High Mountain Echiniscid (Heterotardigrada) Fauna of Taiwan

© 2021 Academia Sinica, Taiwan Open Access High Mountain Echiniscid (Heterotardigrada) Fauna of Taiwan Piotr Gąsiorek1,*, Katarzyna Vončina1, Reinhardt Møbjerg Kristensen2, and Łukasz Michalczyk1 1Department of Invertebrate Evolution, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland. *Correspondence: E-mail: piotr[email protected] (Gąsiorek). E-mail: [email protected] (Vončina); [email protected] (Michalczyk) 2Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark. E-mail: [email protected] (Kristensen) Received 3 August 2021 / Accepted 13 September 2021 / Published 13 December 2021 Communicated by Daniel Stec Taiwan lies at the transitional zone between the East Palaearctic and Oriental regions, which translates into both Palaearctic and Indomalayan taxa being present on the island. Furthermore, large habitat heterogeneity and high mountains contributed to the rise of conditions favouring allopatric speciation and the emergence of endemic species. The tardigrade fauna of Taiwan is poorly studied, and the aim of this contribution is to provide new data on the members of the family Echiniscidae, the largest limno-terrestrial group of the class Heterotardigrada, found at high elevations in central Taiwan. We report 11 species grouped in 5 genera: Claxtonia (1 species), Echiniscus (3 species), Hypechiniscus (1 species), Nebularmis (2 species), and Pseudechiniscus (4 species). All are new to Taiwan, including 5 species that are new to science, 4 or which are described herein by means of integrative taxonomy: Hypechiniscus crassus sp. nov. (the exarmatus morphogroup), Pseudechiniscus (Meridioniscus) dreyeri sp. nov., Pseudechiniscus (Pseudechiniscus) formosus sp. nov., and Pseudechiniscus (Pseudechiniscus) totoro sp. nov. The new findings also help to clarify the description of Echiniscus clevelandi Beasley, 1999, and supplement the phylogenies of the Echiniscus virginicus complex and of the genera Hypechiniscus, Nebularmis and Pseudechiniscus. Key words: Biogeography, Endemism, Integrative taxonomy, Oriental, Palaearctic, Phylogeny. BACKGROUND Taiwan is a large continental island separated from mainland Asia by the Taiwan Strait. The biogeographic history of Taiwan has been a subject of intense research because of the transitional character of its fauna, which comprises both East Palaearctic and Oriental (Indomalayan) taxa (Päckert et al. 2012; He et al. 2018). The mixed origin of the Taiwanese fauna, coupled with numerous isolated habitats in Taiwanese mountains that favoured speciation (Shih et al. 2006), led to the emergence of endemic biota (e.g., Yu 1995). Considering the potential significance in unravelling biodiversity and biogeographic patterns, not enough attention has been paid to the tardigrade fauna of Taiwan. There are only three reports from the 20th century (Mathews 1936–37; Ito 1990; Séméria 1994), followed by four works from the current century (Li and Li 2008; Yin and Li 2011; Gąsiorek et al. 2019a b). Out of the eight Taiwanese echiniscid records, only four can be considered trustworthy: Echiniscus lineatus Pilato et al., 2008a, Kristenseniscus tessellatus (Murray, 1910), Stellariscus pseudelegans (Séméria, 1994), and Viridiscus perviridis (Ramazzotti, 1959). The remaining four records are most likely misidentifications in the light of present taxonomic knowledge: Pseudechiniscus Citation: Gąsiorek P, Vončina K, Kristensen RM, Michalczyk Ł. 2021. High mountain echiniscid (Heterotardigrada) fauna of Taiwan. Zool Stud 60:70. doi:10.6620/ZS.2021.60-70. Zoological Studies 60:70 (2021) doi:10.6620/ZS.2021.60-70 1 © 2021 Academia Sinica, Taiwan (P.) facettalis Petersen, 1951 and Pseudechiniscus (P.) suillus (Ehrenberg, 1853) (see the criticism of their historical records in Grobys et al. 2020), Echiniscus spinulosus (Doyère, 1840) (this West Palaearctic species represents a species complex and it also exhibits one of the most common chaetotaxy morphotypes within Echiniscus, making the verification of older records virtually impossible), and Viridiscus viridis (Murray, 1910) (see the criticism of its historical records in Pilato et al. 2008b). Such a low reported species richness signifies that a large fraction of Taiwanese echiniscid species diversity remains unknown. Therefore, in order to widen our knowledge on the tardigrade fauna of this biogeographically important region, we analysed mixed moss and lichen samples collected at high elevations in Taiwanese mountains. The material contained numerous echiniscid species, including species new to science. All taxa were analysed under phase contrast microscope (PCM) and, if found in sufficiently high numbers, sequenced (DNA barcoding of five genetic markers). Some of them were additionally observed with a scanning electron microscope (SEM). The genetic data were used in new phylogeny reconstructions of the Echiniscus virginicus group, and of the genera Hypechiniscus, Nebularmis and Pseudechiniscus. Our analyses contribute to a better understanding of Taiwanese tardigrade fauna and the biogeographic origin of some of its representatives. MATERIALS AND METHODS Sample collection, animal preparation and microscopy Animals were extracted from seven Taiwanese moss samples (collected by Niklas Dreyer) and an additional Japanese (collected by Szymon Bacher) moss sample (Table 1) according to standard protocols (Dastych 1980; Stec et al. 2015). Isolated specimens were used for the following analyses: (I) imaging in phase contrast microscopy – PCM (morphology and morphometry), (II) imaging in scanning electron microscopy – SEM (ultrastructure), and (III) DNA Table 1. List of examined samples and identified tardigrade species Sample code Coordinates and altitude Locality Species Collection date JP.009 35°24'27''N 139°09'56''E 338 m asl Japan, Kanagawa Prefecture, Tanzawa Mountains, Tanodotoke trail Echiniscus hoonsooi 17.12.2017 TW.004 24°23'00''N 121°13'48''E 3 000 m asl Taiwan, Snow Mountain (Xueshan) EMPTY 17.01.2019 TW.005 24°23'18''N 121°15'39''E 3 200 m asl Taiwan, Snow Mountain (Xueshan), East Peak Echiniscus clevelandi Hypechiniscus crassus sp. nov. Pseudechiniscus dreyeri sp. nov. Pseudechiniscus totoro sp. nov. 10.02.2020 TW.006 24°23'18''N 121°15'39''E 3 200 m asl Taiwan, Snow Mountain (Xueshan), East Peak Echiniscus clevelandi Hypechiniscus crassus sp. nov. 10.02.2020 TW.007 24°23'51''N 121°14'04''E 3 700 m asl Taiwan, Snow Mountain (Xueshan), North Peak Claxtonia sp. nov. Echiniscus blumi Echiniscus clevelandi Nebularmis reticulatus Pseudechiniscus formosus sp. nov. 10.02.2020 TW.008 24°23'51''N 121°14'04''E 3 700 m asl Taiwan, Snow Mountain (Xueshan), North Peak Echiniscus clevelandi Hypechiniscus crassus sp. nov. Nebularmis crebraclava Pseudechiniscus dreyeri sp. nov. 10.02.2020 TW.009 24°23'51''N 121°14'04''E 3 700 m asl Taiwan, Snow Mountain (Xueshan), North Peak Echiniscus blumi Echiniscus semifoveolatus Nebularmis reticulatus 10.02.2020 TW.010 24°10'51''N 121°18'36''E 2°500 m asl Taiwan, Joy Mountain (Hehuanshan) Pseudechiniscus ehrenbergi 31.12.2020 page 2 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan sequencing. Specimens for PCM were mounted on microscope slides in Hoyer’s medium and secured with cover slips. Slides were examined under an Olympus BX53 PCM associated with an Olympus DP74 digital camera. Specimens for SEM were processed in accordance with the protocol from Stec et al. (2015) and examined under high vacuum in a Versa 3D DualBeam SEM at the ATOMIN facility of the Jagiellonian University. All figures were assembled in Corel PhotoPaint X8. A stack of 2–10 images were taken with an equidistance of ca. 0.1 μm for some structures and assembled manually into a single deep-focus image in Corel. Morphometry and terminology All measurements were made under ×1000 magnification with immersion oil and are given either in micrometres (μm) or as relative values presented in the text in italics (sp – the ratio between a length of a given structure and the scapular plate length; Dastych 1999). Structures were measured only if suitably oriented, undamaged and untwisted. