Zoosyst. Evol. 96 (2) 2020, 527-536 | DOI 10.3897/zse.96.53324 yee BERLIN Pseudechiniscus in Japan: re-description of Pseudechiniscus asper Abe et al., 1998 and description of Pseudechiniscus shintai sp. nov. Katarzyna Von¢ina', Reinhardt M. Kristensen’, Piotr Gasiorek! 1 Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Krakéw, Poland 2 Section for Biosystematics, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, Copenhagen 0 DK-2100, Denmark http://zoobank.org/F79B0B2D-728D-4A3D-B3C3-06A1C3405F00 Corresponding author: Piotr Gasiorek (piotr.lukas.gasiorek@gmail.com) Academic editor: Pavel Stoev # Received 16 April 2020 Accepted 2 June 2020 Published | September 2020 Abstract The classification and identification of species within the genus Pseudechiniscus Thulin, 1911 has been considered almost a Sisyphe- an work due to an extremely high homogeneity of its members. Only recently have several contributions made progress in the tax- onomy feasible through their detailed analyses of morphology and, crucially, by the re-description of the ancient, nominal species P. suillus (Ehrenberg, 1853). Herein, we focus on the Japanese representatives of this genus: P. asper, a rare species originally described from Hokkaido, and a new species P. shintai. Both taxa belong to the widespread swillus-facettalis complex. Detailed descriptions entailing DNA barcoding of four markers and illustrations of the ventral pillar patterns are indispensable for an accurate delineation of species within this genus. Key Words biodiversity, Echiniscidae, Heterotardigrada, morphology, sculpturing Introduction Tardigrades are poorly known micrometazoans famous for their ability to enter cryptobiosis (Mobjerg et al. 2011). This phylum is now widely accepted as a lineage within the superclade Ecdysozoa (Campbell et al. 2011) and related to the Onychophora and Arthropoda within the Panarthropoda (Giribet and Edgecombe 2017). In the last decade, tens of new species have been described, which reflects limited understanding of tardigrade diver- sity (Bartels et al. 2016). Studies on the Japanese tardi- grades have a long history, resulting in over 150 species reported from this archipelago (Suzuki 2017). Amongst them, ca. 40 spp. (> 20%) belong to the limno-terrestri- al heterotardigrade family Echiniscidae (Gasiorek et al. 2018a, Suzuki et al. 2018), a distinct group characterised by the development of cuticular plates on the dorsal sur- face of the body (Kristensen 1987). Recent advances in the taxonomy of one of the echiniscid genera, Pseudechiniscus Thulin, 1911, are a good illustra- tion of the progress currently being made in the classifica- tion of tardigrades. Firstly, Tumanov (2020) discussed and re-organised the morphological nomenclature after a me- ticulous analysis of various members of Pseudechiniscus and he concluded that several species are unidentifiable, ac- cording to current taxonomic standards. Cesari et al. (2020) demonstrated high genetic variability amongst members of the speciose suillus-facettalis complex, implying that the Species richness in the genus may be underestimated. Final- ly, the ability to confidently describe new Pseudechiniscus Species was enabled by the modern diagnosis of P. suillus (Ehrenberg, 1853), one of enigmatic tardigrade taxa de- scribed in the 19" century (Grobys et al. 2020). In summary, better understanding of morphology, genetic disparities and ontogenetic shifts (Gasiorek et al. 2019, Morek et al. 2019) has facilitated intensification in tardigrade research. Copyright Katarzyna Voncina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 528 Katarzyna Voncina et al.: Japanese Pseudechiniscus Table 1. Primers and references for specific protocols for amplification of the four DNA fragments sequenced in the study. DNA fragment Primer name Primer direction 18S rRNA 18S_Tar_Ffl forward 18S_Tar_Rr2 reverse 28S rRNA 28S_Eutar_F forward 28SRO0990 reverse ITS-1 ITS Echi_ F forward ITS: leEchi"R reverse Col bcdFO1 forward bcdRO4 reverse * — All PCR programmes are also provided in Stec et al. (2015). In this contribution, we concentrate on the Japanese Pseudechiniscus species. Pseudechiniscus asper Abe et al., 1998 is re-described and P. shintai sp. nov. is described, based on specimens from Aomori Prefecture (Northern Honshu). A brief review of Japanese Pseudechiniscus re- cords is provided, concluding that they should be treated as unreliable and require formal confirmation through a new, large-scale sampling effort undertaken throughout Japan. Such a conclusion is in line with new discoveries of spe- cies complexes in numerous tardigrade genera (e.g. Gui- detti et al. 2019, Stec et al. 2020, Roszkowska et al. 2020). Materials and methods Sample collection and processing Specimens belonging to two species of the genus Pseude- chiniscus were extracted from four moss samples (JP.012— 5) collected from trees in Asamushi, Northern Honshu, Japan (ca. 40°54'03.6"N, 140°51'58"E, 30 m as.l.; R.M. Kristensen leg. on 24 July 2019). Samples were processed according to the protocol developed by Dastych (1980) with further amendments by Stec et al. (2015). The animals were