An integrative description of Macrobiotus shonaicus sp. nov. (Tardigrada: Macrobiotidae) from Japan with notes on its phylogenetic position within the hufelandi group

Tardigrade research in Japan dates back over 100 years, and to date, 167 species of this ecdysozoan phylum have been reported from the country. Of these species, the Macrobiotus hufelandi complex has been represented only by the nominal taxon of this group, Macrobiotus hufelandi. In this article, a new species of the hufelandi group from Japan, Macrobiotus shonaicus sp. nov., is described using integrative taxonomy. In addition to the detailed morphological and morphometric data, obtained using phase contrast light microscopy (PCM) and scanning electron microscopy (SEM), we provide DNA sequences of four molecular markers (both nuclear and mitochondrial). The new species belongs to the persimilis subgroup and is most similar to M. anemone from USA, M. naskreckii from Mozambique, and M. patagonicus from Argentina, but it can be easily distinguished from these species by the presence of thin flexible filaments on terminal discs of the egg process. By the latter character, the new species is most similar to M. paulinae and M. polypiformis, but it can be easily distinguished from them by having a solid egg surface between egg processes (i.e., without pores or reticulum). A phylogenetic analysis of available DNA sequences of the COI marker for the hufelandi group revealed that the new species clusters with the two other species that exhibit filaments on egg process discs (M. paulinae and M. polypiformis) and with two species that have entire egg processes modified into filaments (M. kristenseni and M. scoticus). All five species form a clade distinct from all other sequenced species of the hufelandi group with typical mushroom- or inverted goblet-shaped egg processes, which may suggest that the ancestor of the five species with atypical egg processes had a mutation allowing derivations from the mushroom or inverted chalice-like shape of egg processes.


Introduction
Terrestrial tardigrades are micrometazoans most commonly found in mosses, lichens, leaf litter and soil [1]. The phylogenetic position of the phylum Tardigrada is still uncertain, with PLOS  some studies placing this phylum as a sister group of Arthropoda and Onychophora within the megaclade Panarthropoda, and some others placing tardigrades as a sister group to Nematoda [2 and the literature cited therein]. Until now, over 1200 species have been known to reside within this phylum [3], and approximately twenty new species are described each year [4]. The first report on tardigrades from Japan come from a zoology textbook by Iijima [5]. Currently, Japanese tardigrade fauna include 167 species, of which 26 were originally described from Japan [6]. The family Macrobiotidae is represented by 16 species, but some recordssuch as Mesobiotus harmsworthi (Murray, 1907) [7], Minibiotus intermedius (Plate, 1888) [8], Paramacrobiotus areolatus (Murray, 1907) [7], Paramacrobiotus richtersi (Murray, 1911) [9] and Macrobiotus hufelandi C.A.S. Schultze, 1834 [10]-should be treated with great caution since they are nominal taxa for species complexes for which the original descriptions are incomplete and outdated, making the exact identification almost impossible. The globally distributed Macrobiotus hufelandi complex has been represented until now only by the nominal taxon of this group, M. hufelandi, and no other hufelandi species has been reported from Japan to date [6].
In this article, we describe a new tardigrade species of the hufelandi group, Macrobiotus shonaicus sp. nov., from Japan. The integrative description and species delineation involved morphological and morphometric data obtained using phase contrast light microscopy (PCM) and scanning electron microscopy (SEM) as well as molecular data in the form of DNA sequences for four molecular markers (nuclear: 18S rRNA, 28S rRNA, ITS-2, and mitochondrial COI). The new species belongs to the persimilis subgroup (with a solid egg surface between processes), but it has modified egg processes with some teeth of terminal discs elongated to flexible filaments, such as those found in two other recently described species: Macrobiotus paulinae Stec, Smolak, Kaczmarek & Michalczyk, 2015 [11] from Africa and Macrobiotus polypiformis Roszkowska, Ostrowska, Stec, Janko & Kaczmarek, 2017 [12] from South America.

