bs) support. Black circle and no value = full support in both analyses, i.e. 1.00 for BI or 100 for ML; grey circle = node supported in both analyses but not fully in at least one of them (* = full support, BI/ML values lower than 1.00/100 shown). Newly sequenced taxa/populations are bolded. Neotype or type populations are underlined.   6,7,8,9,10,11,12).
Adorybiotidae fam. nov., by the combination of morphological characters of animals and eggs is unique within the superfamily Macrobiotoidea, and it differs specifically from the family: • Macrobiotidae Thulin, 1928 15 by: large comb-like lunulae under claws on each leg equipped with long and evenly distributed teeth (lunulae smaller, often without teeth and when equipped with teeth, they are short and not as regularly arranged as in Adorybiotidae fam. nov.); and by the system of internal septa within claws on each leg as described by Lisi et al. 13 (the system of internal septa absent in Macrobiotidae). • Richtersiidae Guidetti et al., 2016 11 by: the presence of tubercles/cushions with aggregations of microgranules on their surfaces on all legs and on the dorso-caudal cuticle (granulation on legs and on the dorso-caudal cuticle absent in Richtersiidae); the presence of the microplacoid in the bulbus (microplacoid absent in Richtersiidae); and by egg process morphology (processes in the shape of cones with wide bases and very narrow elongated apices or processes with concave bases in the shape of cooling towers and apices divided into 2-4 thick horizontal branches in Adorybiotidae fam. nov. vs egg process in the shape of elongated, thin, conical spikes in Richtersiidae). • Murrayidae Guidetti et al., 2005 22 by: cuticular pores (absent in Murrayidae); large comb-like lunulae under claws on each leg equipped with long and evenly distributed teeth (lunulae without teeth in Murrayidae); the system of internal septa within claws on each leg as described by Lisi et al. 13 (the system of internal septa absent in Murrayidae); the presence of tubercles/cushions with aggregations of microgranules on their surfaces on all legs and on the dorso-caudal cuticle (only regular granulation present but tubercles/cushions absent in Murrayidae); and by claw morphology (the common tract of the claw longer than the half of the entire claw height in Adorybiotidae fam. nov. vs the common tract of the claw shorter than the half of the entire claw height in Murrayidae).     www.nature.com/scientificreports/ granulation band on the caudal cuticle that extends across the terminal body segment from the left body side, through dorsal surface, to the right side (Figs. 7c, 8d). Beside these dense granulation patches, very fine granulation is present and evenly distributed on the entire body surface but visible only under SEM ( Fig. 8a-f). Remarks: The large elliptical pores reported by Lisi et al. 13 as located laterally to the mouth in C. revelator are also present in C. crenulatus, however their exact position and shape could not be determined as the mouth was retracted in almost all analysed specimens.  www.nature.com/scientificreports/ Claws slender, of the Richtersiidae type. Primary branches with distinct accessory points, a long, constricted in the middle, common tract with a system of internal septa, and with an evident stalk connecting the claw to the lunula ( Fig. 9a-h). The common tract apparently longer than the half of the entire claw height (Fig. 9a,d,g-h). Large, comb-like, triangular lunulae with long and evenly distributed teeth present on all legs ( Fig. 9a-h). Under PCM, the lower portion of the lunulae just above the dentation is evidently darker and visible as a dark arc ( Fig. 9a-b, d-e). The lunulae are curved and clamped around the cuticular swelling/thickening present under them ( Fig. 9g-h) what is well visible in SEM whereas in PCM on the lower focal plane it is visible as a darkening beneath the lunules (Fig. 9c,f). Paired muscle attachments present just below lunulae on legs I-III (Fig. 9a).