used in two analyses: (I) qualitative and quantitative morphology, investigated under phase contrast microsco- py (PCM) and (II) DNA sequencing (see descriptions for details). Each specimen was observed in a drop of distilled water on a temporary slide under a 400 magnification to confirm its identification prior to analysis. Microscopy, imaging and morphometrics Permanent microscope slides were made using Hoyer’s medium and examined using an Olympus BX53 PCM associated with an Olympus DP74 digital camera. All figures were assembled in Corel Photo-Paint X7. All measurements are given in micrometres (um) and were performed under PCM. Structures were measured only when not broken, deformed or twisted and their orienta- tions were suitable. Body length was measured from the anterior to the posterior end of the body, excluding the hind legs. The sp ratio is the ratio of the length of a given structure to the length of the scapular plate expressed as a percentage (Dastych 1999). Morphometric data were zse.pensoft.net Primer sequence (5’-3’) AGGCGAAACCGCGAATGGCTC CTGATCGCCTTCGAACCTCTAACTT TCG ACCCGCTGAACTTAAGCATAT CCTTGGTCCGTGTTTCAAGAC CCGTCGCTACTACCGATTGG GTTCAGAAAACCCTGCAATTCACG CATTTTCHACTAAYCATAARGATATTGG TATAAACY TCDGGATGNCCAAAAAA Primer source PCR programme* Stec et al. (2017) Gasiorek et al. (2017) Gasiorek et al. (2018b) Mironov et al. (2012) Gasiorek et al. (2019) Zeller (2010) Mironov et al. (2012) Wetnicz et al. (2011) Dabert et al. (2008) Wetnicz et al. (2011) handled using the Echiniscoidea ver. 1.3 template, avail- able from the Tardigrada Register, www.tardigrada.net (Michalezyk and Kaczmarek 2013). Importantly, all spe- cies designated as dubious or with insufficient descrip- tions (Grobys et al. 2020, Tumanov 2020), were discard- ed from the differential diagnoses. Genotyping and genetic comparisons DNA was extracted from individual animals following a Chelex 100 resin (Bio-Rad) extraction method (Casquet et al. 2012, Stec et al. 2015). Hologenophores were obtained for both species (Pleijel et al. 2008). Four DNA fragments were sequenced: three nuclear and one mitochondrial (Ta- ble 1) in the case of P. shintai sp. nov.; and three for P. asper. The COI fragment was amplifiable for P. asper but, due to a high number of double peaks, effective sequence cleaning was not possible. All fragments were amplified and sequenced, according to the protocols described in Stec et al. (2015). The obtained alignments were edited and checked manually in BioEdit v7.2.6.1 (Hall 1999) and ClustalW Multiple Alignment tool (Thompson et al. 1994) was used in the alignment of COI for P. shintai sp. nov. and other confidently identified species (Grobys et al. 2020, Roszkowska et al. 2020). MEGA7.0.26 (Kumar et al. 2016) was used for calculation of uncorrected pairwise distances (Srivathsan and Meier 2012). Results Systematic account Phylum: Tardigrada Doyere, 1840 Class: Heterotardigrada Marcus, 1927 Order: Echiniscoidea Richters, 1926 Family: Echiniscidae Thulin, 1928 Genus: Pseudechiniscus Thulin, 1911 Pseudechiniscus asper Abe, Utsugi & Takeda, 1998 Figures 1, 2, 5A, Tables 2, 3 Locus typicus and type material. ca. 42°46'N, 141°24'E, 250 m as.l.; vicinity of the Lake Shikotsu (Chitose, Zoosyst. Evol. 96 (2) 2020, 527-536 Figure 1. Habitus of Pseudechiniscus asper (PCM): A, B — fe- males; C — male hologenophore. Insert shows claws III. Arrow- heads indicate thickenings at the lateral positions C and D. List of abbreviations: c — caudal plate, cA — cirrus A, ce — cirrus ex- ternus, Ci — cirrus internus, cp — cephalic plate, m1—3 — median plates, ps — pseudosegmental plate IV’, sI—II — paired segmental plates, sc — scapular plate. Scale bars: in um. South-western Hokkaido, Japan); foliose lichen Phaeo- Physcia imbricata (Physciaceae) on the trunk of a maple (Acer japonicum). Collector: Kazuo Utsugi. Holotype: adult male on the slide NSMT-Tg 44 deposited in the Na- tional Museum of Nature and Science in Tokyo. Additional material. Four females on the slides JP.012.01, JP.013.01—2, JP.014.01 and a male on the slide JP.012.04. Hologenophores: JP.012.01, 4, JP.013.02. Etymology. From Latin asper = rough, referring to the irregular surface of dorsal plates. Adjective in the nom- inative singular. 529 Figure 2. Sculpturing of Pseudechiniscus asper (PCM): A — dorsal; B — ventral. White arrowheads indicate thickenings at the lateral positions C and D, black arrowheads indicate claw spurs and empty arrows indicate papillae IV. Scale bars: in wm. Description. Mature females (i.e. from the third instar onwards; measurements in Table 2). Body dark orange, with round black eyes present or dissolving soon after mounting (Fig. 1A, B). Member of the swillus-facettalis complex: dome-shaped (hemispherical) cephalic papillae (secondary clavae) and minute (primary) clavae; peri- buccal cirri with poorly developed cirrophores. Cirrus A short, with cirrophore. Dorsal plates well-sclerotised as for a Pseudechiniscus species, clearly demarcated from each other, with Pseudechiniscus-type sculpturing, 1.e. large endocuticular pillars protruding through the epicuticle and visible as dark dots in PCM (Fig. 2A). Striae