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Gąsiorek, Piotr (2023): Eventually tested: Phylogenetic position of "Testechiniscus" meridionalis (Murray, 1906) (Heterotardigrada) revealed. Zoologischer Anzeiger 304: 49-60, DOI: 10.1016/j.jcz.2023.02.004, URL: http://dx.doi.org/10.1016/j.jcz.2023.02.004

4.3. Re-defining Echiniscus Currently, the definition says “Echiniscids with red eyes composed of pigment granules. Rigid buccal tube lacking stylet supports or with fine, fibrillar stylet supports. Two pairs of segmental plates. Two or three median plates, sometimes transversally subdivided. Incisions(notches) on caudal (terminal) plate. No pseudosegmental plates. Ventral subcephalic and/or genital plates may be present.” (G&aogon;siorek et al., 2017). The necessary modification obviously concerns the last sentence, which is rephrased as “Ventral plates may be present, usually only in the form of subcephalic and/or genital plates.”. The affinity of E. becki (Schuster and Grigarick, 1966) remains problematic and thus, consequently, I left its classification unchanged. However, it is clear that this species does not belong in Echiniscus because of the presence of black crystalline eyes (Fig. 9B), which, together with the dorsal plate sculpturing dominated by polygonal endocuticular pillars, make it most similar to Diploechiniscus. Ultimately, E. becki does not exhibit dorsolateral spicules and is comparatively larger (adults 290–420 μm in length) than Diploechiniscus oihonnae (adults < 275 μm in length). The last record of this species, endemic to the Western Nearctic, came from Mexico (Nú˜ nez et al., 2021), yet it is clearly erroneous as the reported individuals had reticulum in their dorsal plates, absent in the paratypes of E. becki (Fig. 9A). Therefore, re-sampling the Californian localities is necessary to obtain DNA barcodes and clarify where this species should be classified., Published as part of Gąsiorek, Piotr, 2023, Eventually tested: Phylogenetic position of " Testechiniscus " meridionalis (Murray, 1906) (Heterotardigrada) revealed, pp. 49-60 in Zoologischer Anzeiger 304 on page 58, DOI: 10.1016/j.jcz.2023.02.004, http://zenodo.org/record/8164274, {"references":["Gasiorek, P., Stec, D., Morek, W., Michalczyk, L., 2017. An integrative redescription of Echiniscus testudo (Doy`ere, 1840), the nominal taxon for the class Heterotardigrada (Ecdysozoa: Panarthropoda: Tardigrada). Zool. Anz. 270, 107 - 122. https: // doi. org / 10.1016 / j. jcz. 2017.09.006.","Schuster, R. O., Grigarick, A. A., 1966. New Tardigrada from Western North America: II, Echiniscus. Proc. Biol. Soc. Wash. 79, 127 - 130."]}

3.2. Redescription of Echiniscus meridionalis stat. rev. Figs. 2 – 7, Tables 2–4, Supplementary Material 4 with raw morphometric data. ZooBank registration number: urn:lsid:zoobank.org:pub:8A36A0BF- AA4F–427F–980B-E70DE960695E. 3.2.1. Terra typica South Orkney Islands. Material examined. South Shetland Islands (see Table 1 for detailed data). 3.2.2. Etymology From Latin meridionalis = southern, referring to the distribution of the species in Antarctic and sub-Antarctic archipelagos. An adjective in nominative singular. 3.2.3. Adult females (i.e. from the third instar onwards; measurements and statistics in Table 2) Body small and plump (Figs. 2A and 3A), light yellow to dark orange in alive specimens. Eyes dark red, minute and dissolving immediately in Hoyer’ s medium. Peribuccal cirri relatively long with respect to the body length (Figs. 2A, 3A and 4A); cirrus A of a moderate length, i.e. reaching approximately 1/3 of the body length; all cephalic cirri with well-delineated cirrophores. Cephalic papillae (secondary clavae) slightly longer than (primary) clavae, both barrel-shaped (Fig. 4A). Body appendage configuration A-C-C d -D-D d -E , with most appendages formed as long, flexible cirri. Short, but robust spines present in positions D and D d (Figs. 2A and 3A). Dorsal plate sculpturing comprises: (I) poorly elevated epicuticular granules, with a strong tendency to merging in all plate portions but the anterior portions of paired segmental plates I–II, median plate 2, and the much reduced median plate 3, where they are separate (Figs. 2A and 3A), (II) pseudopores in the form of delicate depressions distributed irregularly throughout the dorsum, and (III) pores, present mainly in the cephalic, scapular (Figs. 2A and 3A), and occasionally in the caudal (terminal) plates. Pores may be absent as well. Cephalic plate narrow, with the usual chalice-like anterior incision. Cervical plate even narrower, but clearly present and rectangular, tightly joined with the scapular plate. Scapular plate with a distinct faceting-like pattern formed by a system of poorly elevated ridges (Figs. 2A, 3A and 4A); lateralmost plate portions weakly demarcated from the central portion by a similar ridge (Fig. 4A) that replaced a usual suture/incision. Median plate 1 unipartite, m2 bipartite and much larger, m3 developed as a triangular sculptured surface dominated by the caudal plate. Paired segmental plates I–II identical in size and shape. Caudal plate with large triangular posterior incisions (Fig. 2A) that are joined by a central semicircular ridge (Figs. 2A, 3A and 4A). A weakly marked ridge perpendicular to the former may be developed, delineating a cross-like epicuticular structure. Ventral plate armature variously developed, as plates in some individuals may be almost indiscernible, but the general plate formula stands as VIII:2-1-1-2-2-2-2-2 (Figs. 4B and 5A). Particularly the plate in the second row can be hardly identifiable in PCM (Fig. 5A). Ventral plates are smooth (Fig. 4B), but the endocuticular pillars are easily observable in PCM (Fig. 5A). Besides, no granulation present on venter. Hexapartite gonopore and tripartite, large anus (Figs. 4B and 5B). Legs with evident pulvini embedded in proximal portions of limbs and pedal plates in median portions, developed as wrinkled areas with sparse and occasional pores (Figs. 4A and 6B). Long spine I present (Figs. 4A and 6A); papilla IV large and barrel-shaped, dentate fringe on leg IV with numerous irregular teeth, typically interconnected at their bases (Figs. 2A, 4A and 6B). Claws with branches typically curved, those of legs IV slightly higher than the remaining ones. Claw spurs heteromorphic, as spurs IV are more divergent from the branches compared with spurs I– III. The size and position of spurs on claw branches (ca. 1/3–2/5 of the claw height) identical on all claws (Fig. 6). 3.2.4. Adult males (i.e. from the second/third instar onwards, see remarks; measurements and statistics in Table 3) Sexual dimorphism evinces in several characters of males: thinner and shorter body (Figs. 2B and C, 3B, 4C–D), prominent and swollen clavae (Figs. 2B and C, 3B, 4C–D and 5B), and often better visible (darker in PCM) ventral plates (Fig. 5B). Rarely, all appendages but A and E may be formed as spines (Fig. 2B). Gonopore circular. 3.2.5. Juveniles (i.e. the second instar; measurements and statistics in Table 4) Qualitatively similar to sexually mature females, and smaller than both sexes. Gonopore absent. 3.2.6. Larvae (i.e. the first instar; measurements and statistics in Table 4) Qualitatively similar to later life stages, but smaller than juveniles. Body appendage formula: A-Cd- E , all formed as cirri (Fig. 7). Two claws per leg. Gonopore and anus absent. 3.2.7. DNA markers Single haplotype was found in 18S rRNA (OQ 472387), 28S rRNA (OQ 472389), ITS-1 (OQ 476878), ITS-2 (OQ 476879), and two in COI (OQ 472391–2; p -distance = 0.2%). 3.2.8. Differential diagnosis A strong sexual dimorphism, the unusual dorsal plate sculpturing (in some specimens, epicuticular granules may be barely elevated above the surface of plates, and in an initial observation in PCM such exemplars seem to be sculptureless), and the ventral armature covering an entire venter, replacing the endocuticular granulation typical for Echiniscus, together with the geographic range embracing solely Antarctic and sub-Antarctic islands (Fig. 8), make E. meridionalis stat. rev. easily recognisable and well-differentiated morphologically from all other Echiniscus spp. 3.2.9. Remarks The species can be frequently encountered on King George Island, being one of the two commonest echiniscids in that location (the other is Barbaria jenningsi, often co-inhabiting the same mosses and lichens, see Table 1). An undescribed Pseudechiniscus species, eutardigrades Acutuncus antarcticus, Diphascon rudnickii, and unidentified species of the Mesobiotus furciger group (Short et al., 2022) are other relatively common taxa that co-occur with E. meridionalis stat. rev. However, irrespective of its ubiquity in the area, the population density must be low given that typically few representatives were found in samples (Table 1). Ventral armature is already developed in E. meridionalis stat. rev. at sexually immature life stages (i.e. in larvae and juveniles), which often does not take place in the Echiniscidae., Published as part of Gąsiorek, Piotr, 2023, Eventually tested: Phylogenetic position of " Testechiniscus " meridionalis (Murray, 1906) (Heterotardigrada) revealed, pp. 49-60 in Zoologischer Anzeiger 304 on pages 52-54, DOI: 10.1016/j.jcz.2023.02.004, http://zenodo.org/record/8164274, {"references":["Short, K. A., Sands, C. J., McInnes, S. J., Pisani, D., Stevens, M. I., Convey, P., 2022. An ancient, Antarctic-specific species complex: large divergences between multiple Antarctic lineages of the tardigrade genus Mesobiotus. Mol. Phylogenet. Evol. 170, 107429 https: // doi. org / 10.1016 / j. ympev. 2022.107429."]}