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. Morphological terminology follows Kristensen (1987) with subsequent modifications introduced in Gąsiorek et al. (2019a 2021a b c). Body appendages are all appendages in lateral, dorsolateral and dorsal positions (i.e., A, B, C, D and E), whereas trunk appendages exclude appendages A, which are situated at the border of head and trunk. Morphometric data were handled using the “Echiniscoidea” ver. 1.4 template available from the Tardigrada Register (Michalczyk and Kaczmarek 2013). Raw morphometric data for analysed species are provided as supplementary materials (SM.1–5) and in the Tardigrada Register. Tardigrade taxonomy is presented in accordance with the latest edition of the checklist by Degma et al. (2021). Genotyping Individual DNA extractions were made from animals and cysts following a protocol by Casquet et al. (2012) modified in Stec et al. (2020). Hologenophores (Pleijel et al. 2008) were mounted on permanent slides for post-hoc observations. Five DNA fragments were sequenced: the small ribosome subunit (18S rRNA, nDNA), the large ribosome subunit (28S rRNA, nDNA), the internal transcribed spacers (ITS-1 and ITS-2, nDNA), and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified using the primers and PCR programmes listed in SM.6. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University. Sequences were processed in BioEdit ver. 7.2.5 (Hall 1999) and submitted to GenBank (for the accession numbers please see RESULTS). Phylogenetics The sequences were aligned using the default settings of BioEdit (in the case of ITS and COI) and the Q-INS-I method (in the case of ribosomal markers: 18S rRNA, 28S rRNA) of MAFFT7 (Katoh et al. 2002; Katoh and Toh 2008) and manually checked against non-conservative alignments in BioEdit. All COI sequences were translated into protein sequences in MEGA7 (Kumar et al. 2016) to check against pseudogenes. Concatenation was done in SequenceMatrix (Vaidya et al. 2011). Details on the phylogenetic reconstructions for each specific dataset are provided below. The Echiniscus virginicus complex A dataset of ITS-1, ITS-2 and COI from Gąsiorek et al. (2020) was used. The final alignment length was 1725 bp. Using PartitionFinder v.2.1.1 (Lanfear et al. 2017) under the Bayesian information criterion (BIC), the best scheme of partitioning and substitution models for posterior phylogenetic analysis were chosen. The analysis was run to test all possible models implemented in MrBayes. As COI is a protein-coding gene, before partitioning, we divided our alignments of this marker into three data blocks constituting three separate codon positions. GTR+G was inferred to be the best-fit model for the first coding site of COI and a joined ITS-1+ITS-2 partition, GTR+I – for the second coding site of COI, and HKY+G – for the third coding site of COI. Bayesian inference (BI) marginal posterior probabilities were calculated using MrBayes v.3.2 (Ronquist and Huelsenbeck 2003). Random starting trees were used and the analysis was run for ten 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 v.1.6 (Rambaut et al. 2014) was then used to ensure that 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 summarizing the resulting topologies and discarding the ‘burn-in’. All final consensus trees were visualised by FigTree v.1.4.3, available from http://tree.bio.ed.ac.uk/ software/figtree. The parameters and programmes were page 3 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan identical in the latter datasets if not specified otherwise. Hypechiniscus phylogeny A dataset of 18S rRNA, 28S rRNA and ITS-1 from Gąsiorek et al. (2021a) was used. The final alignment length was 2363 bp. PartitionFinder indicated the following models for predefined partitions: TRN+I+G (18S rRNA), GTR+G (28S rRNA) and TVM+I (ITS1). These models were used in BI reconstructions in MrBayes. ModelFinder (Kalyaanamoorthy et al. 2017) was used to choose the best-fit models for Maximum Likelihood (ML) analyses—K2P+I+G4 (18S rRNA), TVMe+G4 (28S rRNA) and K3Pu+F+I (ITS-1)— according to the Bayesian information criterion. W-IQTREE was used for ML reconstruction (Nguyen et al. 2015; Trifinopoulos et al. 2016). One thousand ultrafast bootstrap (UFBoot) replicates were applied to provide support values for branches (Hoang et al. 2018). Nebularmis phylogeny and biogeography A dataset of 18S rRNA, 28S rRNA, ITS-1 and ITS-2 from Gąsiorek et al. (2021b) was used. The final alignment length was 2825 bp. PartitionFinder indicated GTR+I+G for two separate partitions (18S rRNA + 28S rRNA and ITS-1 + ITS-2). The original concatenated matrix was analysed using BEAST (Drummond and Rambaut 2007). Four combinations of clock and tree priors were chosen and run in parallel, analogously to the analyses from Gąsiorek et al. (2021b): (a) a random local clock (Drummond and Suchard 2010) with the coalescent tree prior, (b) a random local clock with speciation: Yule process as the tree prior, (c) a strict clock (Ferreira and Suchard 2008) with the coalescent tree prior, and (d) a strict clock with speciation: Yule process as the tree prior. Tree searches were run for 10 million generations, sampling the tree every 1000 steps. The trees were summarized with TREEANNOTATOR software (distributed with BEAST), with the first 1000 trees removed. Tracer was then used to check the stationarity of Markov chains and determine the ‘burnin’. Consensus trees constructed from all datasets shared identical topologies (consistent with the variant b from Gąsiorek et al. 2021b). Consequently, the first 9000 trees were removed from the set of trees b, and the remaining 1000 trees were used in independent statistical dispersal-vicariance analyses (S-DIVA; Ronquist 1997; Yu et al. 2015), implemented in RASP (Yu et al. 2020), with phylogenetic uncertainty considered in the calculations. Nebularmis records were assigned to the zoogeographic realms (Ficetola et al. 2017) and records of N. reticulatus (Murray, 1905) outside the Palaearctic were discarded as unreliable (Gąsiorek et al. 2019c 2021b). The maximum number of areas at a node was set to 3. Pseudechiniscus phylogeny A dataset of 18S rRNA, 28S rRNA and ITS1 from Gąsiorek et al. (2021c) was used. The final alignment length was 2307 bp. PartitionFinder indicated GTR+I+G for all three partitions treated separately. These models were used in BI reconstructions in MrBayes. ModelFinder indicated the following models: SYM+I+G4 (18S rRNA), SYM+G4 (28S rRNA) and GTR+F+G4 (ITS-1). They were applied in ML reconstruction in W-IQ-TREE. RESULTS Taxonomic account Phylum: Tardigrada Doyère, 1840 Class: Heterotardigrada Marcus, 1927 Order: Echiniscoidea Richters, 1926 Family: Echiniscidae Thulin, 1928 Genus: Claxtonia Gąsiorek & Michalczyk, 2019 in Gąsiorek et al. 2019a Claxtonia sp. nov. (Fig. 1) Material examined: One adult male (slide TW.007.21) with evident U-shaped, granulated subcephalic plates. Remarks: The scarce material and the lack of DNA data prevent a formal description of this new species, most closely resembling the following Claxtonia species: C. wendti (Richters, 1903), C. pardalis (Degma & Schill, 2015), and C. goni Degma et al., 2021. However, in none of the aforementioned echiniscids were males recorded, and the new species differs from these species by minute differences in dorsal plate sculpturing. In the light of this discovery, the record of C. wendti from Hainan (Li et al. 2008), another continental island nearby the East Asian coast, is more likely to represent the new Claxtonia species than C. wendti. Genus: Echiniscus C.A.S. Schultze, 1840 Echiniscus blumi Richters, 1903 sensu lato Material examined: 18 adult females on slides TW.007.20, TW.009.02–5. Three