absent. The cephalic plate pentapartite, with the anterior bi-halved portion and three posterior portions, roughly equal in size (Fig. 1A, B). The cervical (neck) plate absent. The scapular plate with a transverse suture, separating a broader anterior portion and narrower posterior portion (Figs 1B, 2A). Three median plates: m1—2 bipartite, with much reduced, narrow posterior portions, m3 unipartite and large (Fig. 2A) with two pairs of lateral intersegmental platelets flanking the borders of ml—2. Two pairs of large segmental plates, their posterior portions exhibiting thickenings at positions C and D — the latter usually more pronounced (Figs 1A, B, 2). The pseudosegmental plate IV’ divided by a median longitudinal suture; the posterior margin of the plate with a pair of short triangular projections (Figs 1A, B, 2A). The caudal (terminal) plate with short incisions that may be sclerotised (compare Fig. 1A with Fig. 1B). Ventral cuticle with a faint species-specific pattern reaching the lateroventral sides of the body (Figs 2B, 5A), zse.pensoft.net 530 Katarzyna Voncina et al.: Japanese Pseudechiniscus Table 2. Measurements [in um] of selected morphological structures of mature females of Pseudechiniscus asper mounted in Hoy- er’s medium (N — number of specimens/structures measured, RANGE refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation). CHARACTER N RANGE MEAN SD ym sp ym sp ym sp Body length 2 178 - 203 712 - 810 191 761 18 70 Scapular plate length 2 25.0 - 25.1 - 25.1 - 0.1 - Head appendages lengths Cirrus internus 3 Oe. - 11.4 42.0 - 45.4 10.4 43.7 1.1 2.4 Cephalic papilla 3 325 - 4.7 L379 - 18.8 41 16.4 0.6 3.4 Cirrus externus 3 14.8 - 19.4 59.2 - 77.3 16.6 68.2 2.5 12.8 Clava 3 4.3 - 5.9 LZ - 20.3 5s. 18.8 0.8 2.2 Cirrus A 3 24.1 - 30.8 96.4 - LAAT! © 273 109.6 3.4 18.6 Cirrus A/Body length ratio 2 14% - 15% - 14% - 1% - Papilla on leg IV length 4 3.0 - 3.6 12.0 - 13.9 3.4 13.0 0.3 1.4 Claw 1 heights Branch 4 8.8 - 10.7 40.2 - 40.4 9.9 40.3 0.8 0.1 Spur 4 12 - 1.8 4.8 - 6.4 1.6 5.6 0.3 Est Spur/branch length ratio 3 12% - 20% - 16% - A% - Claw 2 heights Branch 3 8.5 - 9.8 38.0 - 39.0 9.3 38.5 0.7 0.7 Spur 3 1.2 - 1.4 4.8 - 5.2 1.3 5:0 0.1 0.3 Spur/branch length ratio 3 13% - 16% - 14% - 2% - Claw 3 heights Branch 2 9.2 - 10.1 36.8 - 40.2 9.7 38.5 0.6 2.4 Spur 2 1.0 - 1.8 4.0 - ae 1.4 5.6 0.6 2.2 Spur/branch length ratio 2 11% - 18% - 14% - 5% - Claw 4 heights Branch 2 11.6 - 11.6 46.2 - 46.2 le 6 46.2 0.0 ? Spur 2 2.0 - 2.0 8.0 - 8.0 2.0 8.0 0.0 ? Spur/branch length ratio 2 17% - 17% - 17% - 0% - Table 3. Measurements [in um] of selected morphological structures of mature males of Pseudechiniscus asper mounted in Hoyer’s medium. Measurements of the holotype taken from Abe et al. (1998). CHARACTER 3 Holotype ym sp ym Body length 159 675 166 Scapular plate length 23.5 - ? Head appendages lengths Cirrus internus 11.4 48.5 8.0 Cephalic papilla 4.7 20.0 ? Cirrus externus 14.8 63.0 12.0 Clava 3.5 14.9 1.5 Cirrus A 19.4 82.6 20.0 Cirrus A/Body length ratio 12% - 12% Papilla on leg IV length 327 L5t7 ? Claw 1 heights Branch ? ? ca. 9.0 Spur ? ? 2 Spur/branch length ratio ? - ? Claw 2 heights Branch 11.1 47.2 ca. 9.0 Spur 0.9 3.8 ? Spur/branch length ratio 8% - 2 Claw 3 heights Branch 10.9 46.4 ca. 9.0 Spur ? ? ? Spur/branch length ratio 2 - ? Claw 4 heights Branch 131 5 oR. ca. 11.0 Spur 1.0 4.3 ? Spur/branch length ratio 8% - ? being a typical reticulum composed of large multiangular, longitudinal shapes connected by belts of pillars. Pillars zse.pensoft.net are particularly poorly visible between legs I and II (Fig. 2B). The subcephalic zone with a wide patch of pillars (Fig. 5A). Sexpartite gonopore located anteriorly of legs IV and a trilobed anus between legs IV. Pedal plates and dentate collar IV absent; instead, large patches of pillars are present centrally on each leg (Fig. 1A, B). Pulvini indistinct. No papilla or spine on leg I visible in PCM, a papilla on leg IV present (Figs 1B, 2). Claws IV higher than claws J-II; internal claws with needle-like spurs positioned at ca. 1/4—-1/5 of the claw height (Fig. 1A, insert). Mature males (1.e. from the second instar onwards; measurements in Table 3). Smaller than females, with slender body (Fig. 1C). Cirri externi approaching the length of cirri A. Pseudosegmental projections in the form of teeth or wide lobes. Gonopore circular. Juveniles. Unknown. Larvae. Unknown. Eggs. Unknown. DNA sequences. Single haplotypes in 18S rRNA (MT645083, 843 bp), 28S rRNA (MT645081, 716 bp) and ITS-1 (MT645085, 631 bp) were obtained. Remarks. This is the third record of this very rare spe- cies, which, in addition to the type locality, has also been found on Mount Taibai, Shaanxi, China (Li et al. 2005). In the original description, only one male was found to possess triangular projections, ending with papillate tips, on the pseudosegmental plate (Abe et al. 1998). However, Zoosyst. Evol. 96 (2) 2020, 527-536 the variability in the shape of the pseudosegmental pro- jections has previously been noted (Fontoura et al. 2010), thus the lobate form of these structures in the Chinese and Japanese (Honshu) specimens is not