Mouth antero-ventral. Bucco-pharyngeal apparatus of modified "Macrobiotus type" (Fig. 10a), i.e. with ten peribuccal lamellae, a rigid buccal tube with the ventral lamina which is provided with an additional ventral thickening in its anterior portion, that appears as an elongated trapezoidal structure pointing towards the mouth www.nature.com/scientificreports/ opening in the ventral view ( Fig. 10c) and is visible as a ridge in the lateral view (Fig. 10d). Based on LCM observations, the oral cavity armature is poorly developed and composed only of the third band of teeth (Fig. 10b,c). The first and the second band of teeth are absent or not visible under LCM (Fig. 10b,c). The teeth of the third band are located within the posterior portion of the oral cavity, anteriorly to the buccal tube opening (Fig. 10b,c). The third band of teeth is divided into the dorsal and the ventral portion. Under LCM, both the dorsal and the ventral portions are seen as two distinct transverse ridges and each of them forms a globular thickening at the medial extremity (Fig. 10b,c). Median teeth absent (Fig. 10b,c). Bulbus spherical (Fig. 10a), with triangular apophyses, three anterior cuticular spikes (typically only two are visible in any given plane) and two rod-shaped macroplacoids (2 < 1) and a microplacoid positioned close to the second macroplacoid (Fig. 10a,e). The first macroplacoid is anteriorly narrowed and constricted in the middle whereas the second has a sub-terminal constriction (Fig. 10e). Table 2): Laid freely, yellowish, spherical with conical processes with elongated apices and egg surface without areolation (Figs. 11a-d, 12a-f). The elongated process apices are often multifurcated into short flexible filaments (Figs. 11a-d, 12a-f). These elongated distal portions of the processes seem to be sometimes separated from the lower portion of the process by a faint septum (Fig. 11a) but this is www.nature.com/scientificreports/ caused a circular thickening on the inner process wall (Fig. 11d). The labyrinthine layer between the process walls is visible as a very faint reticular pattern with circular margins under LCM ( Fig. 11a-d). The faint meshes are visible in the lower part of the processes but are not visible in the elongated upper part ( Fig. 11a-d). The entire surface of processes is smooth under SEM ( Fig. 12a-f). Processes attached to the egg by a ring of short thickenings, seen as dark projections visible only under LCM, which gives the process bases a jagged appearance ( Fig. 11b-c). Only rarely some these projections might be elongated making the impression of connection between two neighbouring processes (Fig. 11b), however this character should be treated with a dose of caution as all the eggs were covered with debris, thus these thin connectors may be an artefact. Besides these structures, egg surface between processes appears as smooth under LCM (Fig. 11a-d), whereas it is slightly wrinkled in SEM (Fig. 12a-e).

Eggs (measurements and statistics in
Reproductive mode. The examination of 28 adults freshly mounted in Hoyer's medium revealed no testes or spermathecae filled with spermatozoa, which suggests that the species is (at least facultatively) parthenogenetic.
DNA sequences. We obtained sequences for all four of the above-mentioned molecular markers from one of the two individuals destined for DNA extraction and sequencing, which are as follow: 18S rRNA (Gen-Bank: MT812474), 994 bp long; 28S rRNA (MT812468), 735 bp long; ITS-2 (MT812606), 398 bp long; COI (MT808079), 629 bp long.
Phenotypic differential diagnosis. To date, the genus Crenubiotus comprises only two species: the nominal C. crenulatus and an extremely similar species, C. revelator, recently described from Colombia. Despite the overall similarity, C. crenulatus differs from C. revelator by: the absence of the circular median tooth in the ventral portion of the third band of teeth in the oral cavity (the median tooth present in C. revelator), the presence of only two lateral teeth in the ventral portion of the third band of teeth, which have thickenings in their medial

Discussion
By the analysis of both morphological and molecular data, we explicitly demonstrated the presence of a previously unrecognised phyletic lineage within the superfamily Macrobiotoidea which comprises two genera, Adorybiotus and Crenubiotus, for which genetic data were extremely limited or absent. To accommodate the phylogenetic and morphological distinctiveness of this group from the remaining three families within the Macrobiotoidea, we erected the new family Adorybiotidae fam. nov. Furthermore, in order to enhance taxonomic studies on the recently erected genus Crenubiotus and stabilise its nomenclature, we provided an integrative redescription of C. crenulatus based on the population from original terra typica and replaced the existing, inadequate neotype with the new one that is associated with DNA barcodes. Two genera analysed in this study, Murrayon in the family Murrayidae and Adorybiotus in Adorybiotidae fam. nov., appear to be paraphyletic. As already shown by Bertolani et al. 6 , Murrayon cf. pullari IT.338 is more closely related to Dactylobiotus than to Murrayon dianae. However, the paraphyly should be treated with great caution because the M. dianae branch is exceptionally long, thus it could lead to topological artefacts. The unbalanced sequencing may also be the cause behind the paraphyly of Adorybiotus, as only the 18S rRNA was sequenced for Adorybiotus granulatus in Bertolani et al. 6 , in contrast to the other analysed populations of the Adorybiotidae fam. nov., for which a complete set of four markers was available. Thus, a better sampling within Murrayon and Adorybiotus, both in terms of the number of species and sequenced markers, is needed to verify the phyletic relationships within the two genera.