Sample processing and tardigrade culturing
A sample of moss Bryum argenteum growing on a car park's concrete surface containing the new species was collected from Otsuka-machi, Tsuruoka-City, Japan (38˚44'24"N, 1394 8'26"E; 13 m asl) in May 2016 by KA. The place of collection was a parking lot of an apartment KA rented, and specific permission was not required. The authors confirm that our sampling did not involve endangered or protected species. The sample was collected and examined for terrestrial tardigrades using a stereo microscope SZ61 (Olympus). Ten individuals of the new species were extracted from the sample and placed in an in vitro culture in five separate pairs. Only one of the five cultures successfully proliferated. Specimens of this isogenic strain were reared using a previously described protocol for Hypsibius dujardini (Doyère, 1840) [13,14]. Briefly, tardigrades were fed Chlorella vulgaris (Chlorella Industry) on 2% Bacto Agar (Difco) plates prepared with Volvic water, and the plates were incubated at 18˚C under constant darkness. Culture plates were renewed every 7-8 days.

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 established by Morek et al. [15]. Slides were then dried for five days at 60˚C. Dried slides were sealed with a transparent nail polish and examined under a Nikon Eclipse 50i phase contrast light microscope (PCM) associated with a Nikon Digital Sight DS-L2 digital camera. To obtain clean and extended specimens for SEM, tardigrades were processed according to the protocol established by Stec et al. [11]. In short, specimens were first subjected to a 60˚C water bath for 30 min to obtain fully extended animals, followed by a water/ethanol and an ethanol/acetone series, and then followed by CO 2 critical point drying. Specimens were finally sputter coated with a thin layer of gold. Specimens were examined under high vacuum in a Versa 3D DualBeam Scanning Electron Microscope at the ATOMIN facility of Jagiellonian University, Kraków, Poland. The type population was also examined for the presence of males using aceto-orcein staining following Stec et al. [16]. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For deep structures that could not be fully focused in a single photograph, a series of 2-8 images were taken every ca. 0.2 μm and then assembled manually into a single deep-focus image.

Morphometrics and morphological nomenclature
All measurements are given in micrometers (μm). Sample size was adjusted following recommendations by Stec et al. [17]. Structures were measured only if 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 (OCA) follows Michalczyk & Kaczmarek [18]. Buccal tube length and the level of the stylet support insertion point were measured according to Pilato [19]. Buccal tube width was measured as the external and internal diameters at the level of the stylet support insertion point. Macroplacoid length sequence is given according to Kaczmarek et al. [20]. The lengths of the claw branches were measured from the base of the claw (i.e., excluding the lunula) to the top of the branch, including accessory points [21]. The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage [19]. The distance between egg processes was measured as the shortest line connecting the base edges of the two closest processes [21]. Morphometric data were handled using the "Parachela" ver. 1.2 template available from the Tardigrada Register [4]. Tardigrade taxonomy follows Bertolani et al. [22].

Genotyping
DNA was extracted from individual animals following a Chelex 1 100 resin (Bio-Rad) extraction method established by Casquet et al. [34] with modifications described in detail in Stec et al. [11]. We sequenced four DNA fragments differing in mutation rates (from the most to least conservative): the small ribosome subunit (18S rRNA, nDNA), the large ribosome subunit (28S rRNA, nDNA), the internal transcribed spacer (ITS-2, nDNA), and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified and sequenced according to the protocols described in Stec et al. [11]; primers and original references for specific PCR programs are listed in Table 1. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University, Kraków, Poland. Sequences were processed in BioEdit ver. 7.2.5 [35] and submitted to GenBank.

