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ORDER: PARACHELA SCHUSTER ET AL., 1980 SUPERFAMILY: MACROBIOTOIDEA THULIN, 1928 (IN MARLEY ET AL., 2011), Published as part of Stec, Daniel, Vončina, Katarzyna, Kristensen, Reinhardt Møbjerg & Michalczyk, Łukasz, 2022, The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades, pp. 1067-1099 in Zoological Journal of the Linnean Society 195 on page 1070, {"references":["Thulin G. 1928. Uber die Phylogenie und das System der Tardigraden. Hereditas 11: 207 - 266."]}

MACROBIOTUS ARIEKAMMENSIS GROENLANDICUS SUBSP. NOV. (TABLES 4, 5; FIGS 5–11) Z o o b a n k r e g i s t r a t i o n: u r n: l s i d: z o o b a n k. org:act: 8A12E93C-8729-4B13-BD36-FC507DD117D3 Material examined: Altogether 110 animals and 78 eggs. Specimens mounted on microscope slides in Hoyer’s medium (83 animals + 68 eggs), fixed on SEM stubs (20 animals + ten eggs + four buccal apparatuses), processed for DNA sequencing (three animals). Etymology: The new subspecies is named after Greenland (from Danish Grønland), the territory where it was discovered. Type locality: 69°15’17’’N, 53°30’46’’W; 30 m a.s.l.: western coast of Greenland, Disko Island, Østerlien; moss on rock. Type depositories: Altogether 83 animals [slides: GL.018. 2–3, 9–17, SEM stubs: 9.06, 12.15 (buccal apparatus), 16.19] and 68 eggs (slides: GL.018. 1, 4–8, SEM stub: 16.19) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387, Kraków, Poland. Description of the new subspecies Animals (measurements and statistics in Table 4): Body colourless in juveniles and whitish in adults, after fixation in Hoyer’s medium transparent (Fig. 5A). Eyes present, visible also after mounting the specimens on permanent slides in Hoyer’s medium. The entire cuticle covered with granulation visible in both PCM and SEM, arranged densely on the dorsum, and less densely on the venter and legs (Figs 5B–G, 6A, C, D, F, G, I). Only in some specimens the cuticular granulation can be less evident under PCM (Fig. 5D). Oval cuticular pores present (0.5–1.4 µm in diameter) (Fig. 5E–G). Patches of dense granulation present on internal and external surface of all legs I– III, as well as on legs IV and clearly visible (Fig. 6A, B, D, E). A pulvinus present on the internal surface of legs I– III (Fig. 6D, E). Granulation on legs IV is visible as a single large patch on dorsal and lateral leg surfaces (Fig. 6G–H). Claws slender, with flat and wide common tract, beginning with a visible stalk that connects the claws to the wide lunulae and ending with elongated branches (especially the primary branch; Fig. 7A–E). Primary branches with distinct accessory points, visible in PCM and SEM (Fig. 7A–E). Lunulae I– III smooth (Fig. 7A, C, D), whereas lunulae IV with clear dentation (Fig. 7B, E). A single continuous cuticular bar (Fig. 7A) and double muscle attachments visible on each leg I– III (Fig. 7A, C, D). Mouth anteroventral with ten peribuccal lamellae. Bucco-pharyngeal apparatus of the Macrobiotus - type (Figs 8A, 9A). Oral cavity armature extremely reduced to only one large tooth present in the dorsal portion of the third band of teeth, whereas other bands of teeth are absent (Figs 8A–D, 9F). Pharyngeal bulb spherical, with triangular apophyses, cuticular spikes, two rod-shaped macroplacoids (macroplacoid sequence: 2 Eggs (measurements and statistics in Table 5): Eggs laid freely, whitish, spherical or slightly oval (Figs 10A–D, 11A). The spaces between the processes are small and the surface of the egg between the processes is continuous and smooth, without any pores or reticulum, i.e. persimilis - type (Figs 10A–D, 11A–D). Between the processes on the egg surface, lightrefracting dots are usually visible in PCM, resembling micropores (Fig. 10A–D). Egg processes single-walled (without reticulation caused by labyrinthine