specimens from each sample were preserved for further molecular analyses. Remarks: A cold stenothermic species, thus it is common in the Arctic, often inhabiting lower page 4 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan elevations (< 1000 m asl) in the temperate zone, but in the tropical and subtropical zone found only at high altitudes (McInnes 1994). The still unsolved species discrimination within the Echiniscus blumi-canadensis complex (Guil and Giribet 2009) makes this a sensu lato record. Echiniscus clevelandi Beasley, 1999 (Figs. 2–9, Tables 2–4) Material examined: 23 adult females, 15 adult males, and three juveniles on slides TW.005.06–7, TW.006.06, TW.007.19, TW.008.02–7, 12. Four specimens on SEM stub 21.07. Four specimens from the sample TW.008 were used for DNA sequencing, including two retrieved as hologenophores. Amended description: Females (i.e., from the third instar onwards; measurements and statistics in Table 2): Body cylindrical to plump (Figs. 2, 4), orange to red with dark red eyes; body colour and eyes disappear soon after mounting in Hoyer’s medium. Echiniscustype cephalic papillae (secondary clavae) and (primary) clavae; cirri growing out from bulbous cirrophores (Fig. 8D). The body appendage configuration is A-BC-D-E, with all trunk appendages formed as short and relatively thick, smooth cirri. Instances of asymmetry in chaetotaxy frequent (Fig. 2B), but only rarely are more than one appendage absent. Dorsal plates with the mixed type of sculpturing typical for the Echiniscus virginicus complex, comprising an evident layer of large polygonal endocuticular pillars visible as black dots under PCM (Figs. 2, 6), and a layer of dark uniform epicuticular matrix (Figs. 2A–B, 6A) pierced with large, often irregularly shaped pores (Figs. 2, 6). Epicuticle and pores are typically well-developed and identifiable in SEM (Figs. 4, 8A–C). Rarely, the pores are small and scarce (Figs. 2C, 5A, 6B, 8C). The cephalic plate is narrow and separated from the cervical (neck) plate by smooth cuticle. The cervical plate visible as a dark belt of minute pillars clearly distinct from the scapular plate. The scapular plate clearly smaller than the caudal (terminal) plate, with additional lateral sutures separating narrow trapezoidal lateral portions devoid of pores (Figs. 2, 6). Paired segmental plates divided into a smaller, much narrower anterior and a dominant posterior part by a smooth transverse stripe. Epicuticular ornamentation better developed in the central plate portions compared to the lateral parts. The caudal plate with short incisions and fully developed epicuticle. Median plates 1, 3 unipartite, whereas median plate 2 bipartite, its anterior portion is narrow, but with identical sculpturing as the posterior part. Ventral cuticle with minute endocuticular pillars covering the entire venter; a pair of subcephalic swellings (likely rudimentary plates, Fig. 8D) and a pair of rectangular genital plates present. Sexpartite gonopore placed between genital plates, and a trilobed anus between legs IV. Pedal plates I–III extremely reduced and only rarely identifiable as aggregations of pillars in central leg portions (Fig. 6B), pedal plate IV developed as a sculptured cushion bearing a dentate collar with Fig. 1. Habitus of a male of a new, undescribed species of Claxtonia (PCM, dorsal view). Scale bar in μm. page 5 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Fig. 2. Habitus of females of Echiniscus clevelandi (PCM): A, fully developed sculpturing and chaetotaxy (dorsal view); B, typical sculpturing and asymmetric lack of spine C (dorsolateral view); C, atypical sculpturing with poorly developed pores and full chaetotaxy (dorsal view). Scale bars in μm. page 6 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan numerous teeth (Figs. 4, 6A, 8F). Pulvini present, but weakly visible (Fig. 2B). A small spine on leg I (Fig. 6) and a papilla on leg IV. Internal claws with identical large spurs on all legs (Figs. 8E–F, 9). Claws IV clearly higher than claws I–III (Table 2). Buccal apparatus short, with a rigid, stout tube and a spherical pharynx. Stylet supports absent. Males (i.e., from the third instar onwards; measurements and statistics in table 3): Sexual dimorphism poorly marked. Circular gonopore. Fully falling in the range of morphometric variability of females. Usually slightly slimmer than females (Fig. 3) and with fewer epicuticular pores (Figs. 3B, 5B, 7). Juveniles (i.e., the second instar; measurements and statistics in table 4): Gonopore absent. Smaller than sexually mature specimens of both sexes. Morphometric differences evident also in cephalic appendages and claw heights. Body appendage configuration A-C-E. Dorsal cuticle lacks epicuticular ornamentation. Larvae: Not found. Eggs: Two to three orange eggs per exuvia were found. Molecular markers and phylogenetic position: Single haplotype was found in 18S rRNA (OK048609– 10), ITS-1 (OK048639–40) and COI (OK047271–2), but two haplotypes were revealed in 28S rRNA (OK048627–8) and ITS-2 (OK048620–1), with minor intra-population p-distances (0.1–0.2%). We acquired Table 2. Measurements [in μm] of selected morphological structures of the adult females of E. clevelandi mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 16 145–228 445–583 185 496 22 33 Scapular plate length 16 32.2–43.7 – 37.3 –3.2 – Head appendage lengths Cirrus internus 13 14.4–21.3 36.8–59.6 17.1 45.5 2.2 6.3 Cephalic papilla 16 6.5–8.6 16.7–23.9 7.3 19.7 0.7 2.1 Cirrus externus 15 15.0–23.1 40.5–63.0 18.3 49.3 2.1 5.9 Clava 15 4.7–6.8 14.4–18.2 5.9 15.9 0.5 1.1 Cirrus A12 32.8–48.3 89.6–122.2 38.9 103.3 4.9 10.5 Cirrus A/Body length ratio 12 17%–24% –21% –2% – Body appendage lengths Cirrus B16 11.9–21.4 31.6–53.8 17.0 45.5 2.9 6.8 Cirrus C16 20.5–37.7 54.4–94.7 27.0 72.5 4.5 10.4 Cirrus D16 21.9–35.3 57.0–88.7 26.3 70.5 3.6 9.0 Cirrus E16 23.7–41.6 60.0–109.9 32.3 86.9 5.1 14.1 Spine on leg I length 16 2.4–4.5 7.3–11.5 3.2 8.6 0.5 1.2 Papilla on leg IV length 16 3.9–5.6 10.0–14.6 4.6 12.5 0.5 1.4 Number of teeth on the collar 15 5–15 – 10.4 –2.4 – Claw I heights Branch 16 8.8–11.7 23.8–31.1 10.1 27.1 0.7 2.2 Spur 13 1.9–2.4 4.6–7.5 2.1 5.7 0.2 0.7 Spur/branch height ratio 13 19%–24% –21% –2% – Claw II heights Branch 16 8.6–10.9 22.9–30.4 9.8 26.3 0.6 1.9 Spur 16 1.6–2.9 4.8–7.3 2.0 5.4 0.3 0.6 Spur/branch height ratio 16 18%–27% –21% –2% – Claw III heights Branch 16 8.8–11.1 23.8–29.2 9.9 26.5 0.7 1.7 Spur 14 1.6–3.0 4.6–7.5 2.1 5.5 0.3 0.8 Spur/branch height ratio 14 17%–27% –21% –2% – Claw IV heights Branch 15 10.9–13.4 28.6–38.8 12.1 32.5 0.8 2.6 Spur 4 2.4–3.7 6.4–9.3 3.0 8.1 0.6 1.2 Spur/branch height ratio 4 21%–29% –25% –3% – page 7 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan a set of all five markers (18S rRNA: OK048611, 28S rRNA: OK048629, ITS-1: OK048641, ITS-2: OK048622, COI: OK047273) also for one specimen of E. hoonsooi Moon & Kim, 1990, a species of similar phenotype (Fig. 10), previously reported from Japan (Abe et al. 2000). The BI tree indicates that E. clevelandi and E. hoonsooi are sister species, and constitute a sister clade to the E. lineatus + E. virginicus Riggin, 1962 clade (Fig. 11; see also Gąsiorek et al. 2020). Fig. 3. Habitus of males of Echiniscus clevelandi (PCM): A, typical sculpturing (dorsolateral view); B, atypical sculpturing with poorly developed pores and asymmetric lack of spine B (dorsolateral view). Scale bars in μm. page 8 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Remarks: Taxonomy of the virginicus complex is scrutinised in table 5. The Taiwanese populations greatly broadened the range of intraspecific variability of E. clevelandi presented in the original description. Although Beasley (1999) specified that some type specimens lack dorsal appendages, this variant of chaetotaxy seems to be dominant in Taiwan. Therefore, the body appendage formula for the species is A-B-C- (Cd)-D-(Dd)-E. Moreover, the