surprising. Moreover, Abe et al. (1998) did not illustrate the complete ventral pattern of this species (most likely because of the quality of the microscope used) and omitted the swelling or thick- ening of the armour at position C, which is weakly devel- oped in this species. As Asamushi lies only ca. 200 km southwards from the shores of Lake Shikotsu (however, the Blakiston’s Line was designated to separate faunae of large vertebrates of Honshu and Hokkaido; see Kawamura 2007), the formal amendments to the original description presented here are justified given that DNA barcodes com- pensate the scarcity of specimens used in morphometrics. Phenotypic differential diagnosis. Taxa most similar to P. asper, i.e. those possessing pseudosegmental projec- tions, can be easily distinguished from this species, based on the presence of striae (even rudimentary striae are absent in P. asper; see Fig. 2 in Fontoura et al. 2010 for microphotographs of other species), and/or by the lack of thickenings at the lateral positions (Abe et al. 1998). Pseudechiniscus shintai sp. nov. http://zoobank.org/1 BC6B3B3-16EF-4442-A D6D-CCD357C47C31 Figures 3, 4, 5B, Tables 4, 5 Locus typicus and type material. ca. 40°54'03.6"N, 140°51'58"E, 30 m a.s.l.; Asamushi-Onsen Forest Park (Aomori, Northern Honshu, Japan); mosses from tree trunks. Collector: R.M. Kristensen. Holotype and al- lotype: mature female and male on slide JP.013.01. Eight juveniles on the slides JP.012.02—3, JP.013.03-4, JP.014.01-3, JP.015.01. Hologenophores: JP.012.02-3, JP.013.03-4. Holotype, allotype and the majority of para- types (slides JP.012.02-3, JP.013.01, JP.013.03-4 and JP.015.01) are deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University, Krakow, Poland; other paratypes (slides JP.014.01—3; NHMD-— 669705—7) are deposited in the Natural History Museum of Denmark, University of Copenhagen, Denmark. Etymology. The name is a patronym honouring Shinta Fu- jimoto, an excellent Japanese tardigradologist specialising in marine Heterotardigrada. Noun in the genitive singular. Description. Mature female (i.e. the third or latter in- star; measurements in Table 4). Body orange, with min- ute, round black eyes that are absent after mounting (Figs 3A, 4A). Member of the swillus-facettalis com- plex: dome-shaped (hemispherical) cephalic papillae (secondary clavae) and minute (primary) clavae; peri- buccal cirri with poorly developed cirrophores. Cirrus A short, with cirrophore. Dorsal plates poorly sclerotised, but clearly demar- cated from each other, with the Pseudechiniscus-type Figure 3. Habitus of Pseudechiniscus shintai sp. nov. (PCM): A — female (holotype); B — male (allotype). Insert shows claws I. Scale bars: in um. Figure 4. Sculpturing of Pseudechiniscus shintai sp. nov. (PCM): A — dorsal; B — ventral. Scale bars: in um. sculpturing, i.e. endocuticular pillars protruding through the epicuticle and visible as dark dots in PCM (Fig. 4A). Striae absent; epicuticular ornamentation visible as dark- er belts on all dorsal plates. The cephalic plate pentapar- tite, with the two anterior portions and three posterior portions approximately equal in size (Fig. 4A). The cer- vical (neck) plate absent. The scapular plate with sutures, separating a wide anterior portion from the four posterior portions (Fig. 4A). Three median plates: m1—2 bipartite; zse.pensoft.net 532 Katarzyna Voncina et al.: Japanese Pseudechiniscus Figure 5. Schematic drawings of ventral sculpturing patterns: A — Pseudechiniscus asper;, B — Pseudechiniscus shintai sp. nov. Table 4. Measurements [in um] of selected morphological structures of mature female (holotype) and male (allotype) of Pseudechiniscus shintai sp. nov. mounted in Hoyer’s medium. CHARACTER Holotype ° Allotype 3 ym sp ym sp Body length 196 P35: 178 754 Scapular plate length 26.7 - 23.6 - Head appendages lengths Cirrus internus 6.8 25,5 8.2 34.7 Cephalic papilla 4.0 15.0 35 14.8 Cirrus externus 12.8 47.9 12.5 53.0 Clava 5.0 18.7 4.0 16.9 Cirrus A 234i 86.5 25.3 107.2 Cirrus A/Body length ratio 12% - 14% - Papilla on leg IV length 3.1 11.6 2.3 OF Claw 1 heights Branch 8.7 32.6 Hid 30.9 Spur 2.2 8.2 1.4 5.9 Spur/branch length ratio 25% - 19% - Claw 2 heights Branch 8.7 32.6 7.0 29,7 Spur 2.0 is) 1.6 6.8 Spur/branch length ratio 23% - 23% - Claw 3 heights Branch 8.8 33.0 7.2 30.5 Spur 1-9 7.1 ileal 4.7 Spur/branch length ratio 22% - 15% - Claw 4 heights Branch O22 34.5 8.8 37.3 Spur Les 6.4 1.8 7.6 Spur/branch length ratio 18% - 20% - m3 unipartite (Figs 3A, 4A); four pairs of lateral interseg- mental platelets flanking the borders of m1l—2. Two pairs of large segmental plates. The pseudosegmental plate IV’ divided by a median longitudinal suture; the posterior margin of the plate can be wide (Fig. 4A), but without lobes or teeth (Fig. 3A). The caudal (terminal) plate with short sclerotised incisions (Figs 3A, 4A). zse.pensoft.net Ventral cuticle with a pronounced species-specific pattern reaching the lateroventral sides of the body (Figs AB and 5B), being a typical reticulum composed of large multiangular, longitudinal shapes joined by belts of pil- lars. The subcephalic zone with a wide belt of