layer) with dome-shaped basal part with distal part being thinner and elongated (Figs 10E–P, 11A–D). Internal septa are sometimes visible between basal and distal portion of the process in PCM (Fig. 10E–P). The basal portions of the processes are pierced by pores of uniform size (1.1– 1.8 µm in diameter) that are arranged alternately with dark thickenings around the process base (Figs 10A– D, 11B–D). In SEM, a reticulate internal structure is visible inside the pores and it seems to be a remnant of the reduced labyrinthine layer (Fig. 11B–D). The apical parts of the processes are flat but devoid of terminal discs and are covered with short, thin and flexible filaments (Figs 10E–P, 11E, F). Reproduction: The population is dioecious (the examination of specimens freshly mounted in Hoyer’s medium revealed testes filled with spermatozoa), but no secondary sexual dimorphism has been observed. DNA sequences: All obtained DNA sequences were represented by a single haplotype per each marker: 18S rRNA: MZ 463662, MZ 463663, MZ 463664. 28S rRNA: MZ 463677, MZ 463678, MZ 463679. ITS 2: MZ 463653, MZ 463654, MZ 463655. COI: MZ 461005, MZ 461006, MZ 461007. Differential diagnosis: Macrobiotus a. groenlandicus, known only from its locus typicus in Disko Island, Greenland, shares with M. a. ariekammensis the elongated primary branches of all claws, only one tooth in the third band of teeth in the oral cavity, and single-layer egg processes surrounded by a crown of pores and thickenings around their bases. However, it differs from M. a. ariekammensis, which is known only from a few localities in Svalbard (Norway) and Poland, by: the presence of a strong pronounced constriction in the first macroplacoid (first macroplacoid weakly constricted in M. a. ariekammensis), the presence of light-refracting dots resembling micropores on the egg surface (egg surface smooth in M. a. ariekammensis) and by the presence of fine granulation on the body cuticle visible in PCM and SEM (the body granulation absent or not visible in PCM in M. a. ariekammensis)., Published as part of Stec, Daniel, Vončina, Katarzyna, Kristensen, Reinhardt Møbjerg & Michalczyk, Łukasz, 2022, The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades, pp. 1067-1099 in Zoological Journal of the Linnean Society 195 on pages 1072-1076, DOI: 10.1093/zoolinnean/zlab101, http://zenodo.org/record/6994499

MACROBIOTUS KIRGHIZICUS TUMANOV, 2005 (TABLES 6, 7; FIGS 12–18) Material examined: Altogether 66 animals, and 15 eggs. Specimens mounted on microscope slides in Hoyer’s medium (53 animals + ten eggs), fixed on SEM stubs (ten + five), processed for DNA sequencing (three animals). Population locality: 41°32’37.98’’N, 75°10’2.28’’E; 2288 m a.s.l.: Kyrgyzstan, Chui, Kegeti, moss on rock. Specimens depositories: Altogether 53 animals (slides: KG.062.006. 1, 9–14, SEM stub: 18.07) and ten eggs (slides: KG.062. 5–8, SEM stub: 18.07) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387, Kraków, Poland. Description of the Kyrgyz Republic population Animals (measurements and statistics in Table 6): Body whitish in adults and colourless in smaller individuals, after fixation in Hoyer’s medium transparent (Fig. 12A). After mounting in Hoyer’s medium eyes present in all specimens. Small, oval pores (0.5–0.8 µm in diameter), visible under PCM and SEM (Fig. 12B, C), scattered randomly on the entire body cuticle, including the external and internal surface of all legs (Fig. 13A–F). Extremely fine body granulation (c. 60 nm in diameter), visible only in SEM, present on the entire dorsocaudal cuticle (Fig. 12C). Patches of dense granulation present on the internal and external surfaces of all legs I– III and clearly visible both in PCM and SEM (Fig. 13A–D). A cuticular bulge, resembling a pulvinus, is present on the internal surfaces of legs