Pseudechiniscus in Japan: re-description of Pseudechiniscus asper Abe et al., 1998 and description of Pseudechiniscus shintai sp. nov.

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 suillus-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.


Introduction
Tardigrades are poorly known micrometazoans famous for their ability to enter cryptobiosis (Møbjerg 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 diversity (Bartels et al. 2016). Studies on the Japanese tardigrades 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-terrestrial heterotardigrade family Echiniscidae (Gąsiorek et al. 2018a, Suzuki et al. 2018), a distinct group characterised by the development of cuticular plates on the dorsal surface of the body (Kristensen 1987).
Recent advances in the taxonomy of one of the echiniscid genera, Pseudechiniscus Thulin, 1911, are a good illustration of the progress currently being made in the classification of tardigrades. Firstly, Tumanov (2020) discussed and re-organised the morphological nomenclature after a meticulous analysis of various members of Pseudechiniscus and he concluded that several species are unidentifiable, according 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. Finally, the ability to confidently describe new Pseudechiniscus species was enabled by the modern diagnosis of P. suillus (Ehrenberg, 1853), one of enigmatic tardigrade taxa described in the 19 th century . In summary, better understanding of morphology, genetic disparities and ontogenetic shifts , Morek et al. 2019 has facilitated intensification in tardigrade research.
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 records 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 species complexes in numerous tardigrade genera (e.g. Guidetti et al. 2019, Stec et al. 2020).

Sample collection and processing
Specimens belonging to two species of the genus Pseudechiniscus 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 a.s.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 microscopy (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 (μm) and were performed under PCM. Structures were measured only when not broken, deformed or twisted and their orientations 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 handled using the Echiniscoidea ver. 1.3 template, available from the Tardigrada Register, www.tardigrada.net (Michalczyk and Kaczmarek 2013). Importantly, all species designated as dubious or with insufficient descriptions , Tumanov 2020, were discarded 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 (Table 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 ). MEGA7.0.26 (Kumar et al. 2016) was used for calculation of uncorrected pairwise distances (Srivathsan and Meier 2012).

Systematic account
Phylum: Tardigrada Doyère, 1840 Class: Heterotardigrada Marcus, 1927Order: Echiniscoidea Richters, 1926Family: Echiniscidae Thulin, 1928Genus: Pseudechiniscus Thulin, 1911 Pseudechiniscus asper Abe, Utsugi & Takeda, 1998  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 suillus-facettalis complex: dome-shaped (hemispherical) cephalic papillae (secondary clavae) and minute (primary) clavae; peribuccal 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, i.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 being a typical reticulum composed of large multiangular, longitudinal shapes connected by belts of pillars. Pillars 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 I-III; internal claws with needle-like spurs positioned at ca. 1/4-1/5 of the claw height (Fig. 1A, insert).
Mature males (i.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. Remarks. This is the third record of this very rare species, 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, the variability in the shape of the pseudosegmental projections 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 thickening of the armour at position C, which is weakly developed 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 compensate the scarcity of specimens used in morphometrics. Etymology. The name is a patronym honouring Shinta Fujimoto, an excellent Japanese tardigradologist specialising in marine Heterotardigrada. Noun in the genitive singular.
Description. Mature female (i.e. the third or latter instar; measurements in Table 4). Body orange, with minute, round black eyes that are absent after mounting (Figs 3A, 4A). Member of the suillus-facettalis complex: dome-shaped (hemispherical) cephalic papillae (secondary clavae) and minute (primary) clavae; peribuccal cirri with poorly developed cirrophores. Cirrus A short, with cirrophore. Dorsal plates poorly sclerotised, but clearly demarcated from each other, with the Pseudechiniscus-type sculpturing, i.e. endocuticular pillars protruding through the epicuticle and visible as dark dots in PCM (Fig. 4A). Striae absent; epicuticular ornamentation visible as darker belts on all dorsal plates. The cephalic plate pentapartite, with the two anterior portions and three posterior portions approximately equal in size (Fig. 4A). The cervical (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;

A B
m3 unipartite (Figs 3A, 4A); four pairs of lateral intersegmental platelets flanking the borders of m1-2. 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).
Ventral cuticle with a pronounced species-specific pattern reaching the lateroventral sides of the body (Figs 4B and 5B), being a typical reticulum composed of large multiangular, longitudinal shapes joined by belts of pillars. The subcephalic zone with a wide belt of pillars. 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. 3A). Pulvini indistinct. A papilla on leg I undetectable 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).
Juveniles (i.e. the second instar; measurements in Table 5). A morphometric gap exists between adult females and juveniles. Phenotypically similar to adults. Gonopore absent.
Phenotypic differential diagnosis. The species was compared with the members of the suillus-facettalis complex (with hemispherical cephalic papillae) and other Pseude- 1. P. angelusalas Roszkowska et al., 2020, described from Madagascar, by the shape of its cephalic papillae (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); 2. P. beasleyi Li et al., 2007, described from Qinling Mountains (China), by much shorter claws (5.3-9.2 μm in P. shintai sp. nov. vs. 9.1-13.1 μm in P. beasleyi); 3. P. chengi Xue et al., 2017, described  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 P. angelusalas, P. dastychi and P. indistinctus in the suillus-facettalis complex, such combination is phylogenetically unjustified, as they all exhibit elongated (dactyloid) cephalic papillae, which is a distinguishing trait of P. novaezeelandiae (Richters, 1908) (see Pilato et al. 2005) and of the entire novaezeelandiae lineage (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). Intraspecific distance was equal to 0.2%.

Discussion
The dorsal sculpturing of P. asper is particularly interesting morphologically, as large, roughly circular endocuticular pillars protrude through the epicuticle as isolated, 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 andLisi 2006). Tumanov (2020) suggested that their presence may represent a phylogenetic signal and, as striae are absent in P. suillus , this could mean that the absence of striae is a trait specific to the suillus-facettalis 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 misidentification , Tumanov 2020. This is the case for almost all Pseudechiniscus spp. reported from Japan: P. suillus, P. bartkei Węglarska, 1962, P. facettalis Petersen, 1951, P. pseudoconifer Ramazzotti, 1943and 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 glaciation periods (Motokawa 2017), it cannot be excluded that some of the above-mentioned species inhabit the Japanese 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.