porosity of dorsal plates varies greatly between specimens, from highly porous with irregularly shaped pores (see fig. 4 in Beasley 1999 and Figs. 2–8C herein), through moderately porous with mostly round pores (Figs. 2–8C) to almost completely smooth plates with few small pores (Fig. 5A). If found separately, these morphotypes could be identified as separate taxa, which underlines the importance of integrative analyses carried out on a considerable number of specimens in order to reduce the risk of taxonomic inflation. Echiniscus semifoveolatus Ito, 1993 Material examined: One adult female on slide TW.009.01. Remarks: A likely East Asian endemic species (Qiao et al. 2013; Suzuki 2017); rarely encountered, always in mountain locales. Fig. 4. Habitus of females of Echiniscus clevelandi with fully developed sculpturing (dorsal view, SEM). Scale bars in μm. page 9 of 45 Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Venter weakly granulated and regularly wrinkled, as is typical for Nebularmis (Gąsiorek et al. 2021b); the only areas with evident endocuticular pillars are the pair of trapezoidal subcephalic plates (Fig. 22A–C) and the pair of wing-shaped genital plates (Fig. 22D). A sexpartite gonopore placed between genital plates, and a trilobed anus between legs IV. Pedal plates I–III formed as clear aggregations of pillars in central limb portions (Fig. 22B–C). Pedal plates IV strongly sculptured, with pillars present also on the dentate collar (Fig. 22D). Pulvini absent. Spine I triangular (Fig. 22B–C), papilla IV small and elongated (Figs. 20, 22D). Claws robust, isonych/homomorphic; primary spurs present on all internal branches (Fig. 22C–D). Males: Not found. Juveniles, larvae and eggs: Not found. Molecular markers and phylogenetic position: All five gene fragments were sequenced: 18S rRNA (OK048614), 28S rRNA (OK048632), ITS-1 (OK048642), ITS-2 (OK048623) and COI (OK047274). The updated phylogeny from Gąsiorek et al. (2021b) indicates the presence of sister clades in Nebularmis: one of the Oriental origin, and the second of a mixed Palaearctic and Oriental origin (Fig. 23). Nebularmis crebraclava belongs to the latter clade, being the sister species of N. reticulatus. Table 3. Measurements [in μm] of selected morphological structures of the adult males of E. clevelandi mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 11 145–192 475–598 169 522 17 33 Scapular plate length 11 28.6–37.5 – 32.5 –3.0 – Head appendage lengths Cirrus internus 10 13.8–20.2 42.7–59.2 17.3 53.7 1.9 5.7 Cephalic papilla 11 7.0–9.6 19.5–29.2 8.4 25.8 0.9 2.5 Cirrus externus 9 14.9–22.4 43.5–62.7 18.5 55.9 2.6 6.5 Clava 11 4.9–7.4 16.3–21.8 6.2 19.2 0.8 1.9 Cirrus A10 29.7–45.2 92.0–131.2 35.9 111.7 4.3 13.3 Cirrus A/Body length ratio 10 18%–26% –22% –3% – Body appendage lengths Cirrus B11 10.8–26.8 37.0–75.1 18.0 55.2 4.3 10.1 Cirrus C11 16.9–37.9 57.9–106.2 27.3 83.9 5.7 13.7 Cirrus D10 18.1–37.1 62.0–103.9 27.3 84.4 5.4 12.6 Cirrus E10 21.2–38.1 72.6–106.7 29.8 91.8 5.6 10.8 Spine on leg I length 11 1.9–3.7 6.4–10.7 2.8 8.4 0.7 1.7 Papilla on leg IV length 11 3.5–5.7 12.0–16.5 4.6 14.0 0.7 1.5 Number of teeth on the collar 11 7–13 – 9.8 –1.7 – Claw I heights Branch 11 8.0–10.8 25.7–31.3 9.4 28.8 1.0 2.0 Spur 9 1.6–2.5 5.1–7.1 2.1 6.4 0.3 0.7 Spur/branch height ratio 9 18%–26% –23% –3% – Claw II heights Branch 11 7.4–10.1 24.6–28.9 8.8 27.2 0.9 1.7 Spur 11 1.7–2.2 4.8–6.4 1.9 5.9 0.2 0.6 Spur/branch height ratio 11 17%–25% –22% –2% – Claw III heights Branch 11 7.7–10.4 25.9–30.6 9.2 28.3 0.9 1.9 Spur 10 1.5–2.2 4.6–6.2 1.8 5.5 0.2 0.5 Spur/branch height ratio 10 16%–21% –19% –2% – Claw IV heights Branch 9 9.3–14.1 31.7–40.9 11.4 35.7 1.5 3.1 Spur 1 2.5–2.5 8.3–8.3 2.5 8.3 ?? Spur/branch height ratio 1 22%–22% –22% –?– page 16 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Table 4. Measurements [in μm] of selected morphological structures of the juveniles of E. clevelandi mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 3 121–139 481–495 129 487 97 Scapular plate length 3 25.0–28.1 – 26.6 –1.6 – Head appendage lengths Cirrus internus 3 8.8–11.4 33.1–42.9 9.8 37.1 1.4 5.1 Cephalic papilla 3 4.8–5.2 17.8–20.8 5.0 18.9 0.2 1.7 Cirrus externus 3 10.2–11.7 36.3–44.0 10.9 41.3 0.8 4.3 Clava 2 3.5–4.3 13.2–17.2 3.9 15.2 0.6 2.9 Cirrus A3 22.3–27.1 81.1–101.9 24.1 90.7 2.6 10.5 Cirrus A/Body length ratio 3 16%–21% –19% –2% – Body appendage lengths Cirrus C3 11.5–13.6 40.9–54.4 12.8 48.4 1.1 6.9 Cirrus E3 14.1–19.4 56.2–72.9 16.4 61.9 2.7 9.6 Spine on leg I length 3 2.0–2.4 7.5–8.8 2.2 8.3 0.2 0.7 Papilla on leg IV length 2 2.8–3.5 10.0–14.0 3.2 12.0 0.5 2.9 Number of teeth on the collar 3 7–10 – 8.7 –1.5 – Claw I heights Branch 3 6.6–6.8 23.5–26.4 6.7 25.2 0.1 1.5 Spur 3 1.4–1.9 5.0–7.1 1.6 6.2 0.3 1.1 Spur/branch height ratio 3 21%–28% –24% –3% – Claw II heights Branch 3 6.3–6.5 22.8–25.2 6.4 24.1 0.1 1.2 Spur 3 1.3–1.5 4.6–6.0 1.4 5.3 0.1 0.7 Spur/branch height ratio 3 20%–24% –22% –2% – Claw III heights Branch 3 6.0–6.3 22.4–24.0 6.2 23.4 0.2 0.8 Spur 2 1.1–1.1 3.9–4.1 1.1 4.0 0.0 0.2 Spur/branch height ratio 2 17%–17% –17% –0% – Claw IV heights Branch 3 7.4–7.7 26.3–29.6 7.5 28.3 0.2 1.7 Spur 2 1.6–1.7 6.0–6.0 1.7 6.0 0.1 0.0 Spur/branch height ratio 2 21%–23% –22% –2% – Table 5. Comparison of morphological traits and reproductive modes between the Echiniscus virginicus complex species Character E. cheonyoungi E. clevelandi E. hoonsooi E. lineatus E. masculinus E. virginicus Chaetotaxy A-B-C-Cd-D-Dd-E A-B-C-(Cd)-D-(Dd)-E A-(C)-(D)-E A-(B)-C-Cd-D-Dd-E A-C-D-(Dd)-E A-(B)-C-Cd-D-Dd-E Dorsal plates with pores pores pseudopores pseudopores pseudopores pores Pedal plate sculpturing present, with both pillars and pores present, only with pillars absent absent absent absent Claws homomorphic and spurless homomorphic heteromorphic homomorphic homomorphic homomorphic Males never found* present never found never found present never found *E. cheonyoungi is the only species for which ample population data are lacking, thus it is currently impossible to determine whether males are present in this species. page 17 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Fig. 13. Habitus of Hypechiniscus crassus sp. nov. (PCM): A, female (dorsal view); B, allotypic male (dorsolateral view). Arrowhead indicates papilla IV. Scale bars in μm. Fig. 12. Holotypic female of Hypechiniscus crassus sp. nov. (dorsolateral view, PCM). White arrowhead indicates papilla IV. Scale bar in μm. page 18 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Remarks: The newly found females supplement the original description which was based solely on males (Sun et al. 2014). Nebularmis reticulatus (Murray, 1905) Material examined: 31 adult females, 29 juveniles, and 2 larvae on the slides TW.007.02–19, TW.009.06–8. Eight specimens on the SEM stubs 21.05–6. Four specimens per each of the two samples were used for DNA sequencing, including six retrieved as hologenophores. Remarks: Numerous individuals exhibit large atypical granules (as depicted in fig. 5b in Gąsiorek et al. 2019c) on lateralmost portions of the scapular plate, and some of them also on other plates, e.g., on the caudal plate. However, the genetic distances with respect to European populations (including the neotype population described in Gąsiorek et al. 2019c) are small and clearly fall under intraspecific variation: p = 0.2– 1.2% in ITS-1 (a single new haplotype, OK048643–5), 0.2–3.3% in ITS-2 (two new haplotypes, OK048624–6), 0.7–0.8% in COI (a single new haplotype, OK047275– 7). Genus: Pseudechiniscus Thulin, 1911 Subgenus: Meridioniscus Gąsiorek et al., 2021 Pseudechiniscus (Meridioniscus) dreyeri sp. nov. (Figs. 24–25, Tables 11–12) urn:lsid:zoobank.org:act:B38091F7-3B79-438B-A42AA4E53C25C34B Tardigrada Register: www.tardigrada.net/ register/0112.htm Fig. 14. Habitus of Hypechiniscus crassus sp. nov. (dorsal view, SEM): A, female; B, male. Arrowheads indicate papilla