pillars. Sexpartite gonopore located anteriorly of legs IV and a trilobed anus between legs I'V. Pedal plates and dentate collar IV absent, instead large patches of pillars are present centrally on each leg (Fig. 3A). Pulvini indistinct. A papilla on leg I undetect- able in PCM and a papilla on leg IV present, but scarcely visible. Claws I-IV of similar heights. External claws on all legs smooth. Internal claws with minuscule, thin spurs positioned at ca. 1/5 of the claw height. (Fig. 3A, insert). Mature male (i.e. the second or latter instar; measure- ments in Table 4). No significant differences from females (Fig. 3B). Gonopore circular. Juveniles (1.e. the second instar; measurements in Ta- ble 5). A morphometric gap exists between adult females and juveniles. Phenotypically similar to adults. Gonopore absent. Larvae. Unknown. Eggs. Unknown. DNA _ sequences. Single haplotypes in 18S rRNA (MT645084, 900 bp), 28S rRNA (MT645082, 754 bp) and ITS-1 (MT645086, 622 bp), but two in COI (MT644270- 1,510 bp) were found. Phenotypic differential diagnosis. The species was com- pared with the members of the sui//us-facettalis complex (with hemispherical cephalic papillae) and other Pseude- Zoosyst. Evol. 96 (2) 2020, 527-536 533 Table 5. Measurements [in um] of selected morphological structures of juveniles of Pseudechiniscus shintai sp. nov. mounted in Hoyer’s medium (N — number of specimens/structures measured, RANGE refers to the smallest and the largest structure amongst all measured specimens; SD — standard deviation). CHARACTER N RANGE MEAN SD ym sp ym sp ym sp Body length 6 93 - 172 597 - 730 144 682 27 49 Scapular plate length 7 13.2 - 24.1 - 21.7 - 3.6 - Head appendages lengths Cirrus internus A 5.2 - 8.9 22./ - 37.4 71 See 1.2 5.2 Cephalic papilla 6 2.0 - 4.3 11.9 - 18.4 3.0 14.5 0.8 2.2 Cirrus externus 7 6.1 - 12.1 41.9 - 57 10.0 46.8 1.9 3.6 Clava 2 3.9 - 44 1726 - 18.8 4.2 18.2 0.4 0.8 Cirrus A 4 ial - 26.0 84.2 - BEET 22.2 96.8 3x/ 11.0 Cirrus A/Body length ratio 3 12% - 16% - 14% - 2% - Papilla on leg IV length 4 2.9 - 3.9 2256 - 16.5 3:3 14.3 0.5 1.6 Claw 1 heights Branch 5 5°9 - 7.8 30.5 - 44,7 7.1 34.9 0.7 5.6 Spur 5 0.9 - IEE 5.9 - 7.4 1.4 6.7 0.3 0:5 Spur/branch length ratio 5 15% - 23% - 20% - 3% - Claw 2 heights Branch 6 6.7 - 8.9 30.1 - 37.2 Tet 33.2 0.8 2.3 Spur 6 ileal - 1.7 Oe, - 7.1 1.4 oa) 0.2 0.8 Spur/branch length ratio 6 16% - 20% - 18% - 2% - Claw 3 heights Branch 6 53 - 9.0 29.2 - 40.2 7.2 34.3 1.2 4.0 Spur 6 1.0 - 1.8 55 - 7.6 1.4 G7 0.3 0.9 Spur/branch length ratio 6 18% - 21% - 19% - 1% - Claw 4 heights Branch 5 7.2 - 9.0 3.575 - SO 8.4 37.2 0.7 1.4 Spur 5 1.4 - 2.1 6.0 - 9.2 We Yess) 0.3 1.2 Spur/branch length ratio 5 16% - 23% - 20% - 3% - chiniscus species lacking pseudosegmental projections. P. shintai sp. nov. is differentiated from: 6. P. indistinctus Roszkowska et al., 2020, described from Norway, by the shape of its cephalic papillae I. P. angelusalas Roszkowska et al., 2020, described from Madagascar, by the shape of its cephalic papil- lae (hemispherical in P. shintai sp. nov. vs. dactyloid, elongated in P. angelusalas) and by the presence of striae (striae absent in P. shintai sp. nov. vs. present, but poorly developed in P. angelusalas); . P. beasleyi Li et al., 2007, described from Qinling Mountains (China), by much shorter claws (5.3—9.2 um in P. shintai sp. nov. vs. 9.1-13.1 um in P. beasleyi), . P. chengi Xue et al., 2017, described from Ningxia (China), by body colour (orange in P. shintai sp. nov. vs. brown in P. chengi) and by the distribution of pil- lars in the dorsal plates (sparsely distributed in P. shin- tai sp. nov. vs. densely arranged in P. chengi); . P. dastychi Roszkowska et al., 2020, described from the Argentine Islands (maritime Antarctic), by the shape of the cephalic papillae (hemispherical in P. shintai sp. nov. vs. dactyloid, elongated in P. dastychi) and by the presence of striae (striae absent in P. shintai Sp. nov. vs. present in P. dastychi); . P. ehrenbergi Roszkowska et al., 2020, described from Northern Italy and reported from Mongolia (Cesari et al. 2020), by the subdivision of the scapular plate (with- out the median longitudinal suture in P. shintai sp. nov. vs. with the median longitudinal suture in P. ehrenber- gi) and by the presence of a rudimentary papilla I (ab- sent in P. shintai sp. nov. vs. present in P. ehrenbergi); (hemispherical in P. shintai sp. nov. vs. dactyloid, elongated in P. indistinctus) and by the presence of Striae (striae absent in P. shintai sp. nov. vs. present in P. indistinctus); 7. P. lacyformis Roszkowska et al., 2020, described from Norway, by the length of its cephalic appendages: cirrus internus (5.2-8.9 um in P. shintai sp. nov. vs. 10.6—14.0 um in P. lacyformis), cirrus externus (6.1— 12.8 um in P. shintai sp. nov. vs. 14.1-19.4 um in P. lacyformis) and cirrus A (17.1—26.0 um in P. shintai sp. nov. vs. 26.5—35.0 um in P. lacyformis), 8. P. suillus (Ehrenberg, 1853), reliably recorded only from Italy (Grobys et al. 2020), by the length of cirrus A (17.1—26.0 um in P. shintai sp. nov. vs. 28.4—-34.4 um in P. suillus) and by the presence of males (present in P. shintai