3.3. Revised morphological characters for Echiniscus becki and Echiniscus laterosetosus An analysis of the type material of E. becki revealed new traits crucial in determining its phylogenetic position. The dorsal plate sculpturing comprises tightly arranged, polygonal endocuticular pillars (Fig. 9A); they lack centrally positioned pores typical for the blumi-canadensis complex of species and Testechiniscus (G&aogon;siorek et al., 2018). In contrary to the original description (Schuster and Grigarick, 1966), the species does not have dark red eye spots, which always dissolve in Hoyer’ s medium, but large black crystalline eyes identifiable in all examined paratypes (Fig. 9B). An erroneous ascribing of the red colour most probably resulted from a strong orange-reddish glow enclosing eyes. In fact, already the description in Schuster and Grigarick (1966) raised suspicions as the authors drew well-identifiable eyes in the mounted holotypic female, whereas red carotenoid eyes in echiniscids in the vast majority of cases vanish within an hour from preparing a permanent slide with a tardigrade. Echiniscus becki exhibits also a pair of exceptionally shaped subcephalic plates, which extend from the lateral portions of the head and twist in a boomerang-like manner at the level of pharynx (Fig. 9B). Representatives of E. laterosetosus, collected in the high mountains of Honshu, allowed for providing the morphological details relevant to its taxonomy. Eye spots, drawn in the original description without stating their colour (Ito, 1993), are small and black (Fig. 10A). The dorsal plate sculpturing consists of well-developed endocuticular pillars, each joined by striae with four or five neighbouring pillars, and pores distributed between them (Fig. 10B)., Published as part of Gąsiorek, Piotr, 2023, Eventually tested: Phylogenetic position of " Testechiniscus " meridionalis (Murray, 1906) (Heterotardigrada) revealed, pp. 49-60 in Zoologischer Anzeiger 304 on page 55, DOI: 10.1016/j.jcz.2023.02.004, http://zenodo.org/record/8164274, {"references":["Gasiorek, P., Stec, D., Zawierucha, K., Kristensen, R. M., Michalczyk, L., 2018. Revision of Testechiniscus Kristensen, 1987 (Heterotardigrada: Echiniscidae) refutes the polartemperate distribution of the genus. Zootaxa 4472, 261 - 297. https: // doi. org / 10.11646 / zootaxa. 4472.2.3.","Schuster, R. O., Grigarick, A. A., 1966. New Tardigrada from Western North America: II, Echiniscus. Proc. Biol. Soc. Wash. 79, 127 - 130.","Ito, M., 1993. Taxonomic study on the class Heterotardigrada (Tardigrada) from the northern slope of Mt. Fuji, Central Japan. Edaphologia 50, 1 - 13."]}

4.2. Re-defining Testechiniscus and the composition of Diploechiniscus At present, the definition of Testechiniscus stands as follows: “Medium-sized echiniscids with black, crystalline eyes. Rigid buccal tube with large cuticular stylet supports. Appendaged, i.e. having both cephalic and trunk cirri. Two pairs of segmental plates, unpaired scapular and caudal plates. Three median plates. Cuticular sculpture composed of large true round or polygonal pores that gradually become reticulum. Incisions (notches) on caudal (terminal) plate. Eight rows of ventral plates. No pseudosegmental plates.” (G&aogon;siorek et al., 2018). I would like to add a new character to the diagnosis: the presence of dorsolateral spicules. This trait is an advanced convergence of Diploechiniscus and Testechiniscus, absent in all but three species of Echiniscus: E. laterosetosus, E. polygonalis (Ito, 1993; it is worth noting that both are similar and potentially conspecific), and E. tympanista (Murray, 1911; a species of incomplete description and never found again). Given that eyes of E. laterosetosus are black and crystalline (Fig. 10A; eyes were reported as “present” in Ito, 1993, and drawn in black therein), the dorsal sculpturing comprises densely arranged endocuticular pillars intermingled with pores (Fig. 10B), and the abovementioned dorsolateral spicules are present in the positions Bl , Cl , Dl , and El , I transfer both E. laterosetosus and E. polygonalis to Diploechiniscus as D. laterosetosus (Ito, 1993) comb. nov. and D. polygonalis (Ito, 1993) comb. nov., pending further tests of the probable conspecificity. Such action agrees also with the biogeography of the genus, which is a Holarctic taxon (Vicente et al., 2013). By adding such a character to the diagnosis, Testechiniscus becomes truly separate from both Diploechiniscus and Echiniscus (E. tympanista is probably related to Diploechiniscus oihonnae, as noted by Murray (1911) himself). The next step is the removal of “ Testechiniscus ” macronyx from Testechiniscus, which should be performed with the application of integrative approach by precisely determining its place in the echiniscid evolutionary tree. Its autapomorphies (G&aogon;siorek et al., 2018) make it unclassifiable within either Diploechiniscus, Echiniscus, or Testechiniscus.