IV. Scale bars in μm. page 19 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Description: Females (i.e., from the third instar onwards; measurements and statistics in table 11): Body small and cylindrical (Fig. 24A), light orange with tiny crystalline eyes; body colour and eyes dissolve in Hoyer’s medium. Clavae elongated (dactyloid); cirrophores of peribuccal cirri merged with flagellum, cirrophores A distinct (Fig. 24A–B). Dorsal plate sculpturing of the Pseudechiniscus type, with rudimentary, faint striae present occasionally in central plate portions (Fig. 24B). Endocuticular pillars of similar diameters throughout the dorsum. Pentapartite cephalic plate adjacent to the scapular plate; lacking cervical plate (Fig. 24A). Scapular plate divided by two weakly marked sutures: central longitudinal suture and transversal suture, thus delineating four plate portions: two large anterior ones and two posterior, more narrow and with poorly visible lateralmost subportions (Fig. 24B). Median plates m1, m3 unipartite and large, a pair of lateral intersegmental plates flanking m1 and two pairs of such plates flanking m2; m2 bipartite, with Fig. 15. Sculpturing of Hypechiniscus crassus sp. nov. (PCM): A, dorsal (female); B, ventral (female). Arrowhead indicates papilla IV. Scale bars in μm. page 20 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan narrow triangular posterior part. Paired segmental plates I–II and paired pseudosegmental plate IV’ present (Fig. 24B). Caudal plate with two slightly curved incisions (Fig. 24A). Ventral sculpturing with well-developed and evident reticulum composed of endocuticular pillars solely (Figs. 24C–25). Single dense aggregation of pillars forming an aliform shape in the subcephalic region. Sexpartite gonopore placed between legs III– IV, and a trilobed anus between legs IV. Pedal plates formed as belts of pillars in central limb portions (Fig. 24A). Pulvini faint. Papillae or spines on legs I absent. Papilla IV small and tubby (Fig. 24A). Claws minute and isonych; internal claws with delicate primary spurs positioned at ca. 20% of the branch height and closely adjacent to it (Fig. 24A, insert). Males: Not found. Juveniles (i.e., the second instar; measurements and statistics in table 12): Qualitatively identical to females, beside of the lack of gonopore. No morphometric gap with respect to adult females. Larvae (i.e., the first instar): Cuticle sculpturing developed as in older instars. Gonopore and anus absent. Body length 94–100 μm, scapular plate length 14.4–14.7 μm; cephalic appendages lengths: cirri interni 3.8–6.0 μm, cephalic papillae 2.8–3.0 μm, cirri externi 4.7–6.4 μm, (primary) clavae 3.0–3.1 μm, cirrus A 15.3–15.9 μm. Papilla IV length 1.4–1.6 μm. Claw Fig. 16. Morphological details of Hypechiniscus crassus sp. nov. (SEM): A, cephalic region with peribuccal appendages; B, sculpturing of the scapular plate in close-up (empty incised arrowheads indicate rudimentary striae); C, claws I; D, claws IV. White arrowheads point out pseudoaccessory points, and empty arrowhead – aberrant secondary spur on external claw. Scale bars in μm. page 21 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan branches 4.6–5.0 μm, spurs 1.4–1.8 μm. Eggs: Up to two orange eggs per exuvia were found. Molecular markers and phylogenetic position: Single haplotypes were found in all markers: 18S rRNA (OK048615), 28S rRNA (OK048633), ITS1 (OK048646) and COI (OK047278). In the updated phylogeny from Gąsiorek et al. (2021c), the closest relative of P. (M.) dreyeri sp. nov. within the Meridioniscus clade is P. (M.) cf. saltensis from the Neotropics (Fig. 30). Type material: Holotype (adult female on the slide TW.008.12), 32 paratypes: 18 adult females, 12 juveniles, and two larvae on the slides TW.005.11, 13, 15–16, TW.008.10–13. Four specimens were preserved for molecular analyses. Holotype deposited in the Biodiversity Research Center of the Academia Sinica (ASIZ01000039), the one paratype (NHMD-915766) deposited in the Natural History Museum of Denmark, and remaining material stored at the Jagiellonian University. Type locality: 24°23'51"N, 121°14'04"E, 3 700 m asl: Taiwan, Snow Mountain (Xueshan), North Peak. Mosses from rocks exposed to sun. Etymology: Patronym honouring Niklas Dreyer, a carcinologist and the collector of the Taiwanese moss samples used in this study. Noun in the genitive singular. Differential diagnosis: There are few Meridioniscus species with a smooth posterior margin of the pseudosegmental plate IV’ (or with minute projections) and lacking lateral hemispherical projections. Pseudechiniscus (M.) dreyeri sp. nov. differs from: A B Fig. 17. Schematic depiction of female morphology of Hypechiniscus crassus sp. nov.: A, dorsum; B, venter. page 22 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Fig. 18. Larva of Hypechiniscus crassus sp. nov. Scale bar in μm. Arrowhead indicates papilla IV. Table 6. Measurements [in μm] of selected morphological structures of the adult females of H. crassus sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD Holotype µm sp µm sp µm sp µm sp Body length 11 186–220 888–1024 203 932 10 42 198 888 Scapular plate length 11 20.1 – 21.8 –0.8 –22.3 – Head appendage lengths Cirrus internus 11 12.5–16.2 57.6–70.1 14.1 64.7 1.1 3.7 14.8 66.4 Cephalic papilla 10 4.8–5.8 21.8–26.4 5.4 24.6 0.3 1.2 5.7 25.6 Cirrus externus 11 16.9–21.7 79.0–94.6 18.9 86.7 1.5 5.6 19.6 87.9 Clava 11 3.8–5.6 17.5–25.1 4.7 21.6 0.4 1.9 4.7 21.1 Cirrus A10 16.3–21.4 79.1–96.8 19.1 87.6 1.4 5.2 18.8 84.3 Cirrus A/Body length ratio 10 8%–10% –9% –1% –9% – Body appendage lengths Papilla on leg IV length 6 2.6–3.5 12.1–15.7 3.0 14.1 0.3 1.4 3.5 15.7 Claw I heights Branch 11 10.2–12.1 48.1–55.8 11.0 50.5 0.5 2.3 10.9 48.9 Spur 11 1.5–1.9 6.9–9.0 1.7 8.0 0.1 0.8 1.8 8.1 Spur/branch height ratio 11 14%–18% –16% –1% –17% – Claw II heights Branch 11 9.8–11.9 46.5–53.2 10.9 50.1 0.6 1.8 11.1 49.8 Spur 10 1.6–2.3 7.2–10.0 1.9 8.7 0.3 1.0 1.7 7.6 Spur/branch height ratio 10 14%–20% –17% –2% –15% – Claw III heights Branch 11 10.3–11.5 46.8–53.2 10.9 50.0 0.4 2.1 10.9 48.9 Spur 7 1.6–2.1 7.4–9.5 1.9 8.6 0.2 0.7 1.8 8.1 Spur/branch height ratio 7 14%–19% –17% –2% –17% – Claw IV heights Branch 11 11.6–13.0 52.9–60.7 12.2 56.0 0.5 2.3 12.1 54.3 Spur 2 2.0–2.4 9.7–10.8 2.2 10.2 0.3 0.7 2.4 10.8 Spur/branch height ratio 2 16%–20% –18% –3% –20% – page 23 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan P. (M.) angelusalas Roszkowska et al., 2020, described from Madagascar, by relative lengths of some of the cephalic appendages (cirrus internus 22.0–33.0, cirrus A 96.8–126.8 in P. (M.) dreyeri sp. nov. vs cirrus internus 34.4–36.6, cirrus A 129.2–152.2 in P. (M.) angelusalas). P. (M.) dastychi Roszkowska et al., 2020, known from the maritime Antarctic, by adult female body size (body length 121–152 μm and scapular plate length 18.9–23.7 μm in P. (M.) dreyeri sp. nov. vs 167–202 μm and sc 27.5–33.0 μm in P. (M.) dastychi), lengths of cephalic appendages (cirrus internus 4.7–7.0 μm, cirrus externus 6.8–11.9 μm, cirrus A 21.0–27.0 μm [14–20% of the body length] in P. (M.) dreyeri sp. nov. vs cirrus internus 10.4–12.7 μm, cirrus externus 15.9–19.1 μm, cirrus A 40.0–45.0 μm [22–26% of the body length] in P. (M.) dastychi), and claw heights (5.0–8.1 μm in P. (M.) dreyeri sp. nov. vs 8.7–12.2 μm in P. (M.) dastychi). P. (M.) indistinctus Roszkowska et al., 2020, known from the Scandinavian Peninsula, by the morphology of dorsal pillars (homogeneous in size in P. (M.) dreyeri sp. nov. vs heterogeneous in size in P. (M.) indistinctus) and relatively shorter peribuccal cirri (cirrus internus 22.0–33.0, cirrus externus 31.9–52.2 in P. (M.) dreyeri sp. nov. vs cirrus internus 34.1–38.5, cirrus externus 54.3–59.3 in P. (M.) indistinctus). P. (M.) mascarenensis Kiosya et al., 2021, known from Mauritius, by having smaller adult females (121– 152 μm in P. (M.) dreyeri sp. nov. vs 151–177 μm in