As was pointed out in the Introduction, thanks to the easier acquisition of genetic data and their use in phylogenetic studies, the relationships between major evolutionary lineages within the phylum Tardigrada are www.nature.com/scientificreports/ being gradually resolved. The increasing popularity of integrative taxonomy also contributed to the recognition of considerable and yet undescribed species diversity within this animal group, e.g. 10,11,[24][25][26] . The recent years showed several times that old, outdated and inadequate tardigrade species descriptions or redescription are often the major obstacle in resolving the systematics within genera and species complexes 24,25,27-31 . In the   31 . Importantly, Crenubiotus crenulatus (Richters, 1904) 17 , the type species of its genus, suffers from a similar problem. The species was originally described from the Svalbard Archipelago (specifically from Smeerenburg on Spitsbergen), where it seems to be a common element of the tardigrade fauna, e.g. [34][35][36][37][38] . Nonetheless, the diagnosis of this species has been questioned for many years, specifically concerning its possible synonymy with Macrobiotus echinogenitus Richters, 1904, also originally described from the Svalbard Archipelago 13,17,39 . The issue has been clarified to some extent by Binda 40 41 . This requirement was in force already when Binda 40 made her designation 42 . Second, the neotype series is in a bad condition, which prevents a detailed morphological characterisation of the species 13 . Finally, taking into consideration the morphological similarity between C. crenulatus and C. revelator, which has been highlighted in this study and in Lisi et al. 13 , the future taxonomic studies on the genus Crenubiotus will be challenging without the use of DNA barcodes. Therefore, in order to stabilise the taxonomy and nomenclature within Crenubiotus, in agreement with to the International Code of Zoological Nomenclature, we established a new neotype from a population found at 180 km from the original locus typicus, on the same archipelago. With an integrative redescription that comprises detailed morphological data and associated DNA barcodes, future species identification will be much less problematic that it would have been with Binda types from northern Italy. Furthermore, as the same rule of ICZN that was mentioned above was violated when establishing the neotype of M. echinogenitus based on the population from Algeria, which is ca. 5500 km away from the Svalbard Archipelago, we propose to consider this designation as invalid too. As the original description of M. echinogenitus from Richters (1903) 32 is vague and there has been confusion around its identity 40 , we suggest caution in assigning an individual to this species until it is redescribed with material from the original locus typicus.
As obstacles in the form of incomplete and outdated descriptions of the type species for Richtersius and Crenubiotus (Richtersiidae and Adorybiotidae fam. nov., respectively) have been now removed by Stec et al. 31 and this study, respectively, attention should be paid to similar issues in the remaining two genera, Adorybiotus and Diaforobiotus. In this study, we presented some details of Adorybiotus morphology and genetics, but they were all based on an undetermined species from Japan that cannot be confidently identified until Adorybiotus granulatus (Richters, 1903) 32 , the type and the only species of the genus, is redescribed by means of integrative taxonomy. The species was described originally from Norway (Merok), and later redescribed by Maucci and Ramazzotti 12 based also on a population from Norway (Steinkjer). Importantly, however, this designation can be questioned as Maucci and Ramazzotti 12 did not fulfil properly the condition given in Article 75.3.4 of the Code 41 , which requires mentioning the "reasons for believing the name-bearing type specimen(s) (i.e. holotype, or lectotype, or all syntypes, or prior neotype) to be lost or destroyed, and the steps that had been taken to trace it or them" (this requirement was also in force already when Maucci and Ramazzotti 12 made their designation 42 ). This opens up the possibility of an extensive taxonomic study on A. granulatus that would result in an integrative redescription and a designation of a new neotype, when a suitable population from central Norway is found. The genus Diaforobiotus comprises currently only two subspecies D. islandicus islandicus (Richters, 1904) 43 (the Table 2. Measurements [in µm] of selected morphological structures of the eggs from the neotype population of C. crenulatus s.s. (Richters, 1904) mounted in Hoyer's medium (N-number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD-standard deviation). www.nature.com/scientificreports/ nominal subspecies) and D. islandicus nicaraguensis (Séméria, 1985) 44 . For years, numerous records of D. islandicus islandicus accumulated in the literature from localities all over the world [45][46][47][48] but it has been demonstrated by Guidetti et al. 11 as well as by this study that the genus definitely comprises more than one species. Nonetheless, since the types of D. islandicus islandicus do not exist and morphological details of the species are unknown, we consider descriptions of new taxa highly hazardous until the type species is redescribed by means of integrative taxonomy. The Diaforobiotus population from Iceland analysed in this study is a suitable candidate for the neotype population, which together with two other populations from Norway and Indonesia will be revised by us in the near future and published as an integrative revision of the genus. Such integrative redescriptions of the type taxa have opened and will continue to open the windows for describing species diversity within genera or species groups/complexes, further contributing to our understanding of evolution of microscopic invertebrates, including tardigrades.

Material and methods
Samples and specimens. To reconstruct the phylogeny of Richtersiidae and Murrayidae, along with already published data, we analysed eight new populations representing eight species isolated from moss or pond sediment samples collected from eight distinct localities (see Table 3 for details). In our study, by a population we mean a group of conspecific individuals found in a single sample. All samples were processed following a protocol described in detail in Stec et al. 49 .