Acanthechiniscus goedeni (Grigarick, Mihel&ccaron;i&ccaron; & Schuster, 1964) (Figure1) Material examined and repository Voucher specimens are deposited in the Invertebrate Zoology Collections, Royal BC Museum (RBCM), 675 Belleville Street, Victoria, BC Canada (sample code: S198 HC 2018-14-1), RBCM022-0030 - 001; (sample code: S1904 GSB-1c), RBCM022-00031 - 1; and (sample code: S1913 GSB-7), RBCM022-0032 - 001). Description of the larva. Length 230μm, body plump (Fig. 1A, B). Eyes not visible after mounting. On the head, a full set of appendages is present: cirrus internus (8.5 μm), cephalic papilla (7.0 μm), cirrus externus (10.8 μm). Dorsal plates as described by Grigarick, Mihel&ccaron;i&ccaron; & Schuster (1964) and Kathman & Dastych (1990) (Fig. 1 B, C), but with fewer and less dense granules than the adult (Fig. 1D, E). Cirrus A present (64.4 μm) with clava (5.0 μm). Very small and stout lateral spines in B (7.3 μm), C (9.1 μm), and D (6.8 μm) (in the same positions that would be filaments in the adult) (Fig. 1A). Small dorsolateral spinules are present above the spines in B, C, and D. In D, a pair of additional dorsal spinules are present, which are smaller than the ones in dorsolateral position D. On the pseudosegmental plate, two long spines (Fig. 1 B, D) (29.7 μm) are present in the dorsal position (similarly to the adult, Fig. 1 C, E), in addition to small dorsolateral small spinules and two spines in the lateral position. Two long spines (34.1 μm) on the caudal plate E are present. Leg I with small spine (1.8 μm), leg IV with papilla (5.3 μm) and dentate collar with 6 teeth present. Two claws present, both with spur oriented downward starting at about 30% of the claw length on each leg (Fig. 1B) (adult with four claws and spur on the two internal claws). Lengths of claws from I to IV are: 11.6 μm, 9.5 μm, 10.2 μm, and 11.3 μm, respectively. No anus or gonopore observed. DNA. Four sequences were obtained from one individual: • SSU: GenBank OP730698 • LSU: GenBank OP730697 • COI: GenBank OP729912 • ITS2: GenBank OP734819 Phylogenetic position. The phylogenetic reconstruction (Figure 2) recovered A. goedeni as closely related to A. islandicus. The relatedness of A. goedeni to A. islandicus had already been indicated by Grigarick et al. (1964) based on some similarities in the chaetotaxy and in the plate ornamentation. The phylogenetic reconstruction supports the generic attribution of A. goedeni, as proposed by Vecchi et al. (2016) and G&aogon;siorek & Michalczyk (2020). The discovery of a new population of A. goedeni expands the range of this species to the north in British Columbia (Canada) by about 250 km, which is consistent with all the previous records that point to this species as being endemic to the north-west coast of North America. As pointed out by Bertolani et al. (1984), the ontogeny of echiniscid lateral appendages is variable between species and genera. In another Acanthechiniscus species (A. victor, synonym A. tridentifer), Bertolani et al. (1984) reported that the number of trunk appendages remained the same, but they increased in length during development. An identical developmental pattern was also found in A. islandicus (Piotr G&aogon;siorek, pers. comm.). A similar general pattern (increase in the number and size of trunk appendages during development) has been reported from different echiniscid taxa (see for example species in the genus Echiniscus; Claxton, 1996; G&aogon;siorek et al., 2022). Other developmental pathways found in echiniscids, but not observed in this study of A. goedeni, include the blurred distinction between dorsal plates (in Proechiniscus and Pseudechiniscus; Kristensen, 1987; G&aogon;siorek et a., 2022) and a simultaneous increase in size in lateral trunk appendages with a reduction in both size and number of dorsal trunk appendages (in Mopsechiniscus; Dastych, 2001). We are grateful to the Hakai Institute, Tula Foundation- Eric Peterson and Christina Munck for making research on Calvert Island possible. We also wish to thank Gustav Paulay (University of Florida), Matt Lemay (Hakai), and Matt Whalen (UBC-Hakai) for facilitating collecting opportunities, Gillian Sadlier-Brown (Hakai) for collecting part of the samples used in this study, and Hugh MacIntosh (Royal BC Museum) for assistance with the processing of field samples. We are grateful to Daniel Stec (Institute of Systematics and Evolution of Animals, Polish Academy of Sciences) for acquiring microphotographs of the specimens belonging to the population examined in this study. We also wish to thank Piotr G&aogon;siorek and an anonymous reviewer for their constructive comments and suggestions. This study was supported by an Academy of Finland Fellowships to S.C. (#314219 and #335759)., Published as part of Matteo, Vecchi, Sara, Calhim & Henry, Choong, 2023, Larva description and phylogenetic position of the armoured tardigrade Acanthechiniscus goedeni (Grigarick, Mihelčič & Schuster, 1964), pp. 295-300 in Zootaxa 5227 (2) on pages 296-298, DOI: 10.11646/zootaxa.5227.2.9, http://zenodo.org/record/7518712, {"references":["Grigarick, A. A., Mihelcic, F. & Schuster, R. O. (1964) New Tardigrada from western North America: I, Pseudechiniscus. Proceedings of the Biological Society of Washington, 77, 5 - 8.","Kathman, R. D. & Dastych, H. (1990) Some Echiniscidae (Tardigrada: Heterotardigrada) from Vancouver Island, British Columbia, Canada. Canadian Journal of Zoology, 68 (4), 699 - 706. https: // doi. org / 10.1139 / z 90 - 102","Vecchi, M., Cesari, M., Bertolani, R., Jonsson, K. I., Rebecchi, L. & Guidetti, R. (2016) Integrative systematic studies on tardigrades from Antarctica identify new genera and new species within Macrobiotoidea and Echiniscoidea. Invertebrate Systematics, 30 (5), 521 - 521. https: // doi. org / 10.1071 / IS 15033 _ CO","Gasiorek, P. & Michalczyk, L. (2020) Revised Cornechiniscus (Heterotardigrada) and new phylogenetic analyses negate echiniscid subfamilies and tribes. Royal Society Open Science, 7 (6), 200581. https: // doi. org / 10.1098 / rsos. 200581","Bertolani, R., De Zio, S. G., Gallo, M. D. A. & Morone De Lucia, M. R. (1984) Postembryonic development in heterotardigrades. Monitore Zoologico Italiano - Italian Journal of Zoology, 18 (4), 307 - 320.","Jorgensen, A. & Kristensen, R. M. (2004) Molecular phylogeny of Tardigrada - investigation of the monophyly of Heterotardigrada. Molecular Phylogenetics and Evolution, 32 (2), 666 - 670. https: // doi. org / 10.1016 / j. ympev. 2004.04.017","Jorgensen, A., Mobjerg, N. & Kristensen, R. M. (2011) Phylogeny and evolution of the Echiniscidae (Echiniscoidea, Tardigrada) - an investigation of the congruence between molecules and morphology. Journal of Zoological Systematics and Evolutionary Research, 49 (S 1), 6 - 16. https: // doi. org / 10.1111 / j. 1439 - 0469.2010.00592. x","Voncina, K., Kristensen, R. M. & Gasiorek, P. (2020) Pseudechiniscus in Japan: re-description of Pseudechiniscus asper Abe et al., 1998 and description of Pseudechiniscus shintai sp. nov. Zoosystematics and Evolution, 96 (2), 527 - 536. https: // doi. org / 10.3897 / zse. 96.53324","Claxton, S. K. (1996) Sexual dimorphism in Australian Echiniscus (Tardigrada, Echiniscidae) with descriptions of three new species. Zoological Journal of the Linnean Society, 116, 13 - 33. https: // doi. org / 10.1111 / j. 1096 - 3642.1996. tb 02330. x","Gasiorek, P., Voncina, K., Bochnak, M., Surmacz, B., Morek, W. & Michalczyk, L. (2022) Echiniscidae (Heterotardigrada) of South Africa. Zootaxa, 5156 (1), 1 - 238. https: // doi. org / 10.11646 / zootaxa. 5156.1.1","Kristensen, R. M. (1987) Generic revision of the Echiniscidae (Heterotardigrada), with a discussion of the origin of the family. In: Bertolani, R. (Ed.), Biology of Tardigrades. Selected Symposia and Monographs U. Z. I., 1, pp. 261 - 335.","Dastych, H. (2001) Notes on the revision of the genus Mopsechiniscus (Tardigrada). Zoologischer Anzeiger - A Journal of Comparative Zoology, 240, 299 - 308. https: // doi. org / 10.1078 / 0044 - 5231 - 00037"]}

Matteo, Vecchi, Sara, Calhim, Henry, Choong (2023): Larva description and phylogenetic position of the armoured tardigrade Acanthechiniscus goedeni (Grigarick, Mihelčič & Schuster, 1964). Zootaxa 5227 (2): 295-300, DOI: https://doi.org/10.11646/zootaxa.5227.2.9

There are two predominant sources of taxonomically useful morphological variability in the diverse tardigrade family Echiniscidae: the internal structure and surface sculpture of the cuticular plates covering the dorsum (sculpturing) and the arrangement and morphology of the trunk appendages (chaetotaxy). However, since the appendages often exhibit intraspecific variation (they can be reduced or can develop asymmetrically), sculpturing has been considered more stable at the species level and descriptions of new echiniscid species based solely on morphology are still being published. Here, we present a case study in which a detailed analysis of the morphology and multiple genetic markers of several species of the genus Viridiscus shows that cuticular sculpture may also exhibit considerable intraspecific variation and lead to false taxonomic conclusions. In a population collected from the eastern Nearctic, in the type locality of the recently described species V. miraviridis, individuals with transitional morphotypes between those reported for V. viridissimus and V. miraviridis were found. Importantly, all morphotypes within the viridissimus–miraviridis spectrum were grouped in a single monospecific clade according to rapidly evolving markers (ITS-1, ITS-2 and COI). Given the morphological and genetic evidence, we establish V. miraviridis as a junior synonym of V. viridissimus. This study explicitly demonstrates that a lack of DNA data associated with morphological descriptions of new taxa jeopardizes the efforts to unclutter tardigrade systematics. Additionally, V. perviridis and V. viridissimus are reported from Lâm Đồng Province in southern Vietnam, which considerably broadens their known geographic ranges.

