Description of a new species of Tardigrada Hypsibius nivalis sp. nov. and new phylogenetic line in Hypsibiidae from snow ecosystem in Japan

Snow ecosystems are an important component of polar and mountainous regions, influencing water regime, biogeochemical cycles and supporting snow specific taxa. Although snow is considered to be one of the most unique, and at the same time a disappearing habitat, knowledge of its taxonomic diversity is still limited. It is true especially for micrometazoans appearing in snow algae blooming areas. In this study, we used morphological and molecular approaches to identify two tardigrade species found in green snow patches of Mt. Gassan in Japan. By morphology, light (PCM) and scanning electron microscopy (SEM), and morphometry we described Hypsibius nivalis sp. nov. which differs from other similar species by granular, polygonal sculpture on the dorsal cuticle and by the presence of cuticular bars next to the internal claws. Additionally, phylogenetic multilocus (COI, 18S rRNA, 28S rRNA) analysis of the second taxon, Hypsibius sp. identified by morphology as convergens-pallidus group, showed its affinity to the Hypsibiidae family and it is placed as a sister clade to all species in the Hypsibiinae subfamily. Our study shows that microinvertebrates associated with snow are poorly known and the assumption that snow might be inhabited by snow-requiring tardigrade taxa cannot be ruled out. Furthermore, our study contributes to the understanding subfamily Hypsibiinae showing that on its own the morphology of specimens belonging to convergens-pallidus group is insufficient in establishing a true systematic position of specimens.

www.nature.com/scientificreports/ ca. 1400 species of tardigrades have been reported 17 . However, an increasing number of new taxa descriptions is a robust indicator of many water bears still awaiting discovery across the globe 17 (https:// www. tardi grada. net). Owing to the ability of cryptobiosis that is a latent state under which tardigrade metabolism is undetectable, many limnoterrestrial species can withstand unfavourable conditions e.g., freezing 18,19 , high pressure 20 and radiation 21,22 . However, active tardigrade species are found in extreme habitats such as ice or snow 4,16 . Tardigrades play multitrophic roles in ecosystems i.e. some of them may effectively control the population of other metazoans in soil ecosystems, tardigrades on snow feed on algae 4 . The question how many species inhabit snow ecosystems and how they differ from other tardigrades remains open. New taxa of tardigrades in cold environments, like glaciers and ice sheets, have been described in recent years. For example, Cryoconicus with dark-brown pigment and claws of Ramazzotius type were reported from cryoconite granules (dark, oval, biogenic structures on ice; mixture of organic and mineral particles 23,24 ) in glaciers of central Asia 25 , or Cryobiotus with dark pigment, big eyes and modified claws of Hypsibius type from cryoconite holes (water-filled reservoirs on glacial ice 26,27 ) in mountainous glaciers of Europe and Asia [28][29][30][31] . These glacier genera have dark-coloured pigment, which is thought to protect from a high dose of UV radiation 25,32 . Cryoconite holes in the Arctic and Antarctica are also inhabited by transparent tardigrades, some of them representing new species, most probably glacier obligates 33 . Extensive field sampling revealed that some glacier tardigrades are unique and adapted to live only on glaciers 34 . Taking into account all findings from glacial ecosystems and recent findings of tardigrades on snow 4,35 , we decided to identify and provide the description of snow tardigrades to give a baseline for answering a question whether as on glaciers, tardigrades on snow are represented by unique, snow-requiring species.
In this paper, we report two taxa of tardigrades belonging to the Hypsibiidae family from snow ecosystems in Japan, one of which is formally described as a new species. We have analyzed green snow samples collected over two seasons (2019 and 2020) from seasonal snow patches at 750 m a.s.l. in Mt. Gassan in the north of Japan where active tardigrades have already been observed 4 . The first tardigrade found was described by classical methods combining light and scanning electron microscopy imaging, the second was diagnosed by combing light imaging with sequencing of nuclear and mitochondrial DNA fragments, two conservative (18S rRNA, 28S rRNA) and one more variable (COI).