Comparative analysis
For molecular comparisons, all the published sequences of the four abovementioned markers for species of the hufelandi group were downloaded from GenBank (listed in Table 2). The sequences were aligned using the default settings in MAFFT version 7 [42,43] and manually checked against non-conservative alignments in BioEdit ver. 7.2.5 [35]. Then, the aligned sequences were trimmed to 773 (18S rRNA), 711 (28S rRNA), 307 (ITS-2), and 621 (COI), bp. All COI sequences were translated into protein sequences in MEGA7 [44] to check against pseudogenes. Uncorrected pairwise distances were calculated using MEGA version 7.0 [44]. Despite the fact that genetic distances in barcoding studies are frequently calculated in accordance with the Kimura 2 parameter model, as proposed by Hebert et al. [45], the more recent work by Srivathsan & Meier [46] showed that this model of nucleotide evolution is poorly justified. Moreover, Srivathsan & Meier [46] showed that uncorrected p-distances may provide a comparable or even a higher success rate of taxon delimitation than distances computed under the K2P. Therefore, we used basic p-distances in all our analyses.

Phylogenetic analysis
To verify the phylogenetic position of the new species, a phylogenetic tree was constructed on published COI sequences of species from the hufelandi group with three Milnesium species as the outgroup (see Table 2 for references). Since the COI is a protein-coding gene, before partitioning, we divided our alignment into three data blocks constituting three separate codon positions using PartitionFinder version 2.1.1 [47] under the Bayesian Information Criterion (BIC). The best schemes for partitioning and substitution models were chosen for posterior phylogenetic analysis. First, we ran the analysis to test all possible models implemented in the program. As a best-fit partitioning scheme, PartitionFinder suggested retaining three predefined partitions separately. The best-fit models for these partitions were F81+I for the first codon position, TRN+G for the second codon position and SYM+I for the third codon position. Since RAxML [48] allows only a single model of rate heterogeneity (of the GTR family) in partitioned analyses, we additionally tested GTR, GTR+I, GTR+G and GTR+I+G using Parti-tionFinder. The best-fit model for all partitions in this analysis was GTR+G.
Maximum-likelihood (ML) topologies were constructed using RAxML v8.0.19 [48]. The strength of support for internal nodes of ML construction was measured using 1000 rapid  [65]. Random starting trees were used, and the analysis was run for eight 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 [66] 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. A consensus tree was obtained after summarizing the resulting topologies and discarding the 'burn-in'. Based on the BI consensus tree, clades recovered with a posterior probability (PP) between 0.95 and 1 were considered well supported, those

Data deposition
Raw morphometric measurements underlying the description of Macrobiotus shonaicus sp. nov. are given in supplementary materials (S1 File) and are additionally deposited in the Tardigrada Register [4] under www.tardigrada.net/register/0051.htm. The DNA sequences for the type population are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank). Uncorrected pairwise distances are given in the supplementary materials (S2 File).

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:FE5D4FE7-11F9-48C9-B1E2-9EA516CCBD08. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.  Table 3). Body white in juveniles and slightly yellowish in adults, transparent after fixation in Hoyer's medium (Fig 1A). Eyes present in live animals (dissolved 33% of specimens mounted in Hoyer's medium). Small round and oval pores (0.2-0.4 μm in diameter), invisible under PCM, but clearly visible under SEM, scattered randomly on the entire dorso-lateral cuticle (Fig 1B-1E), including the external and internal surface of all legs (Fig 2C, 2D and 2F, indented arrowheads). Cuticular granulation present on external surface of all legs (Fig 2A-2D). A cuticular bulge/fold resembling a pulvinus is present on the internal surface of all legs I-III (Fig 2E and 2F, flat filled arrowhead), whereas just above the claws, a faint cuticular fold is also present (Fig 2E and 2F, flat empty arrowhead). Both structures are visible only if the legs are fully extended and well oriented on the slide, especially the cuticular fold above the claws.

