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.

Funding: This work was supported by the Sonata Bis programme of the Polish National Science Centre (grant no. 2016/22/E/NZ8/00417 to ŁM): study design, data collection and analysis, decision to publish and preparation of the manuscript; KAKENHI Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) (grant no. 17H03620 to KA): data collection; Yamagata Prefectural Government and Tsuruoka City, Japan (to KA): data collection.

Copyright: © 2018 Stec et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

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 records—such 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 ].

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 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 ].

Materials and methods

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, 139°48’26”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].

Comparative material The taxonomic key for the hufelandi group by Kaczmarek & Michalczyk [21] was used to determine whether the isolated species had already been described. After the species could not be identified with the key, we compared it with the original descriptions of the most similar hufelandi group species, which have solid egg surfaces (persimilis group, 11 species): M. anemone Meyer, Domingue & Hinton, 2014 [23], M. halophilus Fontoura, Rubal & Veiga, 2017 [24], M. hyperboreus Biserov, 1990 [25], M. kazmierskii Kaczmarek & Michalczyk, 2009 [26], M. kristenseni Guidetti, Peluffo, Rocha, Cesari & Moly de Peluffo, 2013 [27], M. marlenae Kaczmarek & Michalczyk, 2004 [28], M. naskreckii Bąkowski, Roszkowska, Gawlak & Kaczmarek, 2016 [29], M. patagonicus Maucci, 1988 [30], M. persimilis Binda & Pilato, 1972 [31], M. polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003 [32], and M. recens Cuénot, 1932 [33]. Moreover, two species that like the new species exhibit flexible filaments at the terminal disc edge of the egg process were used as comparative material in this study: M. paulinae and M. polypiformis.

Genotyping DNA was extracted from individual animals following a Chelex® 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. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Primers and references for PCR protocols for amplification of the four DNA fragments sequenced in the study. https://doi.org/10.1371/journal.pone.0192210.t001

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. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Sequences used for molecular comparisons and phylogenetic analyses of Macrobiotus shonaicus sp. nov. with all other species of the Macrobiotus hufelandi group for which DNA sequences are currently available. https://doi.org/10.1371/journal.pone.0192210.t002

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 PartitionFinder. 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 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 [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 with a PP between 0.90 and 0.94 were considered moderately supported, and those with a lower PP were considered unsupported. All final consensus trees were viewed in and visualized by FigTree v.1.4.3, available from http://tree.bio.ed.ac.uk/software/figtree.

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).