Results and discussion
We identified two taxa of tardigrades found in the blooming of green algae (Chloromonas spp.) on snow surface in Japan (Fig. 1a,b). Their intestine were green which suggest that they feed on Chloromonas spp. Both taxa belong to the one of the most species-rich tardigrade family of Hybsibiidae. According to morphology, both species belong to the Hypsiibinae subfamily. By morphology alone, we described here Hypsibius nivalis sp. nov., and by morphology and DNA we diagnosed the second taxon Hypsibius sp., which according to its morphology (claws of Hypsibius type, two macroplacoids, the lack of cuticular bars, hook-shaped AISM) belongs to the convergens-pallidus group. However, phylogenetic analysis (Bayesian) based on concatenated mitochondrial (COI) and nuclear (18S rRNA, 28S rRNA) molecular markers placed Hypsibius sp. from Mt. Gassan as a sister clade to Hypsibiinae subfamily (Fig. 2). This taxon was found by DNA during two sampling seasons in 2019 and 2020, which suggests its link with green algae blooming on the snow surface.  19, Japan snow no. 1/10" is deposited at the Graduate School of Science and Engineering, Chiba University, Chiba, Japan; 31 paratypes, slide codes: "April, Japan snow no. 1/4, 1/6-1/9, 1/15, 2/1-2/2, R/2, R/4-R6"; SEM stubs codes: "2005GA no. R-1, R" are deposited at the Graduate School of Science and Engineering, Chiba University, Chiba, Japan; and four paratypes, slide codes: "Japan snow 2/1, 2/3, 3/2" are deposited at Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznań.
Etymology. Name nivalis refers to the environment where the species was found -nival in a latin means related to snow.
Teeth in the oral cavity armature absent or not visible under PCM. AISM blunt hook-shaped (Fig. 5a), similar to Mixibius 41 . Stylet supports located in posterior position of the buccal tube. Typical Hypsibius type stylet furcae (Fig. 5a,b). Pharynx with apophyses and with two rod-shaped macroplacoids. Apophyses are big, triangular or square in shape, clearly separated from macroplacoids. Macroplacoid length sequence 2 < 1. The first macroplacoid with constriction, clearly separated from the second one. Microplacoid and septulum absent ( Fig. 5a-d).
Claws of the Hypsibius-type, internal and anterior claws smaller than external and posterior claws respectively ( Fig. 6a-f). Claws with widened bases and with obvious accessory points on the primary branches. Near the border between accessory points and primary claw branch, a thick line is visible along entire branch length        (Fig. 7a,b). The buccal apparatus of the Hypsibius type (Fig. 8a,b). Oral cavity armature either absent or not visible in the PCM (Fig. 8b). The pharyngeal bulb with apophyses, with two rod-shaped macroplacoids ( Fig. 8a-d). Stylet supports located in the posterior position. AISM hook-shaped, as presented for Hypsibius in Pilato 41 (Fig. 8a). Hypsibius type furcae present. The macroplacoid length sequence 2 < 1, microplacoid and septulum absent (Fig. 8a-d). The apophyses clearly separated from the 1st macroplacoids. All macroplacoids clearly separated (Fig. 8c,d). All main branches with accessory points (Fig. 9a-d). Cuticular bars under and between the claws absent. However the thickening under the claw base IV clearly visible (Fig. 9c,d) Remarks on the species. This species belongs to a large group of hypsibiids with smooth cuticle, two macroplacoids and the lack of cuticular bars under the claws I-III 15,33,53 . Although phylogenetic analysis placed Hypsibius sp. from Mt. Gassan as a sister lineage to species of Hypsibiinae (Fig. 2), the formal erection of the species as a new taxa without integrative redescription of the most similar by morphology Hypsibius convergens and Hypsibius pallidus, made an exact morphological differential diagnosis dubious. As it has already been shown by using molecular approaches, the genus Hypsibius is polyphyletic, representing similar morphology but distant genetics among its taxon [53][54][55] . According to DNA, it could be erected as a separate genus, however morphological obstacles do not allow for a proper description in contrast to Cryobiotus or Borealibius which are nested among with other Hypsibius species but differ from them by morphology 29,56 . Therefore, we decided to only present data on the morphology, morphometry and DNA of a potentially new species from snow.