Description of the new species Animals (measurements and statistics in
Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type, with the ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes (Figs 3A, 4A and 4B). An irregular ring of pores, visible only in SEM, is present around the mouth opening, immediately behind the peribuccal sensory lobes (Fig 4B, arrow). Under PCM, the oral cavity armature is of the maculatus type (only the third band of teeth visible under PCM) in smaller specimens and of the patagonicus type (only the second and third band of teeth visible under PCM) in larger specimens. However, in SEM the oral cavity is always composed of three bands of teeth, i.e., under PCM only the third band or second and third bands of teeth are visible ( Fig  3B-3D), whereas all three bands are always detectable in SEM (Fig 4C and 4D). The first band of teeth is composed of numerous extremely small cones arranged in one to two rows situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae (Fig 4C and 4D, flat filled arrowhead). The second band of teeth is situated between the ring fold and the third band of teeth and comprises 4-5 rows of small cones, slightly bigger than those of the first band ( Fig 4C and 4D, empty flat arrowhead). Under PCM, only the bigger teeth of second band are visible, and they often appear as a single smudge rather than separate teeth (Fig 3D  empty flat arrowhead). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Fig 4C and  4D). The third band of teeth is discontinuous and divided into the dorsal and the ventral portions. Under PCM, the dorsal teeth form a single transversal ridge with poorly visible thickenings, whereas the ventral teeth appear as two separate lateral transversal ridges between which a roundish median tooth is sometimes visible (Fig 3B-3E). However, in SEM, both dorsal and ventral teeth form two single ridges (Fig 4C and 4D), although with peaks. The dorsal ridge has two larger lateral peaks and several smaller median peaks and indentations (Fig 4C), which correspond to thickenings sometimes visible in this band under PCM (Fig 3B and 3D). The ventral ridge has two evident peaks corresponding with two lateral teeth, between which the ventral ridge is reduced/indented and only slightly serrated (Fig 4C). This median portion can be seen as roundish median tooth under PCM, especially in larger specimens (Fig 3E). Pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids and a triangular small microplacoid (Fig 3A). Macroplacoid length sequence 2<1. The first and the second macroplacoid have a constriction, central and subterminal, respectively (Fig 3A, upper insert). Claws small and slender, of the hufelandi type (Fig 5A-5D). Primary branches with distinct accessory points, a long common tract, and with an evident stalk connecting the claw to the lunula (Fig 5A-5D). Lunulae on legs I-III smooth (Fig 5A and 5C), whereas on legs IV, the lunules are sparsely dentate (Fig 5B and 5D). Cuticular bars under claws absent.
The population type is dioecious. Males were discovered using aceto-orcein staining, which revealed testicles filled with spermatozoa. The dioecism (gonochory) was also confirmed experimentally by isolating virgin individuals that have never reproduced, suggesting that females cannot reproduce parthenogenetically. No morphological secondary sexual dimorphism, such as gibbosities on legs IV in males, was identified.
Eggs (measurements and statistics in Table 4). Laid freely, white/light yellow, spherical or slightly oval (Figs 6A, 6B, 7A and 7B). The surface between processes of the persimilis type, i.e., chorion surface solid, without pores or reticulum, covered by irregularly shaped and sized convex cushion-like platforms (Fig 7C and 7D). Superimposed on these platforms, and at their Table 4  margins, are occasional vein-like folds and extensions (Fig 7E). The complex and intricate intersection of these platforms (Fig 7D and 7E) forms the dark dots seen under PCM (Fig 6F).