I– III (Fig. 13C, D). Cuticular granulation on legs IV present and always clearly visible both in PCM and SEM (Fig. 13E, F). Claws slender, with flat and wide common tract, beginning with an evident stalk that connects the claws to the wide lunulae and ending with extremely elongated branches (especially the primary branch; Fig. 14A, B, D, E). Primary branches with indistinct accessory points, barely visible in PCM, but clearly visible in SEM (Fig. 14A, B, D, E). Lunulae I– III smooth (Fig. 14A, D), whereas lunulae IV with clear dentation (Fig. 14B, E). Mouth anteroventral with ten peribuccal lamellae (Fig. 16A, B). Bucco-pharyngeal apparatus of the Macrobiotus - type (Fig. 15A). Under PCM, only the second and third band of teeth visible, with the second band being faintly marked (Fig. 15B, C). However, under SEM, all of the three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of several irregular rows of small granular teeth surrounding the oral cavity (Fig. 16A, C). The second band of teeth is situated between the ring fold and the third band of teeth, and is comprises of small cones, barely visible in PCM (Figs 15B, 16B; note: in Fig 16B, only distal portion of these teeth are visible from behind the ring fold; due to unsuitable positioned specimen it was impossible to get better image in SEM). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 15B–D, 16A, B). The third band of teeth is discontinuous and divided into a dorsal and a ventral portion. Under PCM, the dorsal teeth form a transversal ridge weakly divided into two granular teeth, whereas the ventral teeth are smaller and faintly visible as two separate lateral transverse ridges with granular/roundish thickening at their medial extremities (Fig 15B–D). In SEM, both the dorsal and the ventral portion of the third band of teeth are visible as one fused ridge with two evident teeth extending from the medial portion of the ridge (Fig 16A, B). Pharyngeal bulb spherical, with triangular apophyses, cuticular spikes, two rodshaped macroplacoids (macroplacoid sequence: 2 Eggs (measurements and statistics in Table 7): Eggs laid freely, whitish, spherical or slightly oval (Figs 17A, B, 18A). Although the spaces between processes are small, the surface between processes is of the persimilis - type, i.e. with the continuous smooth chorion, with few, randomly distributed pores (Figs 17A, B, 18B–D). Egg processes single-walled (without reticulation caused by labyrinthine layer) with domeshaped basal part and rigid spine-like distal part (Figs 17A–F, 18A–F). In PCM, the basal and distal portions are clearly separated from with single internal septum (Fig 17C–F). The bases of egg processes are pierced with pores of uniform size (0.3–0.7 µm in diameter), distributed evenly around the base and most often arranged in two rows (Figs 17A, B, 18B–F). In PCM, short, dark thickenings are sometimes visible around the process bases below or at the same level as the lower ring of pores (Fig 17A, B). The apical part of the processes is devoid of terminal discs and is covered with short, thin and flexible filaments (Figs 17C–F, 18A–F). Reproduction: The population is dioecious (the examination of specimens freshly mounted in Hoyer’s medium revealed testes filled with spermatozoa), but no secondary sexual dimorphism has been observed. DNA sequences: All obtained DNA sequences were represented by a single haplotype per each marker: 18S rRNA: MZ 463665, MZ 463666, MZ 463667. 