Tardigrades on snow.
Tardigrades have previously been found in snow called "Akashibo" in blooms of algae Hemitoma sp. 57 , and in red snow consisting of algae Chloromonas spp. and Sanguina spp. in North America 5 . In spite of that, they have not been identified. Here, we present the taxonomic description of tardigrades from snow for the first time. Our study shows that snow ecosystems are overlooked for studies on the diversity of microinvertebrates. Although tardigrades are most probably wind-blown, delivered to the snow surface in forests from tree canopies or tree trunks 58,59 , they establish a stable population in snow algae blooming, www.nature.com/scientificreports/ have persisted for multiple seasons, representing different instars and laying eggs 4 . Whether specific species of tardigrades need snow (a low-temperature habitat) for their growth and reproduction or they can be only found in a habitat providing them with food (green algae), and without a high number of competitors (compared to mosses) is an open question and requires future findings. Nevertheless, an increasing number of evidence indicates that tardigrades reproduce and feed on snow algae 4,5 . However, the fate of tardigrades from snow during summer time is unknown and both scenarios, tardigrades are active and reproduce on the snow as well as in mosses after snow melt cannot be ruled out. If these animals need snow ecosystems as a part of their life cycle as was suggested 4 , and like their glacial counterparts 34 , the global disappearance of snow ecosystems 2,60 may trigger negative changes for snow algae blooming associated metazoans.

Material and methods
Sample processing. Snow sampling was conducted at Yumiharidaira park (38°30′N, 140°00′E: altitude 770 m above sea level (a.s.l.)) on Mt. Gassan, Yamagata prefecture in Japan (Fig. 1a), details on sampling sites are provided in Ono et al. 4 . Green snow samples were collected in April and May, 2019 and May, 2020 from seasonal snow in forest area surrounded by beech trees (Fig. 1b). The samples, dimension with 10 × 10 × 2 cm (length × width × depth), were collected using sterile stainless-steel scoop. After sampling, all the samples were   , and three times in 100% for 20 min. After maintained in 100% t-butyl alcohol for 3 h at refrigerator (5℃), specimens were lyophilized by using JFD-320 (JEOL, Tokyo, Japan), then coated with gold and examined using a scanning electron microscope JSM-6010PLUS/LA (JEOL, Tokyo, Japan).

Morphometrics and nomenclature.
Sample size for morphometrics was chosen following recommendations by Stec et al. 63 . All measurements are given in micrometers (μm) and were performed under PCM with Body length was measured from the anterior to the posterior end of the body, excluding the hind legs. The pt ratio is the ratio of the length of a given structure to the length of the buccal tube, expressed as a percentage 64 . Macroplacoid length sequence was determined according to Kaczmarek et al. 65 . Claws were measured following Beasley et al. 66 . Tardigrade taxonomy and systematics is presented according to Bertolani et al. 67 and Degma et al. 17 . Morphometric data were handled using the "Parachela" ver. DNA extraction and amplification. Total genomic DNA was extracted individually from 11 specimens using the DNAeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture instruction. In order to get exoskeletons, after 48 h of digestion and then lysis, 300 ml of a mixture (i.e., ATL buffer-Qiagen, proteinase K, Lysis buffer-Qiagen and 96% ethyl alcohol) with a tardigrade in a 1.5 ml Eppendorf tube was centrifuged at 7000 min −1 . Then, from each tube, 290 ml of the above mixture was carefully removed using a pipette remaining the tardigrade specimen on the bottom in 10 ml of the mixture. The exoskeleton was preserved and then mounted in Hoyer's medium for morphological analysis. A fragment of the cytochrome c oxidase subunit I (COI) gene of mtDNA was amplified with