DNA sequencing. Genomic DNA was extracted from individual animals following a Chelex 100 resin (Bio-Rad) extraction method by Casquet et al. 50 with modifications described in detail in Stec et al. 51 . Each specimen was mounted in water on a temporary microscope slide and examined under light microscope prior to DNA extraction. We sequenced four DNA fragments, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) from 2-4 individuals per each of the six newly analysed populations. All fragments were amplified and sequenced according to the protocols described in Stec et al. 51 ; primers with their original references are listed in Table 4 28 and Ramazzottius subanomalus (Biserov, 1985) 55 as outgroups. We choose isolates from the families Richtersiidae and Murrayidae with all four sequenced markers (18S rRNA, 28S rRNA, ITS-2 and COI) and isolates that overlap with the sequences produced in this study in the cases of 18S rRNA and 28S rRNA. However, there were four exceptions to this: two Adorybiotus granulatus (Richters, 1903) 32 isolates, with only 18S rRNA sequences (HQ604961 and HQ604962), that were included as they are the only available sequences for the nominal species of the genus Adorybiotus (although the species identification is uncertain; see the Discussion for details). The other two exceptions were Murrayon dianae (Kristensen, 1982) 54 and Dactylobiotus ovimutans Kihm et al., 2020 56 that were included to www.nature.com/scientificreports/ have a better representation of the Murrayidae. For the family Macrobiotidae, one to two species of each genus, for which sequences are available in GenBank, were included in the analysis (see Table 5 for GenBank accession numbers). In the analysed dataset, 82% (31/38) terminals had sequences for all four markers. The 18S rRNA, 28S rRNA and ITS-2 sequences were aligned using MAFFT ver. 7 57,58 with the G-INS-i method (thread = 4, threadtb = 5, threadit = 0, reorder, adjustdirection, anysymbol, maxiterate = 1000, retree 1, globalpair input). The COI sequences were aligned according to their aminoacid sequences (translated using the invertebrate mitochondrial code) with the MUSCLE algorithm 59 in MEGA7 60 with default settings (all gap penalties = 0, max iterations = 8, clustering method = UPGMB, lambda = 24). Alignments were visually inspected and trimmed in MEGA7. Aligned sequences were concatenated with an in-house R script provided by MV. Model selection and phylogenetic reconstructions were done on the CIPRES Science Gateway 61 . Model selection was performed for each alignment partition (6 in total: 18S rRNA, 28S rRNA, ITS-2 and three COI codons) with PartitionFinder2 62 , partitions and models selection process and results are present in Supplementary Data S1. The BI phylogenetic reconstruction was done with MrBayes v3.2.6 63 without BEAGLE. Four runs with one cold chain and three heated chains were run for 20 million generations with a burning of 2 million generations, sampling a tree every 1000 generations. Posterior distribution sanity was checked with the Tracer v1.7 64 and with the R package RWTY 65  Microscopy and imaging. Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's medium and secured with a cover slip, following the protocol by Morek et al. 69 . Slides were examined under an Olympus BX53 light microscope with phase and Nomarski differential interference contrasts (PCM and NCM, respectively; named collectively as light contrast microscopy, LCM), associated with an Olympus DP74 digital camera. In order to obtain clean and extended specimens for SEM, tardigrades were processed according to the protocol by Stec et al. 49 . Specimens were examined under high vacuum in a Versa 3D DualBeam Scanning Electron Microscope (SEM) at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For structures that could not be satisfactorily focused in a single LCM photograph, a vertical stack of 2-6 images were taken with an equidistance of ca. 0.2 μm and assembled manually into a single deep-focus image in Corel Photo-Paint.
Morphometrics and nomenclature. All measurements are given in micrometres (μm). Sample size was adjusted following recommendations by Stec et al. 70 . Structures were measured only if undamaged and their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The terminology used to describe oral cavity armature and egg shell morphology follows Michalczyk and Kaczmarek 71 and Kaczmarek and Michalczyk 72 respectively. Macroplacoid length sequence is given according to Kaczmarek et al. 73 . Buccal tube length and the level of the stylet support insertion point were measured according to Pilato 74 . The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage 74 . Measurements of buccal tube widths, heights of claws and eggs follow Kaczmarek and Michalczyk 72 . Morphometric data were handled using the "Parachela" ver. 1.7 template available from the Tardigrada Register 75 and are given in Supplementary Data S4. Tardigrade taxonomy follows Bertolani et al. 6 with updates from Guidetti et al. 11 , Vecchi et al. 76 and Morek et al. 21 .

Data availability
All data generated and analysed during this study are included in the article (and its Supplementary Information files). DNA sequences are deposited and available in GenBank. Table 4. Primers with their original references used for amplification of the four DNA fragments sequenced in the study. COI sequences for all population were amplified with primer set LCO1490-JJ + HCO2198-JJ except Adorybiotus population (JP.008) for which LCO1490 + HCOoutout set was used.