sp. nov. vs. absent in P. suillus), 9. P. xiai Wang et al., 2018, described from Ningxia (Chi- na), by the contrasting dorsal sculpturing (epicuticular ornamentation darker and more pronounced in P. xia) and by the morphology of the pseudosegmental plate IV’ (paired in P. shintai sp. nov. vs. unpaired in P. xiai). Moreover, the ventral pattern distinguishes P. shintai sp. nov. from all other species for which this character has been described. We used morphometric differences for comparisons only as a last resort as sample sizes for the majority of the specimens in the type series were small. Importantly, although Roszkowska et al. (2020) included zse.pensoft.net 534 P. angelusalas, P. dastychi and P. indistinctus in the suil- lus-facettalis complex, such combination is phylogeneti- cally unjustified, as they all exhibit elongated (dactyloid) cephalic papillae, which is a distinguishing trait of P. no- vaezeelandiae (Richters, 1908) (see Pilato et al. 2005) and of the entire novaezeelandiae \ineage (Cesari et al. 2020). Genotypic differential diagnosis. p-distances between the new species and the remaining Pseudechiniscus spp., for which COI sequences are available, ranged between 18.6% (P. suillus) and 29.3% (P. lacyformis). Intraspecif- ic distance was equal to 0.2%. Discussion The dorsal sculpturing of P. asper is particularly inter- esting morphologically, as large, roughly circular endo- cuticular pillars protrude through the epicuticle as isolat- ed, solid bumps, unconnected by thin ridges — striae. In many other Pseudechiniscus species, striae are typical elements of the armour (e.g. Pilato et al. 2004, Pilato and Lisi 2006). Tumanov (2020) suggested that their presence may represent a phylogenetic signal and, as striae are ab- sent in P. suillus (Grobys et al. 2020), this could mean that the absence of striae is a trait specific to the swillus-fac- ettalis lineage (Cesari et al. 2020). The hypothesis would necessitate a comprehensive analysis of the sculpturing amongst the entire suite of species. The recent studies on Pseudechiniscus imply that all previous records of putatively cosmopolitan species should be questioned and verified to ensure against misi- dentification (Grobys et al. 2020, Tumanov 2020). This is the case for almost all Pseudechiniscus spp. reported from Japan: P. suillus, P. bartkei Weglarska, 1962, P. facettalis Petersen, 1951, P. pseudoconifer Ramazzotti, 1943 and P. ramazzottii Maucci, 1952 (see Suzuki 2017). As the Japanese fauna of the four main islands is considered to be a part of the Palaearctic with high levels of endemism in many animal groups due to the isolation during glacia- tion periods (Motokawa 2017), it cannot be excluded that some of the above-mentioned species inhabit the Japa- nese archipelago (all but P. bartkei were described from the Western Palaearctic and Greenland). To confirm their status as native to Japan, re-descriptions must be prepared and an enhanced sampling effort is required in Japan. Acknowledgements Brian Blagden kindly proofread the manuscript. Diane Nelson, Atsushi Suzuki and Harry Meyer are gratefully acknowledged for the improvements made on the manu- script. The study was performed within the scope of the Preludium grant funded by the National Science Centre (grant no. 2019/33/N/NZ8/02777 to PG supervised by LM) and the Sonata Bis programme of the National Sci- ence Centre (grant no. 2016/22/E/NZ8/00417 to LM). zse.pensoft.net Katarzyna Voncina et al.: Japanese Pseudechiniscus Lukasz Michalczyk is acknowledged for advice and con- stant support. We owe our sincere thanks to the Muse- um fur Naturkunde, Berlin, for covering the publication charge. The Authors declare no conflict of interest. References Abe W, Utsugi K, Takeda M (1998) Pseudechiniscus asper, a new Tar- digrada (Heterotardigrada: Echiniscidae) from Hokkaido, Northern Japan. Proceedings of the Biological Society of Washington 111: 843-848. Bartels PJ, Apodaca JJ, Mora C, Nelson DR (2016) A global biodi- versity estimate of a poorly known taxon: phylum Tardigrada. Zo- ological Journal of the Linnean Society 178: 730-736. https://doi. org/10.1111/zoj.12441 Campbell LI, Rota-Stabelli O, Edgecombe GD, Marchioro T, Long- horn SJ, Telford MJ, Philippe H, Rebecchi L, Peterson KJ, Pisani D (2011) MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda. PNAS 108: 15920-15924. https://doi.org/10.1073/ pnas.1105499108 Casquet J, Thebaud C, Gillespie RG (2012) Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders. Molecular Ecology Resources 12: 136-141. https://doi.org/10.1111/j.1755-0998.2011.03073.x Cesari M, Montanari M, Kristensen RM, Bertolani R, Guidetti R, Rebecchi L (2020) An integrated study of the biodiversity within the Pseudechiniscus suillus—facettalis group (Heterotardigrada: Echiniscidae). Zoological Journal of the Linnean Society 188: 717— 732. https://doi.org/10.1093/zoolinnean/z1z045 Dabert J, Ehrnsberger R, Dabert M (2008) Glaucalges tytonis sp. nov. (Analgoidea: Xolalgidae) from the barn owl 7yto alba (Strigiformes: Tytonidae): compiling morphology with DNA barcode data for taxa descriptions in mites (Acari). Zootaxa 1719: 41-52. Dastych