One of the most extensive species radiations in the tardigrade evolution took place in the Echiniscus phylogenetic line, which contains the genus Echiniscus with over 120 species described to date. The internal classification of Echiniscus is historically based on two major sources of variability: chaetotaxy, that is the arrangement and the shape of trunk appendages, and the sculpturing of dorsal plates. These two criteria allowed for the delineation of species complexes, such as the blumi-canadensis or the spinulosus group. Recent phylogenetic analyses confirmed that they represent monophyletic lineages. However, the affinities of some species that have not been sequenced are not straightforward given their uncommon morphologies. In this paper, we present five Echiniscus species found in Argentina, two of which constitute such exemplary cases. Echiniscus aonikenk sp. nov. exhibits extraordinarily elongated cirri A, exceeding 50% of the body length, and dorsoventrally flattened trunk spines, including particularly wide spines E. The species is a probable sister group of the spinulosus complex. Furthermore, Echiniscus evelinae de Barros, 1942, reported here only third time ever, is characterised by an extreme elongation of cirri E, often exceeding the body length, and by autapomorphic dorsal sculpturing comprising epicuticular granules, pseudopores, distinct endocuticular reticulum and pillars. Analogously to E. aonikenk sp. nov., E. evelinae apparently constitutes a separate evolutionary lineage. Finally, Echiniscus pellucidus sp. nov. (the merokensis complex), Echiniscus peruvianus Binda & Pilato, 1994 syn. nov. (the testudo complex) and Echiniscus manuelae da Cunha & do Nascimento Ribeiro, 1962 (the spinulosus complex) are analysed. Morphological peculiarities revealed in this study are discussed in the light of the Echiniscus taxonomy.

Genus: Cornechiniscus Maucci & Ramazzotti, 1981. Diagnosis (from Kristensen, 1987; amended): Echiniscids with black crystalline eyes, flexible buccal tube and pseudosegmental plate IV’. Appendages A horn-shaped. Secondary clavae (cephalic papillae) ovoid, domed; peribuccal cirri typically bulbous (onion-shaped), but exceptionally may be formed typically for the vast majority of echiniscids, i.e. with antenniform, flexible flagellum and well-delimited cirrophore., Published as part of Gąsiorek, Piotr, 2022, Water bear with barbels of a catfish: A new Asian Cornechiniscus (Heterotardigrada: Echiniscidae) illuminates evolution of the genus, pp. 47-64 in Zoologischer Anzeiger 300 on page 49, DOI: 10.1016/j.jcz.2022.06.007, http://zenodo.org/record/8164002, {"references":["Maucci, W., Ramazzotti, G., 1981. Cornechiniscus gen. nov.: nuova posizione sistematica per i cosiddetti ' Pseudechiniscus gruppo cornutus ', con descrizione di una nuova specie (Tardigrada, Echiniscidae). Mem. Ist. Ital. Idrobiol. 39, 147 - 151.","Kristensen, R. M., 1987. Generic revision of the Echiniscidae (Heterotardigrada), with a discussion of the origin of the family. In: Bertolani, R. (Ed.), Biology of Tardigrades. Selected Symposia and Monographs, Modena, vol. 1. U. Z. I., pp. 261 - 335"]}

3.4. Updated description of Cornechiniscus holmeni (Petersen, 1951) Figs. 7D and 12–16, Tables 6–8. 3.4.1. Type locality 82 Ǫ 12 ′ N, 31 Ǫ 30 ′ W, 75 m asl: North Greenland, Peary Land, Brønlund Fjord. 3.4.2. New material 46 Ǫ 30 ′ 29 ′′ N, 11 Ǫ 49 ′ 41 ′′ E, ca. 3000 m asl: Italy, Province of Trento, Dolomites, Piz Bo`e. 15 adult females, 14 juveniles and 18 larvae on slides IT.339.06–25. All larvae hatched from eggs deposited in four exuvia that were maintained on a glass cube in a laboratory. 12 females on SEM stub no. 20.11. Five hologenophores preserved on slide IT.339.01. Found together with Echiniscus granulatus (Doy`ere, 1840), Diaforobiotus hyperonyx (Maucci, 1982), and members of Pseudechiniscus and Richtersius that will be addressed in future studies. All deposited in the Department of Invertebrate Evolution of the Jagiellonian University. 3.4.3. Adult females (i.e. from the third instar onwards; measurements and statistics in Table 6) Body dark orange to red, massive (Figs. 12 and 13). Spines E can be doubled (Fig. 12), or elongated as far as they resemble filamentous cirri (Fig. 14A). Dorsal sculpturing consists of epicuticular granules with a surrounding ring of thin wrinkles (Fig. 15A), the latter imperceptible in PCM (Fig. 14A). Large and robust spine I and tooth IV (Figs. 12–14A, 15B). Claws strongly heteronych and robust (Fig. 15B and C, 16). 3.4.4. Juveniles (i.e. the second instar; measurements and statistics in Table 7) Dorsal spiniform appendages can be particularly massive (Fig. 14B). Smaller than adult females. 3.4.5. Larvae (i.e. the first instar; measurements and statistics in Table 8) All trunk appendages formed as in older life stages (Fig. 14C). Larvae were pale yellow alive, crawling vigorously in a caterpillar-like manner. 3.4.6. DNA sequences Single haplotypes were obtained in all four markers (Table 2). 3.4.7. Comparison between populations from the Holarctic and concluding remarks The species has preferences towards polar/high mountain habitats in the Holarctic (Jørgensen and Kristensen, 1991; G&aogon;siorek and Michalczyk, 2020), what inclined Maucci (1982) to hypothesise that it is a tardigrade postglacial relic. When examining specimens originating from Caucasus, Dastych (1979b) concluded that slight disparities in the dorsal sculpturing between the population from Axel Heiberg Island (Canadian Arctic; W&eogon;glarska and Kuc, 1980) and Western Palaearctic representatives, and sporadic spurs directed upwards found in the Caucasus population, could warrant dividing C. holmeni into subspecies. Although the level of inter-population diversification remains to be investigated with the application of molecular techniques in this case, a relatively low genetic diversity has already been demonstrated for a member of the genus Cryoconicus inhabiting Central Asian glaciers (Zawierucha et al., 2018), which is consistent with other widely distributed tardigrade species (Jørgensen et al., 2007; G&aogon;siorek et al., 2019a). Consequently, it could be expected that C. holmeni is another tardigrade stenothermic species with poorly differentiated genetic structure. Irrespectively of the factual genetic diversity within the species, its morphological variability does not deviate from that of an average echiniscid (e.g. G&aogon;siorek et al., 2019b). Two other Cornechiniscus species, C. lobatus and C. madagascariensis, for which at least two populations originating from distant locales were shown as conspecific, exhibit comparably minor dorsal plate sculpturing variability (Maucci, 1993; Abe and Takeda, 2000; G&aogon;siorek and Michalczyk, 2020). This would mean that the differences in the size of granules and development of striae caught by Dastych (1979b) constitute a significant intraspecific variation as for the genus. Both Dastych (1979b) and Beasley and Miller (2007) collated the dorsal ornamentation of C. holmeni with that of the Echiniscus blumi-canadensis complex, suggesting their similarity. In fact, epicuticular granules of C. holmeni fall within the morphotype variability known for the genus (G&aogon;siorek and Michalczyk, 2020), and do not resemble large pores with thick polygonal edges typical for e.g. E. blumi Richters, 1903, but rather sculpturing described in E. weisseri from Afghanistan (Maucci, 1978). To summarise, recorded populations of C. holmeni form a morphological continuum and most probably are an evidence of relictual character of this species, which means a low dispersal potential., Published as part of Gąsiorek, Piotr, 2022, Water bear with barbels of a catfish: A new Asian Cornechiniscus (Heterotardigrada: Echiniscidae) illuminates evolution of the genus, pp. 47-64 in Zoologischer Anzeiger 300 on pages 55-59, DOI: 10.1016/j.jcz.2022.06.007, http://zenodo.org/record/8164002, {"references":["Petersen, B., 1951. The tardigrade fauna of Greenland. Medd. Gronl. 150, 1 - 94.","Maucci, W., 1982. Sulla presenza in Italia di Cornechiniscus holmeni (Petersen, 1951), e descrizione di Macrobiotus hyperonyx sp. nov. (Tardigrada). Boll. Mus. Civ. Stor. Nat. Verona 9, 175 - 179.","Jorgensen, M., Kristensen, R. M., 1991. Meiofauna investigations from Igloolik, N. W. T. Arctic Canada. In: Jorgensen, M. (Ed.), Arctic Biology Course 1989, Igloolik, N. W. Territories. Canada, Museum Tusculanum, pp. 60 - 80.","Gasiorek, P., Michalczyk, L., 2020. Revised Cornechiniscus (Heterotardigrada) and new phylogenetic analyses negate echiniscid subfamilies and tribes. R. Soc. Open Sci. 7, 200581 https: // doi. org / 10.1098 / rsos. 200581.","Dastych, H., 1979 b. Some Tardigrada from the Caucasus with a description of Isohypsibius zierhofferi sp. nov. Bull. Acad. Polon. Sci., Ser. Sci. Biol. 27, 941 - 947.","Weglarska, B., Kuc, M., 1980. Heterotardigrada from Axel Heiberg Island. Zesz. Nauk. Uniw. Jagiell. 529, 53 - 66.","Zawierucha, K., Stec, D., Lachowska-Cierlik, D., Takeuchi, N., Li, Z., Michalczyk, L., 2018. High mitochondrial diversity in a new water bear species (Tardigrada: Eutardigrada) from mountain glaciers in Central Asia, with the erection of a new genus Cryoconicus. Ann. Zool. 68, 179 - 201. https: // doi. org / 10.3161 / 00034541 ANZ 2018.68.1.007.","Jorgensen, A., Mobjerg, N., Kristensen, R. M., 2007. A molecular study of the tardigrade Echiniscus testudo (Echiniscidae) reveals low DNA sequence diversity over a large geographical area. J. Limnol. 66 (s 1), 77 - 83. https: // doi. org / 10.4081 / jlimnol. 2007. s 1.77.","Gasiorek, P., Jackson, K., Meyer, H., Zajac, K., Kristensen, R. M., Nelson, D. R., Michalczyk, L., 2019 a. Echiniscus virginicus complex: the first case of pseudocryptic allopatry and pantropical distribution in tardigrades. Biol. J. Linn. Soc. 128, 789 - 805. https: // doi. org / 10.1093 / biolinnean / blz 147.","Gasiorek, P., Blagden, B., Michalczyk, L., 2019 b. Towards a better understanding of echiniscid intraspecific variability: a redescription of Nebularmis reticulatus (Murray, 1905) (Heterotardigrada: Echiniscoidea). Zool. Anz. 283, 242 - 255. https: // doi. org / 10.1016 / j. jcz. 2019.08.003.","Maucci, W., 1993. Prime notizie su tardigradi ' terrestri' del Madagascar con descrizione di tre specie nuove. Boll. Mus. Civ. Stor. Nat. Verona 17, 381 - 392.","Abe, W., Takeda, M., 2000. A new record of Cornechiniscus madagascariensis Maucci, 1993 (Tardigrada: Echiniscidae) from India. Proc. Biol. Soc. Wash. 113, 480 - 485.","Beasley, C. W., Miller, W. R., 2007. Tardigrada of Xinjiang Uygur autonomous region, China. J. Limnol. 66 (s 1), 49 - 55. https: // doi. org / 10.4081 / jlimnol. 2007. s 1.49.","Richters, F., 1903. Nordische Tardigraden. Zool. Anz. 27, 168 - 172.","Maucci, W., 1978. Una nuova specie di Echiniscus dall' Afganistan (Tardigrada, Echiniscoidea). Mem. Ist. Ital. Idrobiol. 36, 117 - 120."]}