28S rRNA: MZ 463671, MZ 463672, MZ 463673. ITS 2: MZ 463659, MZ 463660, MZ 463661. COI: MZ 461002, MZ 461003, MZ 461004. PHYLOGENY The phylogenetic reconstruction (Fig. 19) shows three well-supported distinct lineages constituting three separate genera within superclade I (sensu Stec et al., 2021a) of the family Macrobiotidae: the clade comprising Macrobiotus species, and further two monophyletic groups: one corresponding to the genus Mesobiotus Vecchi et al., 2016, and the other representing Sisubiotus Stec et al., 2021a (Fig. 19). Macrobiotus is divided into three well-supported subclades: A, B and C, sensu Stec et al. (2021a). All of the three newly found populations investigated in this study, M. a. ariekammensis, M. a. groenlandicus and M. kirghizicus, are nested in subclade A, which contains species of the Macrobiotus hufelandi morphogroup sensu Stec et al. (2021a) and Macrobiotus basiatus Nelson et al., 2020, which exhibits unique egg morphology. Subclade B comprises three species complexes delineated by Stec et al. (2021a). As in Stec et al. (2021a) and Vecchi & Stec (2021), the Macrobiotus pallari complex and the Macrobiotus pseudohufelandi complex are monophyletic also in the present study (Fig. 19). However, the Macrobiotus persimilis complex, which was monophyletic in the two earlier studies, appears to be paraphyletic in the current analysis (Fig. 19). Thus, further studies are needed to clarify the phyletic character of the latter species complex. Subclade C comprises species of the Macrobiotus hufelandi morphogroup. SPECIES DELIMITATION AND GENETIC DISTANCES The PTP analysis identified 49 and 55 putative species in ML and BI approach, respectively. The ASAP analysis, on the other hand, identified 48 putative species. These results are in line with the general inspection of the tree terminals and the morphological information that would suggest also 48 species among the ingroup taxa. However, for two out of the three newly found populations analysed in this study, both PTP approaches were not congruent with ASAP results. The PTP approaches indicated that M. a. ariekammensis and M. a. groenlandicus constitute a single species, whereas the ASAP analysis identified them as separate entities. Uncorrected pairwise distances between the three newly found populations analysed in this study are as follows: • 18S rRNA: 0.2% for M. a. ariekammensis and M. a. groenlandicus; 0.1% for M. a. ariekammensis and M. kirghizicus; 0.3% for M. a. groenlandicus and M. kirghizicus. • 28S rRNA: 0.1% for M. a. ariekammensis and M. a. groenlandicus; 0.3% for M. a. ariekammensis in PCM (E) and SEM (F). Filled flat arrowheads indicate granulation patch on the external leg surface, empty indented arrowheads indicate cuticular bulge (pulvini), filled indented arrowhead indicates cuticular bar, empty flat arrowheads indicate granulation patch on the internal leg surface. Scale bars in µm. and M. kirghizicus; 0.1% for M. a. groenlandicus and M. kirghizicus. • ITS2: 0.3% to 0.8% for M. a. ariekammensis and M. a. groenlandicus; 6.1% for M. a. ariekammensis and M. kirghizicus; 6.3% for M. a. groenlandicus and M. kirghizicus. • COI: 3.3% for M. a. ariekammensis and M. a. groenlandicus; 16.3% for M. a. ariekammensis and M. kirghizicus; 16.4% for M. a. groenlandicus and M. kirghizicus. Given the discrepancies between the PTP and ASAP species delineation results, shallow genetic divergence and low p -distances in COI and ITS2 between M. a. ariekammensis and M. a. groenlandicus, we interpreted the morphological differences between the two taxa as intraspecific variability, hence the later taxon is described here as a subspecies rather than a separate species., Published as part of Stec, Daniel, Vončina, Katarzyna, Kristensen, Reinhardt Møbjerg & Michalczyk, Łukasz, 2022, The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades, pp. 1067-1099 in Zoological Journal of the Linnean Society 195 on pages 1076-1090, DOI: 10.1093/zoolinnean/zlab101, http://zenodo.org/record/6994499, {"references":["Tumanov DV. 2005. Two new species of Macrobiotus (Eutardigrada, Macrobiotidae) from Tien Shan (Kirghizia), with notes on Macrobiotus tenuis group. Zootaxa 1043: 