. Measurements [in μm] of selected morphological egg structures of Macrobiotus shonaicus sp. nov. mounted in Hoyer's medium (N-number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; and SD-standard deviation
Processes are in the shape of inverted goblets with slightly concave conical trunks and welldefined terminal discs (Figs 6B, 6D and 7C-7F). Terminal discs are indented (cog-shaped) with a concave central area and with 10-15 small irregular teeth (Figs 6E and 7C-7F). Almost all teeth on the terminal disc are elongated into thin flexible filaments, less than 0.2 μm in diameter and 2-5 μm in length (Figs 6C-6E, flat filled arrowhead and 7C-7F). The filaments are hair-like under PCM, but under SEM, they are covered with fine granulation (Fig 7E and  7F), so they probably enhance the adhesive function of egg processes. Under PCM the filaments are sometimes barely visible or even invisible. The most likely explanation for this is that the filaments are very fragile and easily broken; thus, they are not present on some eggs due to mechanical damage. The filaments are very thin, so they could be overlooked and/or misinterpreted as debris attached to eggs. Thus, extreme care must be taken when examining the eggs to avoid incorrect conclusions. DNA sequences. We obtained very good quality sequences for all four molecular markers from all four analyzed specimens (paragenophores). The 18S rRNA and 28S rRNA sequences were represented by single private haplotypes, whereas the ITS-2 and COI were represented by two private haplotypes differing in two (p-distance: 0.9%) and eight variable sites (p-distance: 1.0%), respectively: The 18S rRNA sequence (GenBank: MG757132), 1038 bp long :  TAGATCGTAATCTTACACGGATAACTGTGGTAATTCTAGAGCTAATACGTGCAACCAGCTC  GTTCCCTTGTGGAGCGAGCGCAGTTATTAGAACAAGACCAATCCGGCCTTCGGGTCGGTAC  AATTGGTGACTCTGAATAACCGAAGCGGAGCGCATGGTCTCGTACCGGCGCCAGATCTTTC  AAGTGTCTGACTTATCAGCTTGTTGTTAGGTTATGTTCCTAACAAGGCTTCAACGGGTAAC  GGGGTATCAGGGTCCGATACCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGG  CAGCAGGCGCGCAAATTACCCACTCCTAGCACAGGGAGGTAGTGACGAAAAATAACGATGCG  AGGGCTAATAGCTTCTCGTAATCGGAATGGGTACACTTTAAATCCTTTAACGAGGATCTAT  TGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAG  TTGCTGCGGTTAAAAAGCTCGTAGTTGGATCTGGGCTTCTGAATGGATGGTTCACTTTAC  GGTGTAACTGTTCGTTTGGTGCCACAAGCCGGCCATGTCTTGCATGCCCTTTACTGGGTG  TGCTTGGCGACCGGAACGTTTACTTTGAAAAAATTAGAGTGCTCAAAGCAGGCGTATGG  CCTTGCATAATGGTGCATGGAATAATGGAATAGGACCTCGGTTCTATTTTGTTGGTTTTCG  GAACTCGAGGTAATGATTAAGAGGAACAGACGGGGGCATTCGTATTGCGGCGTTAGAGGT  GAAATTCTTGGATCGTCGCAAGACGAACTACTGCGAAAGCATTTGCCAAGAATGTTTTCAT  TAATCAAGAACGAAAGTTAGAGGTTCGAAGGCGATCAGATACCGCCCTAGTTCTAACCATA  AACGATGCCAACCAGCGATCCGTCGGTGTTTTTTTTATGACTCGACGGGCAGCTTTCCGG  GAAACCAAAGTGCTTAGGTTCCGGGGGAAGTATGGTTGCAAAGCTGAAACTTAAAGGAAT GACGAA.
The 28S rRNA sequence (GenBank: MG757133), 786 bp long: TACTAAGCGGAGGAAAAGAAACCAACGGGGATGCCGAGAGTAACTGCGAGTGAAATCGGCC AAGCCCAGCGCCGAATCCTGTTGcTGGTGACGGTGACAGGAACTGTGGCGTGAAGAACGTCC TTACCGGTACGGTTTGCGTGCGTAAGTTCTCCTGAGTGAGGCTCCATTCCAAGGAGGGTGC AAGACCCGTATCGCGTGCAACCGGTATCGGTGTAAGATGTTCGGAGAGTCGCCTTGTTTGT    Fig 5. Macrobiotus shonaicus sp. nov.-claws (paratypes). A-B-claws I and IV seen in PCM, with smooth and slightly dentate lunules, respectively and C-D-claws I and IV seen in SEM, with smooth and slightly dentate lunules, respectively. Figs A and B assembled from several photos. Scale bars in μm.
Molecular phylogeny. The phylogenetic analysis of the available COI sequences for the M. hufelandi group unequivocally showed that M. shonaicus sp. nov. indeed belongs to the species complex (Fig 8). However, more interestingly, the analysis also revealed that the new species clusters within a single clade with the two other known species of the group that exhibit flexible filaments on terminal discs of egg processes, i.e., M. polypiformis and M. paulinae (Fig  8). Moreover, the clade clusters with two further species with atypical egg processes for which COI sequences are available, i.e., M. scoticus and M. kristenseni. In these two species, egg processes are strongly modified and do not resemble the typical mushroom-shaped processes found most commonly within the hufelandi complex [21] (Fig 8). The remaining sequenced hufelandi group taxa, all exhibiting typical inverted goblet-shaped egg processes (i.e., M. hufelandi, M. cf. hufelandi, M. macrocalix, M. vladimiri, M. terminalis, and M. sandrae), are grouped within a sister clade. The clades are well supported using Bayesian analysis (Fig 8) but weakly supported using the Maximum Likelihood method. Supports of each node on the ML tree were very small (most of the clades have the bootstrap support value <50, indicating a widespread polytomy). Nevertheless, in both analyses, the five species with modified egg  Table 2 for details on species sequences used in the analysis. Scale bar represents substitutions per position.
https://doi.org/10.1371/journal.pone.0192210.g008 processes always grouped together, suggesting their closer affinity compared to any other hufelandi species included in the analysis, even though they come from four different continents (South America, Africa, Europe and Asia).