The tardigrade brain has been the topic of several neuroanatomical studies, as it is key to understanding the evolution of the central nervous systems in Panarthropoda (Tardigrada + Onychophora + Arthropoda). The gross morphology of the brain seems to be well conserved across tardigrades despite often disparate morphologies of their heads and cephalic sensory structures. As such, the general shape of the brain and its major connections to the rest of the central nervous system have been mapped out already by early tardigradologists. Despite subsequent investigations primarily based on transmission electron microscopy or immunohistochemistry, characterization of the different regions of the tardigrade brain has progressed relatively slowly and open questions remain. In an attempt to improve our understanding of different brain regions, we reinvestigated the central nervous system of the heterotardigrade Echiniscus testudo using anti-synapsin and anti-acetylated α-tubulin immunohistochemistry in order to visualize the number and position of tracts, commissures, and neuropils. Our data revealed five major synapsin-immunoreactive domains along the body: a large unitary, horseshoe-shaped neuropil in the head and four neuropils in the trunk ganglia, supporting the hypothesis that the dorsal brain is serially homologous with the ventral trunk ganglia. At the same time, the pattern of anti-synapsin and anti-tubulin immunoreactivity differs between the ganglia, adding to the existing evidence that each of the four trunk ganglia is unique in its morphology. Anti-tubulin labeling further revealed two commissures within the central brain neuropil, one of which is forked, and additional sets of extracerebral cephalic commissures associated with the stomodeal nervous system and the ventral cell cluster. Furthermore, our results showing the innervation of each of the cephalic sensilla in E. testudo support the homology of subsets of these structures with the sensory fields of eutardigrades.

The knowledge of marine tardigrades in the South China Sea (Western Pacific Ocean) is very scarce, with only four species from shallow waters recorded until now. The first deep-sea (1530–1624 m bsl) tardigrade species from this sea, Halechiniscus janus sp. nov. (Arthrotardigrada: Halechiniscidae), is described here. Specimens of the new species have four wrinkled digits without peduncles on each leg, terminated by simple crescent-shaped claws, and primary clavae clearly shorter than cirri A, both inserted on a common cirrophore, as typical for the genus Halechiniscus Richters, 1908. The new species differs from all other Halechiniscus species by its cylindrical body and conical head; by the presence of semispherical secondary clavae, and by sensory organs on legs IV consisting of a short cirrophore followed by a short papilla terminated in a peculiar short bipartite tip. The discovery of this new bathyal species, with its peculiar morphological traits, brings new insights not only to the biogeography and ecology of tardigrades, but also to the understanding of the only partially resolved systematics of the diversified family Halechiniscidae.