33 - 46.","Stec D, Vecchi M, Calhim S, Michalczyk L. 2021 a. New multilocus phylogeny reorganises the family Macrobiotidae (Eutardigrada) and unveils complex morphological evolution of the Macrobiotus hufeland i group. Molecular Phylogenetics and Evolution 160: 106987.","Vecchi M, Cesari M, Bertolani R, Jonsson KI, 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: 303 - 322.","Nelson DR, Adkins Fletcher R, Guidetti R, Roszkowska M, Grobys D, Kaczmarek L. 2020. Two new species of Tardigrada from moss cushions (Grimmia sp.) in a xerothermic habitat in northeast Tennessee (USA, North America), with the first identification of males in the genus Viridiscus. PeerJ 8: e 10251.","Vecchi M, Stec D. 2021. Integrative descriptions of two new Macrobiotus species (Tardigrada, Eutardigrada, Macrobiotidae) from Mississippi (USA) and Crete (Greece). Zoosystematics and Evolution 97: 281 - 306."]}

MACROBIOTUS ARIEKAMMENSIS ARIEKAMMENSIS WĘGLARSKA, 1965 (TABLES 2, 3; FIGS 1–4) Material examined: Seven animals and four eggs. Specimens mounted on microscope slides in Hoyer’s medium (four animals + four eggs), processed for DNA sequencing (three animals). Population locality: 78°40’33’’N, 16°38’49’’E; 208 m a.s.l.: Norway, Svalbard, Fortet; moss on soil. Specimen depositories: Four animals (slides: NO.393.393.2-4) and four eggs (slide: NO.393.01) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387, Kraków, Poland. Description of the Norwegian population from Spitsbergen Animals (measurements and statistics in Table 2): Body transparent in smaller individuals (juveniles) and whitish in adults, after fixation in Hoyer’s medium transparent (Fig. 1A). Eyes present in all specimens, visible after mounting in Hoyer’s medium. Cuticular pores (0.7–1.2 µm in diameter) present, clearly visible under PCM (Fig. 1B–E) and scattered randomly on the entire body cuticle, including the external and internal surface of all legs. Patches of fine granulation present on internal and external surfaces of all legs I– III, as well as on legs IV and visible clearly in PCM (Fig. 1C–E). A pulvinus present on the internal surfaces of legs I– III (Fig. 1D). Granulation on legs IV is visible as a single large granulation patch on dorsal and lateral leg surfaces (Fig. 1E). Claws slender, with flat and wide common tract, beginning with a visible stalk that connects the claws to the wide lunulae and ending with elongated branches (especially the primary branch; Fig. 2A, B). Primary branches with distinct accessory points, visible in PCM (Fig. 2A, B). Lunulae I– III smooth (Fig. 2A), whereas lunulae IV with clear dentation (Fig. 2B, D). A single continuous cuticular bar and double muscle attachments visible on each leg I– III (Fig. 2C). Mouth anteroventral with ten peribuccal lamellae. Bucco-pharyngeal apparatus of the Macrobiotus - type (Fig. 3A). Under PCM, oral cavity armature extremely reduced to only one large tooth present in the dorsal portion of the third band of teeth, whereas other bands of teeth invisible or absent (Fig. 3A, B). Pharyngeal bulb spherical, with triangular apophyses, cuticular spikes, two rod-shaped macroplacoids (macroplacoid sequence: 2 Eggs (measurements and statistics in Table 3): Eggs laid freely, whitish, spherical or slightly oval (Fig. 4A, B). The spaces between processes are small, the surface between processes is of the persimilis - type, i.e. with the continuous smooth chorion, with no pores visible (Fig. 4A, B). Egg processes single-walled (without reticulation caused by the labyrinthine layer) with dome-shaped basal part and thinner and elongated distal portions (Fig. 4C–H). Internal septa are sometimes visible between the basal and the distal portion of the process in PCM (Fig. 4H). The basal portions of the processes are pierced by pores that are arranged alternately with dark thickenings and around the process base (Fig. 4A, B). The apical parts of the processes are flat but devoid of terminal discs, and are covered with short, thin and flexible filaments (Fig. 4C–H). Reproduction: The population is dioecious (the examination of specimens freshly mounted in Hoyer’s medium revealed testis filled with spermatozoa), but no secondary sexual dimorphism has been observed. DNA sequences: All obtained DNA sequences were represented by a single haplotype per each marker: 18S rRNA: MZ 463668, MZ 463669, MZ 463670. 