a bcdF01 forward primer (5'-CAT TTT CHACT AAY CAT AAR GAT ATT GG-3') and bcdR04 reverse primer (5'-TAT AAA CYTCDGGATGNCCA AAA AA-3') 69,70 . A sequence of 18S rRNA gene of nDNA was amplified using the following primers 18s_Tar_1Ff (5'-AGG CGA AAC CGC GAA TGG CTC-3') and 18s_Tar_1Rr (5'-GCC GCA GGC TCC ACT CCT GG-3') 71 . D1-D3 region of 28S rRNA gene nDNA was amplified with 28sEUTAR_F (5'-ACC CGC TGA ACT TAA GCA TAT-3') 53  Amplification of 18S rRNA and 28S rRNA nucleotide genes fragments was conducted in a total volume of 10 ml including 5 ml Type-it Microsatellite PCR Kit (Qiagen), 0.5 ml of each primer (10 ng ml −1 ), 0.5 ml Q-Solution (Qiagen) and 3.5 ml of the DNA template. For the COI gene, a total volume of 5 ml was prepared, including 3 ml Type-it Microsatellite PCR Kit (Qiagen), 0.5 ml of each primer (10 ng ml −1 ) and 1 ml of the DNA template. For amplification 18S rRNA and 28S rRNA gene fragments, a thermocycling profile with one cycle of 5 min at 95 °C followed by 38 steps of 30 s each at 95 °C, 60 s at 60 °C, 1 min at 72 °C, and with a final elongation of 5 min at 72 °C was used. While for COI gene fragment amplification, a hybridization was done at 50 °C for 1 min. After amplification, the PCR products were diluted double-fold with RNase-Free water, after that the diluted PCR product was analysed by electrophoresis on 1% agarose gel. Samples containing visible uniform bands with the expected length of the product were purified with Exonuclease I and Fast alkaline phosphatase (Fermentas). The samples were sequenced using the BigDye Terminator v3.1 kit and the ABI Prism 3130xl Genetic Analyzer (Applied Biosystems), following the manufacturer's instructions.
Phylogeny. The phylogenetic analyses were conducted using concatenated nuclear (18S rRNA, 28S rRNA) and mitochondrial (COI) sequences of 35 Hypsibiidae taxa, one representative of Incerta subfamilia (similar in morphology to Hypsibius sp. from Mt. Gassan genus Acutuncus) with Calohypsibius ornatus 73 as the outgroup to hypsibiids. The phylogenetic pipline follows that of recently published robust phylogenies [74][75][76][77] . Sequences were downloaded from GenBank and the full list of accession numbers is given within Supplementary Material 6.  83 implemented within the software we chose the best scheme of partitioning and substitution models for posterior phylogenetic analysis. As the COI is a protein coding gene, before partitioning, we divided our alignment of this marker into 3 data blocks constituting separated three codon positions. Best-fit partitioning schemes and models suggested by PartitionFinder are given within Supplementary Material 7.
Bayesian inference (BI) marginal posterior probabilities were calculated using MrBayes v3.2 84 . Random starting trees were used and the analysis was run for fifteen million generations, sampling the Markov chain every thousand 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 v1.6 85 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 a consensus tree was obtained after summarising the resulting topologies and discarding the 'burn-in' . All final consensus trees were viewed and visualised with  Species delimitation. The species were identified and compared with other taxa based on the previous descriptions 42,43,46,48,49,52 . If the information on the cuticular bars at the claws was not available either in original descriptions or drawings we assumed these structures were absent. Using data sets for COI which includes sequences newly generated in this study, as well as COI sequences from Hypsibius dujardini 36 and Hypsibius exemplaris 53 we performed a genetic species delimitation analyses named the Assemble Species by Automatic Partitioning (ASAP) 86 . The analyses were run on the respective server (https:// bioin fo. mnhn. fr/ abi/ public/ asap/ asapw eb. html) with default settings. Additionally, we run analysis of p-distance in MEGA7 version 7.0 80 for COI of Hypsibius sp. from Mt. Gassan.