Halechiniscus janus sp. nov. (Figs 2–5, Table 2) Diagnosis: Halechiniscus with a cylindrical body; cuticle finely punctuated; head with a complete set of cephalic cirri. Primary clava and cirrus A with a common cirrophore; cirrus A longer than the primary clava; semispherical secondary clava not fused with a flat, almost indistinct, tertiary clava. Sensory organs on legs I divided, undivided on legs II and III; papillate sensory organs on legs IV with short distal bipartite tips. All legs terminated with four wrinkled digits without peduncles or proximal pads; internal digits slightly longer than external digits; each digit terminates in a large simple crescent-shaped claw. Female gonopore rosette-like. Pair of elongated seminal receptacles and long S-shaped seminal receptacle ducts opening ventrally and latero-posteriorly in relation to the female gonopore. Male gonopore is a circular opening of a short tube. Etymology: The specific epithet janus refers to Janus, the god of transitions in the Roman mythology, depicted as a man with two faces. The specific epithet alludes to the fact that some characters exhibited by the new species can be found in other halechiniscid species also, such as a semispherical secondary clavae and the morphology of seminal receptacles and ducts similar to those of the Euclavartinae, and cirri A and primary clavae sharing a common cirrophore as in the Halechiniscinae. Material examined: Holotype, adult male (slide B6418400009) collected at station 2019MCA-MC04; allotype, adult female (slide B6418400028) collected at station 2019MA-MC02, both mounted in glycerine, and 25 paratypes (6 females, 4 males, 1 second-instar larva, 9 first-instar larvae and 5 specimens of undetermined gender and life stage). Eight paratypes were collected at station 2019MCA-MC01: two females (slide B6418400011 and B6418400017), five first instar larvae (one slide B6418400018 and four SEM stubs: B6418400012, B6418400013, B6418400014 and B6418400015) and one specimen of undetermined gender and life stage (SEM stub B6418400016). Eight paratypes were collected at station 2019MA-MC02: two females (slide B6418400027 and SEM stub B6418400030), one male (slide B6418400026), one second-instar larva (SEM stub B6418400031), three first-instar larvae (SEM stub B6418400034, slides B6418400032 and B6418400033) and one specimen of undetermined gender and life stage (SEM stub B6418400029). Three paratypes were collected at station 2019MA-MC03: two females (slides B6418400023 and B6418400025) and one male (slide B6418400024). One paratype, a specimen of undetermined gender and life stage (slide B6418400010), was collected at station 2019MCA-MC04. Four paratypes were collected at station 2019MA-MC05: two males (SEM stubs B6418400020 and B6418400022), one first-instar larva (SEM stub B6418400019) and one specimen of undetermined gender and life stage (SEM stub B6418400021). One paratype, a specimen of undetermined gender and life stage (slide B6418400035), was collected at station 2021MCA-MC01. Unfortunately, one specimen mounted on slide (B6418400023) and three specimens on SEM stubs (B6418400019, B6418400020 and B6418400034) were lost during the study. Type locality: The South China Sea (Western Pacific Ocean, 21.1934°N, 118.0933°E), 1540 m bsl. Type repository: The type material (slides and SEM stubs) is deposited in Lin’an Base Sample Bank of the Key Laboratory of Marine Ecosystem Dynamics, Second Institute of Oceanography, Ministry of Natural Resources of the People’s Republic of China. Description: Morphometric data are provided in Table 2. The body is elongated and cylindrical. The conical head is separated from the trunk by a slight neck constriction (Fig. 2). The dorsal cuticle, with dorsal metameric folds, is punctuated by fine epicuticular pillars. Ventrally the cuticle is almost smooth. The cuticular punctuation is present also in the coxal portions of legs. Eyes were not observed prior to mounting on slides. 1 Measurements of four specimens, one from the Mediterranean Sea (Banyuls-sur-Mer, France) and three from the Tyrrhenian Sea (Naples, Italy), by Schulz (1955). 2 Measurements of two specimens, one from the Pacific Ocean (New Caledonia) and the other from the Atlantic Ocean (Brest), by Renaud-Mornant (1970). A complete set of cephalic appendages is present (Fig. 3A), consisting of an unpaired median cirrus, and remaining paired organs: internal cirri, external cirri located latero-ventrally, cirri A, primary, secondary, and tertiary clavae. All cephalic cirri with cirrophores (very short in internal and external cirri). Each cephalic cirrus is divided into scapus and flagellum, which is subdivided into a tubular portion and thinner distal portion. Cirrus A and primary clava share a common cirrophore. Primary clavae much shorter than cirri A. Primary clava, inserted ventrally in relation to cirrus A, is sausage-shaped with van der Land’s body at its base. The semispherical secondary clava is located between the internal and external cirrus (Figs 3A–B). Flat tertiary clavae, almost indistinct and not fused with secondary clavae, can be hardly observed ventrally, between external cirri. Although their complete contours are difficult to recognize, tertiary clavae appear as a D-shaped structure (Figs 3A, C). FFIGURE 3. Halechiniscus janus sp. nov. — head structures: A. The head of a paratype B6418400012 (SEM photo, scale bar=4 µm); B. Lateral view of the secondary clava of holotype B6418400009 (DIC photo, scale bar=10 µm); C. Ventral view of the tertiary clava (paratype B6418400035, DIC photo, scale bar=10 µm), insert shows the edge of tertiary clava (dashed line); D. pharyngeal bulb (paratype B6418400028, DIC photo, scale bar=10 µm). Abbreviations: bt—buccal tube; ca—cirrus A; ec—external cirrus; ic—internal cirrus; mc—median cirrus; pb—pharyngeal bulb; pc—primary clava; sc—secondary clava; st—stylet; tc—tertiary clava. Bucco-pharyngeal apparatus (Fig. 3D) consists of the antero-ventral mouth opening surrounded by a thick mouth ring, buccal tube (18–25 µm long), paired stylets (21–38 µm long), three long simple placoids and a spherical muscle pharynx (diameter 14–19 µm). Stylet supports seem to be present but this character needs to be confirmed. Cirri E are inserted on a short cirrophore and have an annulated scapus and long, thin flagellum (Figs 2C, 4B). Four pairs of legs. Each leg consists of coxa, femur, tibia and tarsus, which terminates with four wrinkled digits. Each digit terminated by a large (5–6 µm long), simple crescent-shaped claw, entirely retractable into a membranous sheath. Digits are longer on legs IV than on legs I–III and; on each leg, internal digits are slightly longer than the external ones. Digits without basal pads or peduncles (Figs 4A, E–F). Sensory organs present on all legs. Sensory organs on legs I divided into scapus and flagellum. The flagellum is subdivided into a tubular portion and a distal, shorter and thinner portion (Fig. 4A). Sensory organs on legs II–III are undivided spines (Fig. 2). On legs IV, each sensory organ consists of a proximal portion (cirrophore) followed by a short papilla with van der Land’s body at base and terminated by a short tip (1–2 µm long). A careful observation of the tip reveals that it is bipartite (Figs 4C–D). The female gonopore has the typical six-lobed rosette-like appearance, its diameter is about 6 µm and it is positioned at a 10 µm distance from the anus (Figs 5A–B). The male gonopore (4–5 µm in diameter) is a circular opening of a short tube (Figs 5C–D), close to the anus (distance anus–gonopore about 5 µm). The anus is a sinuous slit between two longitudinally elongated lateral lobes (Fig. 5D). Seminal receptacles are elongated vesicles, continued by long S-shaped seminal receptacle ducts. Each seminal receptacle duct leaves the seminal receptacle posteriorly, then loops immediately frontwards and after a second loop it turns backwards again to open ventrally after a small internal receptacle duct pouch, located latero-posteriorly, but far away from the gonopore (Figs 5A–B). Males are very similar to females in qualitative characters, except for their circular gonopore, which is also located closer to the anus. Two-clawed larvae (first-instar), with the exception of missing external digits, anus and gonopore, and the second-instar larva, with four digits but without visible gonopore, exhibit all other characteristics as in the adults. The sample size is too small and does not allow to draw any conclusions about morphometric differences between the sexes and life stages (Table 2).