H (1980) Niesporczaki (Tardigrada) Tatrzanskiego Parku Narodowego. Monografie Fauny Polski 9: 1—232. Dastych H (1999) A new species of the genus Mopsechniscus Du Bois-Reymond Marcus, 1944 (Tardigrada) from the Venezuelan An- des. Acta biologica Benrodis 10: 91-101. Doyeére M (1840) Mémoire sur les tardigrades. Annales des Sciences Naturelles, Zoologie (Series 2) 14: 269-362. Ehrenberg CG (1853) Diagnoses novarum formarum. Monatsberichte der Koniglich Preussischen Akademie der Wissenschaften zu Berlin 8: 526-533. Fontoura P, Pilato G, Lisi O (2010) First record of Tardigrada from Sao Tomé (Gulf of Guinea, Western Equatorial Africa) and descrip- tion of Pseudechiniscus santomensis sp. nov. (Heterotardigrada: Echiniscidae). Zootaxa 2564: 31-42. https://doi.org/10.11646/zoo- taxa.2564.1.2 Gasiorek P, Blagden B, Michalczyk L (2019) Towards a better un- derstanding of echiniscid intraspecific variability: A redescrip- tion of Nebularmis reticulatus (Murray, 1905) (Heterotardigrada: Echiniscoidea). Zoologischer Anzeiger 283: 242-255. https://do1. org/10.1016/j.jcz.2019.08.003 Gasiorek P, Stec D, Morek W, Michalezyk L (2017) An integrative re- description of Echiniscus testudo (Doyere, 1840), the nominal taxon for the class Heterotardigrada (Ecdysozoa: Panarthropoda: Tardigra- Zoosyst. Evol. 96 (2) 2020, 527-536 da). Zoologischer Anzeiger 270: 107-122. https://doi.org/10.1016/j. jez.2017.09.006 Gasiorek P, Stec D, Zawierucha K, Kristensen RM, Michalczyk L (2018b) Revision of Testechiniscus Kristensen, 1987 (Heterotar- digrada: Echiniscidae) refutes the polar-temperate distribution of the genus. Zootaxa 4472: 261-297. https://doi.org/10.11646/zoot- axa.4472.2.3 Gasiorek P, Suzuki AC, Kristensen RM, Lachowska-Cierlik D, Michal- czyk L (2018a) Untangling the Echiniscus Gordian knot: Stellariscus gen. nov. (Heterotardigrada: Echiniscidae) from Far East Asia. Inver- tebrate Systematics 32: 1234-1247. https://doi.org/10.1071/1S18023 Giribet G, Edgecombe GD (2017) Current understanding of Ecdysozoa and its internal phylogenetic relationships. Integrative and Compar- ative Biology 57: 455—466. https://doi.org/10.1093/icb/icx072 Grobys D, Roszkowska M, Gawlak M, Kmita H, Kepel A, Kepel M, Parnikoza I, Bartylak T, Kaczmarek L (2020) High diversity in the Pseudechiniscus suillus—facettalis complex (Heterotardigrada: Echiniscidae) with remarks on the morphology of the genus Pseude- chiniscus. Zoological Journal of the Linnean Society 188: 733-752. https://doi.org/10.1093/zoolinnean/zlz171 Guidetti R, Cesari M, Bertolani R, Altiero T, Rebecchi L (2019) High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae). Zoological Letters 5: 1. https://do1.org/10.1186/s40851-018-0113-z Hall TA (1999) BioEdit: a user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Kawamura Y (2007) Last glacial and Holocene land mammals of the Japanese islands: their fauna, extinction and immigration. Quaterna- ry Research 46: 171-177. https://doi.org/10.4116/jaqua.46.171 Kristensen RM (1987) Generic revision of the Echiniscidae (Heterotar- digrada), with a discussion of the origin of the family. In: Bertola- ni R (Ed.) Biology of Tardigrades. Selected Symposia and Mono- graphs U.Z.I. 1: 261-335. Kumar S, Stecher G, Tamura K (2016) MEGA 7: Molecular evolution- ary genetics analysis version 7.0 for bigger datasets. Molecular Biol- ogy and Evolution 33: 1870-1874. https://doi.org/10.1093/molbev/ msw054 Li X, Wang L, Liu Y, Su L (2005) A new species and five new records of the family Echiniscidae (Tardigrada) from China. Zootaxa 1093: 25-33. https://do1.org/10.11646/zootaxa.1093.1.2 Li X, Wang L, Yu D (2007) The Tardigrada fauna of China with de- scriptions of three new species of Echiniscidae. Zoological Studies 46: 135-147. Marcus E (1927) Zur Anatomie und Okologie mariner Tardigraden. Zo- ologische Jahrbicher. Abteilung fiir Systematik 53: 487-558. Maucci W (1952) Un nuovo Pseudechiniscus del Carso Triestino (Tar- digrada, Scutechiniscidae). Atti della Societa Italiana di Scienze Naturali 91: 127-130. Michalcezyk L, Kaczmarek L (2013) The Tardigrada Register: a com- prehensive online data repository for tardigrade taxonomy. Journal of Limnology 72: 175-181. https://doi.org/10.4081/jlimnol.2013. slie22 Mironov SV, Dabert J, Dabert M (2012) A new feather mite species of the genus Proctophyllodes Robin, 1877 (Astigmata: Proctophyllo- didae) from the long-tailed tit Aegithalos caudatus (Passeriformes: Aegithalidae): morphological description with DNA barcode data. Zootaxa 3253: 54-61. https://doi.org/10.11646/zootaxa.3253.1.2 535 Morek W, Stec D, Gasiorek P, Surmacz B, Michalezyk L (2019) Mil- nesium tardigradum Doyere, 1840: The first integrative study of interpopulation variability in a tardigrade species. Journal of Zoo- logical Systematics and Evolutionary Research 57: 1—23. https://doi. org/10.1111/jzs.12233 Motokawa M (2017) “Land emergence” and “elevation shift” affect diversification: A new perspective toward understanding the high species diversity of terrestrial animals in Japan. In: M. Motoka- wa, H. Kajihara (Eds) Species Diversity of Animals in Japan. Di- versity and Commonality in