28S rRNA: MZ 463674, MZ 463675, MZ 463676. ITS 2: MZ 463656, MZ 463657, MZ 463658. COI: MZ 460999, MZ 461000, MZ 461001., Published as part of Stec, Daniel, Vončina, Katarzyna, Kristensen, Reinhardt Møbjerg & Michalczyk, Łukasz, 2022, The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades, pp. 1067-1099 in Zoological Journal of the Linnean Society 195 on pages 1070-1072, DOI: 10.1093/zoolinnean/zlab101, http://zenodo.org/record/6994499, {"references":["Weglarska B. 1965. Die Tardigraden (Tardigrada) Spitzbergens. Acta Zoologica Cracoviensia 11: 43 - 52."]}

BARBARIA PAUCIGRANULATA WILAMOWSKI, VONČINA, GĄSIOREK & MICHALCZYK,SP. NOV. (FIGS 1–3, 7I, 8I, 9I, 10I, 11K, L; TABLES 3, 4) Zoobank registration: u r n: l s i d: z o o b a n k. org:act: A11E6BDF-7E2A-4F0A-BD6A-9BC87FCE147A Morphometric data: w w w. t a r d i g r a d a. n e t / register/0109.htm Type material: Holotype (adult female, slide AR.303.02) and 12 paratypes (eight adult females on slides AR.302.02, AR.303.01, 3, 5–6 and four juveniles on slides AR.303.03–4). Found together with B. ollantaytamboensis. Type locality: 24°47′14″S, 65°43 ′30″W, 2150 m asl: Argentina, Salta Province, Rosario de Lerma Department, vicinity of Río Rosario; lichen on rock in a shrubland (see also Table 1). Etymology: From Latin paucus, few, and granulatus, grained, alluding to the scarcity of epicuticular granulation on the dorsal plates. An adjective in nominative singular. Description: Adult females (i.e. from the third instar onwards, measurements in Table 3). Dark orange body with large red eyes; the pigment and eyes dissolve quickly after mounting in Hoyer’s medium. Body massive with stumpy limbs (Figs 1A, B, 2). Cylindrical, Echiniscus - type cephalic papillae (secondary clavae) and (primary) clavae; cirri embedded in bulbous cirrophores (Fig. 3D). Cirrus A is short ( Dorsal plate sculpturing of the bigranulata - type, comprising minute, poorly developed pillars (pseudogranulation) and pores (Figs 1C, 2, 3A–C, 7I, 8I, 9I, 10I). Pillars are densely packed and additionally interconnected by thin striae in the scapular (Fig. 7I) and caudal (terminal) plates (Fig. 10I). Pores are slightly larger in the scapular plate, median plate1, posterior portion of median plate 2 and centroposterior portions of paired segmental plates compared with posterolateral portions of paired segmental plates and the caudal plate (Figs 1, 2); pores of similar diameter are rarely present in the entire dorsum. Pores are always absent in the anterior portion of median plate 2 (Fig. 8I), paired segmental plates (Fig. 9I) and the entirety of median plate 3; and all these areas are covered with epicuticular, multangular granules. Lateralmost portions of the scapular and paired segmental plates can be poreless or with single minute pores (Figs 1A, B, 2). The cephalic plate is large, with a pronounced chalice-shaped anterior incision and with only pillars in the posterior portion of the plate. A broad and strongly sclerotized cervical plate is divided into sculptured anterior portion and smooth posterior portion bordering with the scapular plate (Figs 1A, 2A). Lateral sutures in the scapular plate demarcate lateralmost, trapezoidal portions (Figs 1A, B, 2B). Median plates 1 and 3 are unipartite (the latter with strongly