Echiniscus quadrispinosus quadrispinosus Richters, 1902 Figs 1–7, Tables 1–4, Supp. file 2 Material examined Neotype GERMANY • Taunus Mountains Range, near Königstein im Taunus; 50°11′49″ N, 08°27′15″ E; 485 m a.s.l.; 2 Sep. 2019; moss from stone, mixed forest; Johenn Sholl leg.; slide GR1/5; Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Pozna&nacute;. Paraneotypes GERMANY • 33 &female;&female;, 22 &male;&male;, 35 juvs, 15 undefined, 3 larvae; same collection data as for neotype; slides GR 1/1, 1/2, 1/4, 1/5, 1/7; Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Pozna&nacute; • 9 &female;&female;, 6 &male;&male;, 4 juvs, 2 undefined, 1 larvae; same collection data as for neotype; slides GR 1/3, 1/6; Institute of Systematics and Evolution of Animals, Polish Academy of Sciences. Remarks Animals were mounted on microscope slides in Hoyer’s medium, 45 animals prepared for SEM, and 15 prepared for molecular analyses. However, DNA sequences were obtained from two females and one male only (exoskeleton, No GR5/S, GR8/S and GR10/S) which was later mounted on microscope slide in Hoyer’s medium. Description of the neotypic population Females (measurements and statistics in Table 1, Supp. file 2 and Figs 1–4) Body orange in live specimens and transparent/light yellow after preparation. Eyes red. Apart from the head appendages which include internal and external cirri and cylindrical cephalic papillae (secondary clava), only appendage A with clava (primary clava) near its base is present (Fig. 1D). Dorsal and lateral appendages in the shape of short, and long filaments and/or spines are present at positions A-B-C-Cd- D-Dd- E (Fig. 1A–C). However, appendage B may be absent, at least on one side of the body, for more details see Morphological variability below. Dorsal plates well developed. Head and scapular plates not faceted. Under PCM, lateral portions of the scapular plate appear to be detached from the dorsal plate, forming small additional plates (one on each side of the body) (Fig. 1E, arrowheads) divided from the lateral margin of the scapular plate by a thin bright stripe. This division is formed by the narrow stripe without pores, as it is clearly visible in SEM along with a small additional plate (Fig. 1C, arrowhead). Paired plates I and II are divided into two parts – narrow anterior part and a wider posterior part – by smooth stripe without sculpture (Fig. 2A, arrows). Anterior parts most often divided longitudinally into two parts (Fig. 2A–B, indented arrowheads). Median plate 1 and 2 divided into anterior part (Fig. 2A, asterisks) and posterior part (Fig. 2A, filled arrowhead; which is especially visible in lateral position), m3 undivided (Fig. 2A, empty arrow). The terminal plate with two notches (Fig. 2A, empty arrowhead). All dorsal plates, covered with double sculpture under PCM (Fig. 2A–E), i.e., regular polygonal or roundish black ‘granules’, i.e., endocuticular pillars (0.6–1.0 µm in diameter on scapular plate and similar in size also on other plates) and white roundish or oval pores which especially in larger specimens may be merged (seen as white spots, 1.0–4.2 µm in diameter on scapular plate and similar in size also on other plates); but see below for more details. Pores absent on anterior parts of m1 and m2 (Fig. 2A, asterisks), with typical double sculpture present on the posterior parts (Fig. 2A, filled arrowheads). Central part of terminal and scapular plates with cross-like pattern, and on terminal plate a transverse line of the cross is an extension of notches (Fig. 2C–E). Sometimes, on the scapular plate, the transverse line is absent and then only the longitudinal line visible in the middle of this plate or an additional transverse line is present and the plate is poorly divided into six rectangles (Fig. 2C–D). Under SEM the plates with regularly distributed pores (Fig. 2F), which means that where the white pores visible under PCM are absent (i.e., neck plate, lateral portions of the scapular plate and anterior parts of paired plates) the plates appear to be smooth or with poorly visible granulation (Fig. 2F). Two poorly marked ventral rectangular plates, arranged transversally, are present below the head (Fig. 3A, C, arrowheads). Another two rounded square plates are present on lateral sides of the gonopore (Fig. 3B–C, arrows). Ventral cuticle possesses tiny and regular granulation (due to the presence of dense endocuticular pillars). Granulation is a little larger on the plates around the gonophore (0.3–0.4 µm diameter) than in other parts of the ventral cuticle, 0.1–0.2 µm diameter) (Fig. 3D). Outer cuticle of legs I–III with clearly visible stripes of tiny and regular granulation (0.1–0.4 µm in diameter): a thin frontal stripe on the upper part of the leg (Fig. 4A, empty arrow), a wide stripe in the central part of the leg covering frontal and lateral side of the leg (Fig. 4A, empty arrowhead) and the most distal, thin stripe above claws on the ventral side of the leg (Fig. 4A, filled indented arrowhead); white pores absent. On legs IV only frontal stripe on the upper part of the leg (just above the plate with dentate collar) (Fig. 4C, E, empty arrows) and thin stripe above claws on the ventral side of the leg are present. Triangular spine on leg I (Fig. 4B–C, filled indented arrowhead) and finger-like papilla on leg IV, present (Fig. 4D–E, filled arrow). Legs IV with dentate collar with seven to ten sharp, triangular teeth and the plate with the same sculpture as dorsal plates (Fig. 4D–E). External claws of all legs I–IV smooth, internal with spurs directed downwards (Fig. 4C–F)). The gonopore with the typical six-petal rosette (Fig. 3C, asterisk). Males (measurements and statistics in Table 2, Supp. file 2 and Fig. 5) Males are, in general, similar to females in the morphology of plates and chaetotaxy (Fig. 5A, C–D). However, there are differences in the lengths of some morphological structures (especially slightly shorter head appendages, i.e., cirri internal and external and body appendages) (compare values in Tables 1–2). On the dorsal side, regular polygonal or roundish black ‘granules’ 0.3–0.9 µm in diameter (on scapular plate and similar in size also on other plates) and white roundish or oval pores 1.0–3.1 µm in diameter (on scapular plate and similar in size also on other plates). Gonopore round and without the six-petal rosette (Fig. 5B). Juveniles – four-clawed (measurements and statistics in Table 3, Supp. file 2 and Fig. 6) In general, juveniles were similar to adults of both sexes in the morphology of plates (Fig. 6A). However, there are differences in chaetotaxy (Fig. 6A–D), in general, lack of filaments B. Moreover, appendages are shorter in juveniles than in adult females (compare values in Tables 1–3) (Fig. 6A–D). On the dorsal side, regular polygonal or roundish black ‘granules’ 0.4–0.9 µm in diameter (on scapular plate and similar in size also on other plates) and white roundish or oval pores 1.0–2.7 µm in diameter (on scapular plate and similar in size also on other plates). Gonopore absent. Larvae – two-clawed (measurements and statistics in Table 4, Supp. file 2 and Fig. 7G–H) All head and body appendages much shorter than in adults and juveniles (compare values in Tables 1–4) (Fig. 7G–H). Moreover, morphology and sculpture of plates are also different. Anterior parts of the paired plates not divided. Although, sculpture on dorsal plates composed of regular polygonal or roundish black ‘granules’ and white roundish or oval pores as in adults and juveniles. However, on head plate, anterior parts of paired plates and on entire median plate m3, pores completely absent and black ‘granules’ poorly marked. On scapular plate pores are distributed mainly on the margins of the plate and almost absent in the centre. On the dorsal side, regular polygonal or roundish black ‘granules’ 0.4–0.9 µm in diameter (on scapular plate) and white roundish or oval pores 0.7–1.4 µm in diameter (on scapular plate). Also, chaetotaxy different than adults and juveniles, i.e., A-Cd- Dd- E (Fig. 7G). Gonopore absent (Fig. 7H). Eggs Smooth, light orange and deposited in the exuviae up to 6 in one exuvium. DNA sequences We obtained good quality sequences for the applied molecular markers: 18S rRNA: GenBank: MZ798389 - MZ798396, 771 bp long; 28S rRNA: GenBank: MZ816972 - MZ816979, 715-747 bp long; ITS-2: GenBank: MZ816980 - MZ816987, 464 bp long; COI: GenBank: MZ798397 - MZ798404, 688 bp long. Morphological variability A strict chaetotaxy was analysed in 36 females, 23 males, 35 juveniles, 3 larvae. In all adult specimens, both females and males typical chaetotaxy, i.e., A-B-C-Cd- D-Dd- E was observed (Fig. 1A–B), whereas, in juveniles appendages B are most often absent (chaetotaxy: A-C-Cd- D-Dd- E). The other dorsal and lateral appendages were in general shorter in juveniles than in adults. In all studied larvae, chaetotaxy was always A-Cd- Dd- E and all appendages were much shorter than in juveniles and adults (compare values in Tables 1–4). Moreover, some modifications in chaetotaxy were observed in juveniles and adults. In two juveniles appendages B were present on both sides of the body and in five juveniles appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) (Fig. 6A–B). Moreover, in one juvenile appendages Cd and Dd were present only on one side of the body, and in another one Dd was present on both sides and Cd only on one side (chaetotaxy: A-C-Cd- D-Dd- E) (Fig. 6C–D). In two females appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) (Fig. 7A). Two females had additional small spines near the base of normally developed appendages B (chaetotaxy: A-B-C-Cd- D-Dd- E) (Fig. 7B–D). In other two females only appendage Cd on one side of the body was present and appendages Dd were completely absent (chaetotaxy: A-B-C-Cd- D-E ) (Fig. 7E–F). One female had very short appendage B on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E). In another one a very short appendage B on one side of the body was present and appendages Cd and Dd were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E). Finally, in one female appendages Cd and Dd were present on only one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E). In four males appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) (Fig. 5C–D) and in two males appendages B were absent on both sides (chaetotaxy: A-C-Cd- D-Dd- E) (Fig. 5A). Based on these assumptions chaetotaxy formula for adults and juveniles of this species is in general A-(B)- C-Cd- D-Dd- E and for larvae A-Cd- Dd- E. The other observed aberrations in chaetotaxy are only accidental. Genetic variability The obtained eight COI sequences (GenBank accession numbers: MZ798397 - MZ798404) of E. q. quadrispinosus consisted of four COI haplotypes. Haplotype 1 was found in the Norwegian (169/7 sequence, population code: 169) and German (GR8 and GR10 sequences, population code: GR) populations whereas haplotypes 2, 3 and 4 were identified in different Norwegian populations (haplotype2 – 169/8sequence, population code: 169; haplotype 3– 184/3 and 184/8 sequences, population code: 184; haplotype 4 –187/4 and 187/7 sequences, population code: 187). The value of uncorrected genetic p-distances between obtained COI haplotypes ranged from 0.2% to 0.8%. In turn, the analysis of the p-distances between E. q. quadrispinosus and compared 20 taxa of the genus Echiniscus ranged from the most similar 1.2% for E. quadrispinosus (GenBank accession number: JX683821, Vincente et al. 2013) to the least similar 21.8% for E. tantulus G&aogon;siorek, Bochnak, Von&ccaron;ina & Kristensen, 2020 (GenBank accession number: MT107427, Bochnak et al. 2020), with an average p-distance of 14.4%. In the conservative 18S rRNA gene fragment we observed no differences between our eight sequences from the German and Norwegian populations (GenBank accession numbers: MZ798389 - MZ798396) and sequences of E. quadrispinosus deposited in NCBI (GenBank accession number: MK529684). In turn, the uncorrected genetic p-distances between the other 21 taxa of the genus Echiniscus showed that the least similar was E. belloporus G&aogon;siorek & Kristensen, 2018 (GenBank accession number: MK529674, G&aogon;siorek et al. 2019a) with a genetic distance value of 3.1% and an average p-distance was 1.4%. The analysis of the p-distances between our eight sequences of 28S rRNA from the German and Norwegian populations (GenBank accession numbers: MZ816972 - MZ816979; two groups of sequences, i.e., the first consisted of GR8, GR10, 169/8, 169/9, 184/3, 184/8, 187/1 sequences and the second – one 187/8 sequence) indicated that the genetic distance was 1%. Comparison with other 19 taxa of the genus Echiniscus, for which GenBank sequences are available, are as follows: the most similar was E. quadrispinosus (GenBank accession number: MK529714, G&aogon;siorek et al. 2019a) with 1% value of the p-distance and the least similar was E. belloporus G&aogon;siorek & Kristensen, 2018 (GenBank accession numbers: MK529702, G&aogon;siorek et al. 2019a) – 5.4%, with an average p-distance of 2.5%. No genetic differences were observed between our eight ITS2 sequences from the German and Norwegian populations (GenBank accession numbers: MZ816980 - MZ816987). The ranges of uncorrected genetic p-distances between our sequences and the other 10 species of the genus Echiniscus indicated that the most similar was E. virginicus Riggin, 1962 (GenBank accession number: MN545756, G&aogon;siorek et al. 2019b) – 0.42% and the least similar was E. blumi Richters, 1903 (GenBank accession number: EF620383, Jørgensen et al. 2007) – 34.5%, with an average p-distance of 21.8%. There were no available ITS2 sequences of E. quadrispinosus in the GenBank database. Establishing of the neotype and paraneotypes of E. q. quadrispinosus The search for the type material of E. q. quadrispinosus in various collections did not bring positive results. We can probably assume that the type material of E. q. quadrispinosus no longer exists. Taking into consideration that accurate diagnoses of the species were poorly provided in the past, it is necessary to establish a neotype series of this species. For this reason, we designated the neotype and 108 paraneotypes of E. q. quadrispinosus which agree with the original description and were collected in the terra typica in the Taunus Mountain Range (Germany). The neotype series was deposited at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Pozna&nacute; and Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Poland. All the above-mentioned statements are in accordance with the International Commission on Zoological Nomenclature (ICZN) acts dedicated to the establishment of neotype series., Published as part of Kaczmarek, Łukasz, Kayastha, Pushpalata, Gawlak, Magdalena, Mioduchowska, Monika & Roszkowska, Milena, 2022, An integrative redescription of Echiniscus quadrispinosus quadrispinosus Richters, 1902 (Heterotardigrada, Echiniscidae) from the terra typica in Taunus Mountain Range (Europe; Germany), pp. 102-124 in European Journal of Taxonomy 823 (1) on pages 105-118, DOI: 10.5852/ejt.2022.823.1819, http://zenodo.org/record/6658659, {"references":["Richters F. 1902. Neue Moosbewohner. Bericht der Senckenbergischen Naturforschenden Gesellschaft in Frankfurt am Main 1902: 23 - 26. Available from https: // www. biodiversitylibrary. org / page / 8478822 [accessed 4 May 2022].","Bochnak M., Voncina K., Kristensen R. M. & Gasiorek P. 2020. Continued exploration of Tanzanian rainforests reveals a new echiniscid species (Heterotardigrada). Zoological Studies 59: 18. https: // doi. org / 10.6620 / ZS. 2020.59 - 18","Gasiorek P. & Kristensen R. M. 2018. Echiniscidae (Heterotardigrada) of Tanzania and Uganda. Tropical Zoology 31 (3): 131 - 160. https: // doi. org / 10.1080 / 03946975.2018.1477350","Gasiorek P., Morek W., Stec D. & Michalczyk L. 2019 a. Untangling the Echiniscus Gordian knot: paraphyly of the \" arctomys group \" (Heterotardigrada: Echiniscidae). Cladistics 35 (6): 633 - 653. https: // doi. org / 10.1111 / cla. 12377","Gasiorek P., Jackson K. J., Meyer H. A., Zajac K., Nelson D. R., Kristensen R. M. & Michalczyk L. 2019 b. Echiniscus virginicus complex: the first case of pseudocryptic allopatry and pantropical distribution in tardigrades. Biological Journal of the Linnean Society 128 (4): 789 - 805. https: // doi. org / 10.1093 / biolinnean / blz 147","Jorgensen A., Mobjerg N. & Kristensen R. M. 2007. A molecular study of the tardigrade Echiniscus testudo (Echiniscidae) reveals low DNA sequence diversity over a large geographical area. Journal of Limnology 66 (1 s): 77 - 83. https: // doi. org / 10.4081 / jlimnol. 2007. s 1.77"]}