Animals. Springer, Tokyo. https://doi. org/10.1007/978-4-43 1-56432-4 Mobjerg N, Halberg KA, Jorgensen A, Persson D, Bjorn M, Ramlov H, Kristensen RM (2011) Survival in extreme environments — on the current knowledge of adaptations in tardigrades. Acta Physiologica 202: 409-420. https://doi.org/10.1111/).1748-1716.2011.02252.x Petersen B (1951) The tardigrade fauna of Greenland. Meddelelser om Gronland 150: 5—94. Pilato G, Binda MG, Lisi O (2004) Notes on some tardigrades from Thailand, with the description of two new species. New Zealand Journal of Zoology 31: 319-325. https://doi.org/10.1080/0301422 3.2004.9518385 Pilato G, Binda MG, Lisi O (2005) Remarks on some Echiniscidae (Heterotardigrada) from New Zealand with the description of two new species. Zootaxa 1027: 27-45. https://doi.org/10.11646/zoot- axa.1027.1.2 Pilato G, Lisi O (2006) Notes on some tardigrades from southern Mex- ico with description of three new species. Zootaxa 1236: 53-68. https://doi.org/10.11646/zootaxa.1236.1.4 Pleijel F, Jondelius U, Norlinder E, Nygren A, Oxelman B, Schander C, Sundberg P, Thollesson M (2008) Phylogenies without roots? A plea for the use of vouchers in molecular studies. Molecular Phy- logenetics and Evolution 48: 369-371. https://doi.org/10.1016/j. ympev.2008.03.024 Ramazzotti G (1943) Di alcuni tardigradi italiani con descrizione di una nuova specie. Atti della Societa Italiana di Scienze Naturali e del Museo Civico di Storia Naturale in Milano 82: 27-35. Richters F (1908) Beitrag zur Kenntnis der Moosfauna Australiens und der Inseln des Pazifischen Ozeans. Zoologische Jahrbicher. Abteilung fiir Systematik, Okologie und Geographie der Tiere 26: 196-213. Richters F (1926) Tardigrada. In: Ktkenthal W, Krumbach T (Eds) Handbuch der Zoologie. Berlin und Leipzig, Walter de Gruyter & Co.: 1-68. Roszkowska M, Grobys D, Bartylak T, Gawlak M, Kmita H, Kepel A, Kepel M, Parnikoza I, Kaczmarek L (2020) Integrative description of five Pseudechiniscus species (Heterotardigrada; Echiniscidae; the suillus-facettalis complex). Zootaxa 4763: 451-484. https://doi. org/10.11646/zootaxa.4763.4.1 Srivathsan A, Meier R (2012) On the inappropriate use of Kimura—2—pa- rameter (K2P) divergences in the DNA-barcoding literature. Cladis- tics 28: 190-194. https://doi.org/10.1111/).1096-0031.2011.00370.x Stec D, Krzywanski L, Arakawa K, Michalczyk L (2020) A new rede- scription of Richtersius coronifer, supported by transcriptome, pro- vides resources for describing concealed species diversity within the monotypic genus Richtersius (Eutardigrada). Zoological Letters 6: 2. https://doi.org/10.1186/s40851-020-0154-y Stec D, Smolak R, Kaczmarek L, Michalczyk L (2015) An integrative description of Macrobiotus paulinae sp. nov. (Tardigrada: Eutardi- zse.pensoft.net 536 grada: Macrobiotidae: hufelandi group) from Kenya. Zootaxa 4052: 501-526. https://doi.org/10.11646/zootaxa.4052.5.1 Stec D, Zawierucha K, Michalczyk L (2017) An integrative description of Ramazzottius subanomalus (Biserov, 1985) (Tardigrada) from Poland. Zootaxa 4300: 403-420. https://doi.org/10.11646/zoot- axa.4300.3.4 Suzuki AC (2017) Tardigrade Research in Japan. In: M. Motokawa, H. Kajihara (Eds) Species Diversity of Animals in Japan. Diver- sity and Commonality in Animals. Springer, Tokyo. https://doi. org/10.1007/978-4-431-56432-4 10 Suzuki AC, Heard L, Sugiura K (2018) Tardigrada of Mikurajima. Mi- kurensis 7: 3—8. https://mikura-isle.com/pdf/mikurensis20 1 8/3-8 pdf Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. https://do1. org/10.1093/nar/22.22.4673 Thulin G (1911) Beitrage zur Kenntnis der Tardigradenfauna Schwedens. Arkiv for Zoologi 7: 1-60. https://doi.org/10.5962/bhl. part. 1270 Thulin G (1928) Uber die Phylogenie und das System der Tardigraden. Hereditas 11: 207-266. https://doi.org/10.1111/j.1601-5223.1928. tb02488.x zse.pensoft.net Katarzyna Voncina et al.: Japanese Pseudechiniscus Tumanov DV (2020) Analysis of non-morphometric morphological characters used in the taxonomy of the genus Pseudechiniscus (Tar- digrada: Echiniscidae). Zoological Journal of the Linnean Society 188: 753-775. https://doi.org/10.1093/zoolinnean/z1z097 Wang L, Xue J, Li X (2018) A description of Pseudechiniscus xiai sp. nov., with a key to genus Pseudechiniscus in China. Zootaxa 4388: 255-264. https://doi.org/10.11646/zootaxa.4388.2.7 Welnicz W, Grohme MA, Kaczmarek L, Schill RO, Frohme M (2011) ITS-2 and 18S rRNA data from Macrobiotus polonicus and Milne- sium tardigradum (Eutardigrada, Tardigrada). Journal of Zoological Systematics and Evolutionary Research 49 (Supplement 1): 34-39. https://doi.org/10.1111/j.1439-0469.2010.00595.x Weeglarska B (1962) Die Tardigraden Vietnams. Acta Societatis Zoolog- icae Bohemoslovenicae 26: 300-307. Xue J, Li X, Wang L, Xian P, Chen H (2017) Bryochoerus liupanensis sp. nov. and Pseudechiniscus chengi sp. nov. (Tardigrada: Heterotar- digrada: Echiniscidae) from China. Zootaxa 4291: 324—334. https:// doi.org/10.11646/zootaxa.4291.2.5 Zeller C (2010) Untersuchung der Phylogenie von Tardigraden anhand der Genabschnitte 18S rDNA und Cytochrom c Oxidase Unterein- heit 1 (COX I). MSc Thesis, Technische Hochschule Wildau, Ger- many, 105 pp.