P. (M.) mascarenensis) and a relatively longer cirrus A (14–20% of the body length in P. (M.) dreyeri sp. nov. vs 9–13% in P. (M.) mascarenensis). P. (M.) santomensis Fontoura et al., 2010, a São Tomé endemic, by a relatively longer cirrus A (14–20% of the body length in P. (M.) dreyeri sp. nov. vs 9–14% in P. (M.) santomensis) and dorsal plate sculpturing (striae rarely identifiable in P. (M.) dreyeri sp. nov. vs striae delicate and thin, but clear in all plates in P. (M.) Table 7. Measurements [in μm] of selected morphological structures of the adult males of H. crassus sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD Allotype µm sp µm sp µm sp µm sp Body length 4 178 909–1020 187 943 11 52 178 918 Scapular plate length 4 19.4–20.2 – 19.8 –0.3 –19.4 – Head appendage lengths Cirrus internus 4 10.1–11.6 51.3–57.4 10.6 53.6 0.7 2.9 10.5 54.1 Cephalic papilla 4 4.4–5.3 21.8–27.3 4.9 24.7 0.5 2.8 5.3 27.3 Cirrus externus 4 14.8–15.9 75.1–79.8 15.4 78.0 0.5 2.0 15.2 78.4 Clava 4 3.7–4.5 18.8–23.2 4.2 21.3 0.4 2.1 4.5 23.2 Cirrus A4 16.1–19.2 81.3–95.0 17.4 88.0 1.3 5.6 17.0 87.6 Cirrus A/Body length ratio 4 8%–10% –9% –1% –10% – Body appendage lengths Papilla on leg IV length 3 2.0–2.4 10.2–12.4 2.3 11.5 0.2 1.2 2.4 12.4 Claw I heights Branch 4 9.1–10.2 46.2–51.5 9.8 49.4 0.5 2.3 10.0 51.5 Spur 2 1.6–1.6 8.1–8.2 1.6 8.2 0.0 0.1 1.6 8.2 Spur/branch height ratio 2 16%–16% –16% –0% –16% – Claw II heights Branch 4 9.3–10.7 47.2–53.0 9.9 50.2 0.7 3.0 9.3 47.9 Spur 2 1.7–1.7 8.4–8.6 1.7 8.5 0.0 0.1 ?? Spur/branch height ratio 2 16%–16% –16% –0% –?– Claw III heights Branch 4 9.2–10.0 46.5–50.5 9.7 48.8 0.3 1.7 9.8 50.5 Spur 0 ? ??????? Spur/branch height ratio 0 ? – ? –?–?– Claw IV heights Branch 4 9.6–11.2 48.7–56.6 10.7 54.1 0.7 3.7 10.9 56.2 Spur 1 2.1–2.1 10.6–10.6 2.1 10.6 ???? Spur/branch height ratio 1 19%–19% –19% –?–?– page 24 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan santomensis). Moreover, P. (M.) dreyeri sp. nov. is distinguishable from all abovementioned species by the ventral sculpturing pattern. Subgenus: Pseudechiniscus Thulin, 1911 Pseudechiniscus (Pseudechiniscus) ehrenbergi Roszkowska et al., 2020 Material examined: 37 adult females, eight adult males, and eight juveniles on slides TW.010.01–5. Ten specimens were used for DNA sequencing, including three retrieved as hologenophores. Remarks: This is another record suggesting a wide geographic distribution of P. (P.) ehrenbergi (Cesari et al. 2020; Roszkowska et al. 2020; Gąsiorek et al. 2021c); however, there is a considerable variability in the ventral sculpturing between various populations (Gąsiorek et al. 2021c), thus the distribution and intraspecific variation of the species require new, integrative analyses. Pseudechiniscus (Pseudechiniscus) formosus sp. nov. (Figs. 26–27) urn:lsid:zoobank.org:act:7FDACA7F-5E5F-4220-A668DD8179BD04A6 Tardigrada Register: http://www.tardigrada.net/ register/0113.htm Description: Female (i.e., the third instar): Large (187 μm, sc = 24.9 μm) Pseudechiniscus with cylindrical body (Fig. 26A); pale yellow with black crystalline eyes, body colour disappears, but eyes persist after mounting in Hoyer’s medium. Pseudohemispherical cephalic papillae (3.9 μm) and elongated (primary) clavae (4.0 μm, Fig. 27); cirrophores of peribuccal Table 8. Measurements [in μm] of selected morphological structures of the juveniles of H. crassus sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 4 150–172 896–980 162 927 938 Scapular plate length 4 15.3–19.2 – 17.6 –1.6 – Head appendage lengths Cirrus internus 4 8.1–10.6 46.0–60.1 9.2 52.5 1.1 6.4 Cephalic papilla 4 4.5–5.1 25.6–30.1 4.8 27.5 0.3 1.9 Cirrus externus 3 11.5–13.0 65.3–83.0 12.4 72.0 0.8 9.6 Clava 4 3.6–4.2 21.9–23.5 4.0 22.6 0.3 0.7 Cirrus A4 12.8–15.0 70.7–94.1 14.1 80.6 0.9 9.8 Cirrus A/Body length ratio 4 8%–10% –9% –1% – Body appendage lengths Papilla on leg IV length 4 1.7–2.6 10.2–14.4 2.2 12.3 0.5 2.0 Claw I heights Branch 4 7.9–8.4 43.6–52.9 8.1 46.3 0.2 4.5 Spur 4 1.2–1.8 6.6–9.4 1.4 8.0 0.3 1.2 Spur/branch height ratio 4 15%–21% –17% –3% – Claw II heights Branch 4 7.5–9.1 41.4–53.6 8.1 46.5 0.7 5.3 Spur 4 1.3–1.5 7.2–8.5 1.4 7.7 0.1 0.6 Spur/branch height ratio 4 16%–17% –17% –1% – Claw III heights Branch 4 7.6–9.2 43.1–51.6 8.1 46.5 0.7 4.1 Spur 2 1.4–1.5 8.5–9.2 1.5 8.8 0.1 0.4 Spur/branch height ratio 2 18%–20% –19% –1% – Claw IV heights Branch 4 7.8–10.0 43.1–55.6 8.7 49.8 0.9 5.3 Spur 1 1.6–1.6 9.1–9.1 1.6 9.1 ?? Spur/branch height ratio 1 19%–19% –19% –?– page 25 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan formosus sp. nov. vs brown in P. (P. ) chengi) and the epicuticular ornamentation on the dorsum (absent in P. (P.) formosus sp. nov. vs present in P. (P. ) chengi). P. (P. ) ehrenbergi Roszkowska et al., 2020, by a longer cirrus externus and cirrus A (16.6 μm, 32.1 μm in P. (P.) formosus sp. nov. vs 9.0–11.7 μm, 21.6–26.8 μm in P. (P.) ehrenbergi). P. (P.) lacyformis Roszkowska et al., 2020, by the epicuticular ornamentation on the dorsum (absent in P. (P.) formosus sp. nov. vs present in P. (P. ) lacyformis) and shorter cirrus internus (9.3 μm in P. (P.) formosus sp. nov. vs 10.6–14.0 μm in P. (P. ) lacyformis). Fig. 25. Schematic depiction of female ventral morphology of Pseudechiniscus (M.) dreyeri sp. nov. page 32 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan P. (P. ) shintai Vončina et al., 2020, by body colour (pale yellow in P. (P.) formosus sp. nov. vs orange in P. (P.) shintai) and the epicuticular ornamentation on the dorsum (absent in P. (P. ) formosus sp. nov. vs present in P. (P.) shintai). P. (P. ) suillus (Ehrenberg, 1853), by relative lengths of some cephalic appendages (cirrus internus 37.3, cephalic papilla 15.7, (primary) clava 16.1 in P. (P.) formosus sp. nov. vs cirrus internus 44.0–49.6, cephalic papilla 19.1–24.3, (primary) clava 20.9–26.8 in P. (P.) suillus). P. (P. ) xiai Wang et al., 2018, by body colour (pale yellow in P. (P. ) formosus sp. nov. vs orange in P. (P.) xiai) and the epicuticular ornamentation on the dorsum (absent in P. (P.) formosus sp. nov. vs present in P. (P. ) xiai). Moreover, P. (P. ) formosus sp. nov. is also distinguishable from all abovementioned species by the ventral sculpturing pattern. Pseudechiniscus (Pseudechiniscus) totoro sp. nov. (Figs. 28–29, Tables 13–14) urn:lsid:zoobank.org:act:0BA6B1A7-A383-4648-84321DCD132D6D75 Tardigrada Register: http://www.tardigrada.net/ register/0114.htm Description: Females (i.e., from the third instar onwards; measurements and statistics in table 13): Small, yellow to orange body (Fig. 28A) with minute black eyes; body colour and eyes may dissolve after mounting in Hoyer’s medium. Pseudohemispherical cephalic papillae and elongated (primary) clavae; cirrophores of cephalic cirri merged with flagellum. Cirrus A short. Dorsal plate sculpturing of the Pseudechiniscus type, with heterogeneous pillars forming patches of Table 11. Measurements [in μm] of selected morphological structures of the adult females of P. (M.) dreyeri sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD Holotype µm sp µm sp µm sp µm sp Body length 15 121–152 609–695 139 655 925 151 688 Scapular plate length 15 18.9–23.7 – 21.2 –1.2 –21.9 – Head appendage lengths Cirrus internus 14 4.7–7.0 22.0–33.0 6.0 28.4 0.8 3.8 6.1 27.9 Cephalic papilla 15 3.5–5.3 17.9–22.4 4.3 20.3 0.4 1.2 4.6 21.0 Cirrus externus 14 6.8–11.9 31.9–52.2 9.3 43.8 1.6 6.7 10.8 49.3 Clava 14 3.5–4.7 15.2–21.6 4.0 19.1 0.4 1.8 4.2 19.2 Cirrus A14 21.0–27.0 96.8–126.8 23.1 109.3 2.0 9.3 26.5 121.0 Cirrus A/Body length ratio 14 14%–20% –17% –1% –18% – Body appendage lengths Papilla on leg IV length 15 1.5–2.3 7.6–10.4 1.9 9.1 0.2 0.7 2.0 9.1 Claw I heights Branch 15 5.1–7.5 25.2–34.7 6.7 31.7 0.6 2.3 7.0 32.0 Spur 15 1.4–2.2 6.9–10.3 1.8 8.6 0.2 0.9 1.9 8.7 Spur/branch height ratio 15 23%–31% –27% –2% –27% – Claw II heights Branch 15 5.5–6.6 26.6–33.3 6.2 29.2 0.3 1.8 6.2 28.3 Spur 15 1.4–2.0 