Phenotypic differential diagnosis
By the presence of the maculatus or patagonicus OCA type and eggs of the persimilis type, M. shonaicus sp. nov. is most similar to the following species of the hufelandi group: M. anemone, M. naskreckii, and M. patagonicus. However, the new species can be easily distinguished from these species by a having cuticular bulge/fold (resembling a pulvinus) on the internal surface of legs I-III and by thin flexible filaments at the terminal disc of egg process. Moreover, M. shonaicus sp. nov. differs specifically from the following: • M. anemone, reported from its terra typica (Louisiana, USA) and several localities in southeastern USA [23,71,72,73]   The presence of flexible filaments at the terminal disc of egg processes M. shonaicus sp. nov. is similar to two species of the hufelandi subgroup: M. paulinae and M. polypiformis. However, the new species can easily be distinguished from both species by having a solid egg surface between processes (persimilis egg type) instead of the surface covered by the reticulum

Genotypic differential diagnosis
The ranges of uncorrected genetic p-distances between the new species and species of the Macrobiotus hufelandi complex, for which sequences are available from GenBank, are as follows (from the most to the least conservative):

Phylogenetic position within the hufelandi group
According to our phylogenetic analysis, M. shonaicus sp. nov. forms a distinct clade together with other species with modified egg processes, i.e., with M. paulinae, M. polypiformis, M. scoticus and M. kristenseni. Thus, it may be hypothesized that the ancestor of the mentioned species with atypical egg processes might have exhibited a mutation allowing derivations from the inverted goblet-like shape of egg processes. Considering fewer morphological differences between animals of these species compared to those observed in egg morphology, our results show that chorion can evolve faster than animal anatomy, which is consistent with previous studies [16,27,36]. This phenomenon makes egg ornamentation particularly useful for the delineation of closely related species. Even though the BI tree was much better supported than the ML tree, the number of available COI sequences for the hufelandi group species remains rather small. Therefore, a greater effort should be made to increase the sample size to obtain more reliable (and preferably multilocus) phylogenies that would allow testing to determine whether the two lineages revealed in our study indeed represent biological entities.

Conclusions
Thanks to the integrative approach of combining morphological, morphometric and molecular analysis, Macrobiotus shonaicus sp. nov. has been unambiguously delimited from its congeners as a new species. The most characteristic traits of the new species are cuticular folds on the internal surfaces of all legs I-III and eggs with solid surface between processes on which teeth of terminal discs are elongated to thin flexible filaments. Moreover, our phylogenetic analysis showed that species of the Macrobiotus hufelandi complex that exhibit modified egg processes are closely related and form a distinct clade. This is the first original description of the hufelandi group species from Japan, and now, the number of tardigrade species known from this country has increased to 168.