In the present study, we used an integrative taxonomy approach to redescribe a population of Echiniscus quadrispinosus quadrispinosus Richters, 1902 from the neotype locality in the Taunus Mountain Range (Germany). We found clear differences in the chaetotaxy formula between the life stages of E. q. quadrispinosus. The body appendages B are, in general, absent in juveniles. Moreover, in larvae all body lateral appendages, except for E, are absent. We also obtained DNA sequences of 18S rRNA, 28S rRNA, ITS-2, and COI of E. q. quadrispinosus from the neotype locality and three Norwegian populations. Comparison with the sequences available in GenBank showed low genetic differences between the neotypic population and specimens from other localities. Therefore, we decided to establish our specimens from Taunus Mountain Range as neotype and paraneotypes of E. q. quadrispinosus. We also discussed and amended the taxonomic status of three subspecies E. q. brachyspinosus Bartoš, 1934, E. q. cribrosus Murray, 1907 and E. q. fissispinosus Murray, 1907 and established them as junior synonyms of E. q. quadrispinosus. Finally, we also confirmed E. lichenorum Maucci, 1983 as a valid species, clearly different from E. q. quadrispinosus.

Los tardígrados u osos de agua son un grupo de invertebrados microscópicos que habitan en ambientes acuáticos y terrestres, y de manera frecuente en los briófitos. El grupo ha sido poco estudiado en Cuba, los trabajos de tardígrados son meramente taxonómicos y no evalúan los patrones de abundancia y diversidad de las comunidades. El propósito de esta investigación fue analizar la diversidad de tardígrados asociados a musgos en el Jardín de los Helechos en Santiago de Cuba. Para esto se realizaron curvas de acumulación de especies utilizando diversos estimadores de riqueza, se determinaron las diversidades alfa observada y estimadas, se estableció el orden jerárquico de la comunidad y se evaluó el patrón de abundancia mediante curvas de Whittaker. Se encontraron 34 tardígrados pertenecientes a las clases Heterotardigrada y Eutardigrada, los géneros presentes fueron Echiniscus, Milnesium, Mesobiotus y Macrobiotus con un total de 8 morfoespecies identificadas. El morfo dominante fue Macrobiotus sp. 1, mientras que Echiniscus sp., Mesobiotus sp. 2 y Mesobiotus sp. 3 resultaron ser especies raras. La curva de rango-abundancia indicó una comunidad equitativa.

The classification and identification of species within the genus Pseudechiniscus Thulin, 1911 has been considered almost a Sisyphean work due to an extremely high homogeneity of its members. Only recently have several contributions made progress in the taxonomy 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.