6.6–9.6 1.7 8.1 0.2 0.7 1.8 8.2 Spur/branch height ratio 15 24%–32% –28% –2% –29% – Claw III heights Branch 15 5.0–6.6 23.9–32.8 6.0 28.5 0.5 2.5 6.5 29.7 Spur 15 1.4–2.1 6.6–9.3 1.6 7.6 0.2 0.7 1.5 6.8 Spur/branch height ratio 15 23%–34% –27% –4% –23% – Claw IV heights Branch 11 6.6–8.1 31.1–41.5 7.2 34.0 0.5 2.7 7.4 33.8 Spur 11 1.4–2.5 6.7–12.3 2.0 9.5 0.3 1.5 2.0 9.1 Spur/branch height ratio 11 21%–35% –28% –3% –27% – page 33 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan similar size (Fig. 28C). Striae absent. Pentapartite cephalic plate adjacent to the scapular plate, which is divided into a large anterior portion reaching lateralmost plate margins and two narrow, rectangular posterior portions. Median plates m1–2 bipartite, two pairs of lateral intersegmental plates flanking margins of both m1–2 present; m3 unipartite and rhomboidal. Paired segmental plates I–II and paired pseudosegmental plate IV’ present. Caudal plate small and narrow, with short sclerotised incisions (Fig. 28C). Ventral sculpturing well-developed and reaching lateral body portions (Figs. 28C–D, 29), with larger accumulations of pillars only in the subcephalic and genital areas, and at the level of legs I–III (Fig. 29). A sexpartite gonopore placed anteriorly to legs IV, and a trilobed anus between legs IV. Pedal plates formed as belts of large, widely spaced pillars in the central portions of legs (Fig. 28C). Pulvini absent. Papillae or spines on legs I absent. Papilla IV elongated and small. Claws minute and isonych; internal claws with delicate, but evident primary spurs positioned at ca. 20–25% of the branch height and divergent from it (Fig. 28A, insert). Males: Sexual dimorphism evident. Circular gonopore. Body elongated (123–156 μm in length, sc = 18.2–18.5 μm, n = 2) and slim (Fig. 28B). Cephalic appendages lengths: cirrus internus 8.4–9.1 μm, cephalic papilla 3.5–4.0 μm, cirrus externus 12.0–13.0 μm, (primary) clava 3.9–5.0 μm, cirrus A 25.3–27.5 μm. Clear patches of larger pillars present in the anterior portions of paired segmental plates, in the posterior part of the caudal plate, and on central limb portions. Pulvini clearly marked. Papilla IV length 3.9–4.3 μm. Claws: branch heights 6.4–7.7 μm, spurs 1.8–2.3 μm. Juveniles (i.e., the second instar; measurements and statistics in table 14): Gonopore absent. Smaller than females, but the body length range overlaps with that of males. Table 12. Measurements [in μm] of selected morphological structures of the juveniles of P. (M.) dreyeri sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 10 100–141 557–732 126 650 11 48 Scapular plate length 10 16.0–23.2 – 19.5 –2.0 – Head appendage lengths Cirrus internus 10 4.7–7.1 24.1–38.5 5.9 30.7 0.8 4.7 Cephalic papilla 10 2.9–4.9 15.4–25.4 3.9 20.0 0.7 3.2 Cirrus externus 10 7.2–11.4 41.6–55.6 9.6 49.4 1.1 5.1 Clava 10 3.0–4.1 15.7–21.3 3.7 18.8 0.4 1.8 Cirrus A9 16.4–22.9 96.1–121.8 20.4 107.0 2.2 7.9 Cirrus A/Body length ratio 9 14%–18% –16% –1% – Body appendage lengths Papilla on leg IV length 10 1.3–2.0 8.1–10.2 1.8 9.0 0.2 0.8 Claw I heights Branch 9 5.0–6.3 26.7–33.1 5.9 30.5 0.5 2.5 Spur 9 1.5–2.0 7.3–11.3 1.8 9.4 0.2 1.3 Spur/branch height ratio 9 27%–34% –31% –2% – Claw II heights Branch 9 4.6–6.3 27.2–30.2 5.5 28.6 0.5 1.0 Spur 9 1.4–1.8 6.5–10.0 1.7 8.7 0.1 1.2 Spur/branch height ratio 9 24%–35% –30% –4% – Claw III heights Branch 10 4.5–6.2 25.1–31.3 5.5 28.1 0.4 1.7 Spur 10 1.5–1.9 6.5–10.0 1.7 8.6 0.2 1.0 Spur/branch height ratio 10 24%–36% –31% –4% – Claw IV heights Branch 10 5.2–7.3 29.9–33.3 6.3 32.1 0.7 1.1 Spur 10 1.7–2.5 9.0–12.6 2.0 10.4 0.3 1.2 Spur/branch height ratio 10 27%–40% –33% –4% – page 34 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Fig. 26. Morphology of Pseudechiniscus (Pseudechiniscus) formosus sp. nov. (PCM): A, holotypic female in dorsal view; B, dorsal sculpturing (arrowheads indicate large capituli of pillars); C, ventral sculpturing. Scale bars in μm. page 35 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Larvae: Not found. Eggs: Up to one orange egg per exuvia was found. Molecular markers and phylogenetic position: Single haplotypes were found in 18S rRNA (OK048616–9) and 28S rRNA (OK048635–8) and four haplotypes were uncovered in ITS-1 (intraspecific p-distances = 0.2–1.6%; OK048648–51). In the updated phylogeny from Gąsiorek et al. (2021c), the closest relative of P. (P.) totoro sp. nov. is P. (P.) shintai from Japan (Fig. 30). Type material: Holotype (adult female on the slide TW.005.11), allotype (adult male on the slide Fig. 27. Schematic depiction of female ventral morphology of Pseudechiniscus (P.) formosus sp. nov. page 36 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Fig. 28. Morphology of Pseudechiniscus (Pseudechiniscus) totoro sp. nov. (PCM): A, holotypic female in dorsolateral view (insert shows claws III); B, allotypic male in dorsolateral view; C, dorsal sculpturing; D, ventral sculpturing. Scale bars in μm. page 37 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan TW.005.12), 24 paratypes: 13 adult females, 6 adult males, and 5 juveniles on the slides TW.005.08–16. Six specimens were used for DNA sequencing, including two retrieved as hologenophores. Holotype deposited in the Biodiversity Research Center of Academia Sinica (ASIZ01000035), two paratypes (NHMD-915765) deposited in the Natural History Museum of Denmark, and the remaining material stored at the Jagiellonian University. Type locality: 24°23'18"N, 121°15'39"E, 3 200 m asl: Taiwan, Snow Mountain (Xueshan), East Peak. Mosses from rocks exposed to sun. Fig. 29. Schematic depiction of female ventral morphology of Pseudechiniscus (P.) totoro sp. nov. page 38 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Etymology: The specific epithet is derived from the Japanese animated movie My Neighbour Totoro (Tonari no Totoro) by Hayao Miyazaki (1988) and commemorates Totoro, the forest spirit and the symbol of Studio Ghibli. Noun in apposition. Differential diagnosis: Due to the absence of projections/appendages on the posterior margin of the pseudosegmental plate IV’, the same species must be compared with P. (P.) totoro sp. nov. as for P. (P.) formosus sp. nov. Specifically, Pseudechiniscus (P.) totoro sp. nov. differs from: P. (P.) beasleyi, by a different body colour (yelloworange in P. (P. ) totoro sp. nov. vs red in P. (P.) beasleyi) and shorter claws (6.1–8.0 μm in P. (P. ) totoro sp. nov. vs 9.1–13.1 μm in P. (P.) beasleyi). P. (P.) chengi, by a different body colour (yelloworange in P. (P.) totoro sp. nov. vs brown in P. (P. ) chengi) and shorter claws (6.1–8.0 μm in P. (P.) totoro sp. nov. vs 7.9–12.2 μm in P. (P. ) chengi). P. (P.) ehrenbergi, by the absence of papilla I (present in P. (P.) ehrenbergi). P. (P.) formosus sp. nov., by the dorsal epicuticular ornamentation (present in P. (P. ) totoro sp. nov. vs absent in P. (P. ) formosus sp. nov.) and the spacing of dorsal pillars (widely spaced in P. (P.) formosus sp. nov. vs densely arranged in P. (P.) totoro sp. nov.). P. (P. ) lacyformis, by the lengths of peribuccal cirri (cirrus internus 5.8–11.0 μm, cirrus externus 7.8–14.0 μm in P. (P.) totoro sp. nov. vs cirrus internus 10.6–14.0 μm, cirrus externus 14.1–19.4 μm in P. (P.) lacyformis). P. (P.) shintai, by the density and size of pillars present in leg patches (dense and large in P. (P.) totoro sp. nov. vs more widely spaced and smaller in P. (P. ) shintai). P. (P. ) suillus, by the morphology and position Pseudechiniscus sp. 16 * * * * * ** 0.05 Pseudechiniscus sp. 1 Pseudechiniscus quadrilobatus Pseudechiniscus mascarenensis Pseudechiniscus dreyeri sp. nov. Pseudechiniscus cf. saltensis Pseudechiniscus sp. 2 Pseudechiniscus sp. 3 Pseudechiniscus sp. 4 Pseudechiniscus sp. 6 Pseudechiniscus cf. angelusalas Pseudechiniscus sp. 7 Pseudechiniscus sp. 8 Pseudechiniscus sp. 18 Pseudechiniscus sp. 15 Pseudechiniscus sp. 17 Pseudechiniscus formosus sp. nov. Pseudechiniscus totoro sp. nov. Pseudechiniscus sp. 14 Pseudechiniscus sp. 10 Pseudechiniscus sp. 11 Pseudechiniscus sp. 9 Pseudechiniscus suillus Pseudechiniscus asper Pseudechiniscus shintai Pseudechiniscus cf. ehrenbergi Pseudechiniscus sp. 12 Pseudechiniscus sp. 13 outgroup echiniscids *** * ** * * * # 0.99 ** * 0.99 * 0.90 ** * 0.98 #* * * * #* * * * 1.00 100 * * 92 # * * * * * 99 * * 80 97 96 * * 93 * 99 ** 97 * 83 77 ** 98 * * 92 97 * 86 * 83 81 Pseudechiniscus Meridioniscus Fig. 30. Position of the Taiwanese species on the phylogenetic tree of the genus Pseudechiniscus based on the concatenated matrix (18S rRNA+28S rRNA+ITS-1). Values at nodes separated by forward slashes signify Bayesian posterior probability and bootstrap values (ML), respectively. Maximum supports, i.e., 1.00 for BI and 100 for ML, are indicated by asterisks (*), whereas nodes unsupported in either analysis are marked by hashtags (#). The scale refers to the Bayesian consensus tree and represents substitutions per site. Species numbering preserved from Gąsiorek et al. (2021c). page 39 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan of primary spurs on internal claws (delicate spurs positioned lower on claw branches in P. (P.) totoro sp. nov. vs robust spurs positioned higher on claw branches in P. (P.) suillus, see Grobys et al. 2020). P. (P.) xiai, by the division of the pseudosegmental plate IV’ (divided by a median longitudinal suture in P. (P.) totoro sp. nov. vs uniform in P. (P.) xiai) and slightly shorter claws I–III (6.1–7.5 μm in P. (P.) totoro sp. nov. vs 7.6–10.5 μm in P. (P.) xiai). Moreover, P. (P. ) totoro sp. nov. is distinguishable from all abovementioned species by the ventral sculpturing pattern. DISCUSSION The composition of the Taiwanese tardigrade fauna is of particular interest from the biogeographic point of view, as the island constitutes a transient zone, where the Oriental and Palaearctic elements mix and form a unique fauna with a high fraction of endemics (He et al. 2018). Many animal endemics evolved in isolation among high mountain ranges of Taiwan (Shih et al. 2006), which also exhibit habitats suitable for cold stenothermic species that are widespread in the Palaearctic. In parallel, subtropical evergreen forests growing in the lowlands create favourable conditions for species widely distributed in the tropics. Thus, three kinds of species with broad geographic ranges can be distinguished in the Taiwanese tardigrade fauna: (1) widely distributed species, typically associated with colder habitats (probably cosmopolitan E. blumi and Palaearctic N. reticulatus), (2) pantropical species or species widely distributed in the Pacific area (E. lineatus, Kristenseniscus tessellatus; see Suzuki et al. 2018 and Gąsiorek et al. 2019a b), and (3) probable allochthons/ecdemics introduced by humans (V. perviridis; see Kaczmarek and Michalczyk 2010). Table 13. Measurements [in μm] of selected morphological structures of the adult females of P. (P.) totoro sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD Holotype µm sp µm sp µm sp µm sp Body length 8 141–170 701–809 158 747 935 155 752 Scapular plate length 8 18.9–23.4 – 21.2 –1.7 –20.6 – Head appendage lengths Cirrus internus 8 5.8–11.0 25.6–50.7 8.2 39.1 1.5 8.4 8.9 43.2 Cephalic papilla 8 2.4–4.0 11.5–20.6 3.3 15.6 0.6 3.2 2.4 11.7 Cirrus externus 8 7.8–14.0 41.1–71.4 12.2 57.8 2.0 9.8 13.8 67.0 Clava 8 4.0–5.5 19.9–23.8 4.6 21.6 0.6 1.5 4.1 19.9 Cirrus A8 23.7–30.8 108.8–150.8 27.1 128.8 2.3 16.4 26.9 130.6 Cirrus A/Body length ratio 8 14%–20% –17% –2% –17% – Body appendage lengths Papilla on leg IV length 8 3.2–4.5 15.9–22.2 3.9 18.4 0.4 2.0 3.9 18.9 Claw I heights Branch 6 6.4–7.5 29.5–34.7 6.8 32.6 0.4 2.1 7.1 34.5 Spur 6 1.4–2.3 6.5–11.2 2.0 9.5 0.3 1.7 2.3 11.2 Spur/branch height ratio 6 22%–32% –29% –4% –32% – Claw II heights Branch 6 6.1–6.6 27.4–32.8 6.4 30.8 0.2 2.1 6.5 31.6 Spur 6 1.7–2.0 7.9–10.5 1.8 8.9 0.1 1.0 1.8 8.7 Spur/branch height ratio 6 26%–33% –29% –2% –28% – Claw III heights Branch 7 6.1–7.0 27.8–34.0 6.4 30.9 0.3 2.7 7.0 34.0 Spur 7 1.3–1.9 6.0–10.1 1.7 8.3 0.2 1.4 1.8 8.7 Spur/branch height ratio 7 21%–30% –27% –3% –26% – Claw IV heights Branch 5 6.9–8.0 33.5–38.0 7.5 35.8 0.5 1.9 ?? Spur 5 2.2–2.8 10.7–12.3 2.4 11.6 0.2 0.7 ?? Spur/branch height ratio 5 29%–37% –32% –3% –?– page 40 of 45Zoological Studies 60:70 (2021) © 2021 Academia Sinica, Taiwan Given that species endemic to Taiwan have been identified in other invertebrate groups, it is possible that also tardigrades exhibiting geographic distributions limited to the island may be uncovered with intensified sampling. Lastly, several Taiwanese echiniscid species can be defined as elements distinct for the Eastern Palaearctic (E. clevelandi, E. hoonsooi, E. semifoveolatus, N. crebraclava, and likely Stellariscus pseudelegans not found in our samples; Fig. 31); this set of taxa is supplemented by Echiniscus cheonyoungi Moon & Kim, 1994, Echiniscus laterosetosus Ito, 1993, Echiniscus polygonalis Ito, 1993, Hypechiniscus geminus Gąsiorek et al., 2021, H. flavus Gąsiorek et al., 2021, and Stellariscus elegans (Richters, 1907). There is evidence for close phylogenetic relationships between some of the Taiwanese echiniscids and their continental Palaearctic relatives. The sister relationship between E. clevelandi and E. hoonsooi within the E. virginicus complex (Fig. 11) supports the hypothesis that these two taxa evolved from a common ancestor in the Far East Palaearctic. This also suggests that E. cheonyoungi, described from the Korean Peninsula, is related to this clade. Gąsiorek et al. (2019b) hypothesised allopatric distributions of two other representatives of the complex, E. lineatus and E. virginicus, thus it would be desirable to verify whether sympatry occurs between the Asian members of this group or if they are separated in space by topographic factors, such as elevation (both E. clevelandi and E. lineatus inhabit China and Taiwan, and E. hoonsooi and E. cf. virginicus were reported from Japan (Suzuki 2017; Sato and Suzuki 2020). Moreover, many old Japanese tardigrade reports should be considered dubious, as they represent species with common Echiniscus chaetotaxy morphotypes, and chaetotaxy is usually unreliable when considered without the dorsal sculpturing (see records of E. dreyfusi de Barros, 1942, E. fischeri Richters, 1911, E. spiniger Richters, 1904, or Table 14. Measurements [in μm] of selected morphological structures of the juveniles of P. (P.) totoro sp. nov. mounted in Hoyer’s medium. N, number of specimens/structures measured; Range, refers to the smallest and the largest structure among all measured specimens; SD, standard deviation; sp, the proportion between the length of a given structure and the length of the scapular plate Character NRange Mean SD µm sp µm sp µm sp Body length 5 94–138 602–810 128 703 19 74 Scapular plate length 5 11.6–22.3 – 18.6 –4.1 – Head appendage lengths Cirrus internus 4 5.7–6.3 25.6–32.0 6.1 30.1 0.3 3.0 Cephalic papilla 5 2.5–3.4 11.2–24.1 3.0 16.8 0.4 4.7 Cirrus externus 4 9.8–12.4 49.7–57.9 11.0 53.9 1.2 3.6 Clava 5 3.4–4.6 17.9–29.3 4.0 22.1 0.5 4.7 Cirrus A4 14.1–22.1 106.6–121.6 19.8 113.3 3.8 6.2 Cirrus A/Body length ratio 4 15%–16% –16% –0% – Body appendage lengths Papilla on leg IV length 5 1.5–3.4 10.2–17.3 2.5 13.3 0.7 2.6 Claw I heights Branch 4 6.1–7.3 31.3–33.0 6.5 32.1 0.5 0.9 Spur 4 1.8–2.3 9.1–10.8 2.0 10.0 0.2 0.7 Spur/branch height ratio 4 29%–34% –31% –3% – Claw II heights Branch 5 4.5–6.4 28.4–38.8 5.6 31.0 0.7 4.4 Spur 5 1.0–2.0 7.1–10.3 1.5 8.3 0.4 1.2 Spur/branch height ratio 5 22%–36% –27% –5% – Claw III heights Branch 4 4.1–7.0 27.9–35.3 5.7 31.4 1.2 3.0 Spur 4 0.8–1.9 6.9–8.6 1.5 8.2 0.5 0.8 Spur/branch height ratio 4 20%–31% –26% –5% – Claw IV heights Branch 3 5.1–7.5 33.6–44.0 6.6 38.2 1.3 5.3 Spur 3 1.0–2.0 8.6–10.3 1.7 9.3 0.6 0.9 Spur/branch height ratio 3 20%–28% –25% –4% – page 41 of 45Zoological Studies 60:70 (2021)