developed granules), median plate 2 is bipartite (Fig. 8I). Paired segmental plates are without transverse unsculptured bands, as epicuticular granules of anterior portion transition gradually into pillars of the posterior portions (Fig. 9I). The caudal plate with short, poorly sclerotized incisions and no signs of faceting (Figs 1A, 2, 10I). Ventral cuticle with minute endocuticular pillars (Fig. 11K) distributed evenly throughout the entire venter, lacking plates, beside of rarely developed subcephalic plates (Fig. 3D). Sexpartite gonopore placed anteriorly to a trilobed anus between legs IV. Pedal plates without pores, their sculpturing consists of poorly developed endocuticular pillars formed as belts in the central portions of the legs (Figs 1A, B, 11K, L). Thick pulvini on outer side of all legs (Figs 1A, B, 2B). Dentate collar IV has numerous irregular short teeth (Fig. 11L). A small, elongated spine I and a tubby papilla IV (Figs 1B, 2B, 11K, L). Claws slightly heteronych with claws IV (Fig. 3F) higher and more robust than claws I–III (Fig. 3E). Internal claws IV have needle-like spurs more divergent from branches compared to spurs I– III (Fig. 11K, L). Cuticular bars below claw bases on the inner side of legs present. Buccal apparatus with a rigid tube and round pharynx containing placoids. Flexible stylet supports present. Juveniles (i.e. the second instar, measurements in Table 4). Clear morphometric gap between juveniles and adult females. Qualitatively alike adult females, excluding the lack of gonopore. Adult males, larvae or eggs not found. Differential diagnosis: The new species B. paucigranulata is distinguished from its congeners (alphabetically): • Barbaria bigranulata, by the distribution of pores in plates [absent in the anterior portion of median plate 2 (Fig. 8I), paired segmental plates (Fig. 9I) and the entirety of median plate 3 in B. paucigranulata vs. present in the anterior portion of m2 (Fig. 8A), paired segmental plates (Fig. 9A) and the entirety of m 3 in B. bigranulata], the shape of papilla IV [tubby in B. paucigranulata (Fig. 11L) vs. elongated in B. bigranulata (Fig. 11B)] and the primary spur morphology [needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. robust, hook-shaped and divergent from the claw branch in B. bigranulata (Fig. 11A, B)]. • Barbaria charrua, by the shape of papilla IV [tubby in B. paucigranulata (Fig. 11L) vs. elongated in B. charrua (Fig. 11D)] and by the primary spurs [present in B. paucigranulata (Fig. 11K, L) vs. typically absent in B. charrua (Fig. 11C, D)]. • Barbaria danieli, by the shape of papilla IV [tubby in B. paucigranulata (Fig. 11L) vs. elongated in B. danieli (Fig. 12)] and by the primary spurs [present in B. paucigranulata (Fig. 11K, L) vs. absent in B. danieli (Fig. 11O)]. • Barbaria ganczareki, by the dorsal sculpturing [both pillars and pores easily identifiable in B. paucigranulata (Figs 7I, 8I, 9I, 10I) vs. pillars so poorly developed that pores become the dominant element of the sculpture in B. ganczareki (Figs 7C, 8D, 9C, 10C)], the pores in the subcephalic region (absent in B. paucigranulata vs. present in B. ganczareki) and in pedal plates (absent in B. paucigranulata vs. present in B. ganczareki) and by the primary spur morphology [needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. robust, hook-shaped and divergent from the claw branch in B. ganczareki, fig. 21–22 in Michalczyk & Kaczmarek (2007)]. • Barbaria hannae, by the dorsal sculpturing [no smooth plate portions in B. paucigranulata (Figs 8I, 9I, 10I) vs. thickened plate portions devoid of sculpturing present in B. hannae (Figs 8E, 9D, E, 10D, E)] and the primary spur morphology [needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. robust, hook-shaped and divergent from the claw branch in B. hannae (Fig. 11P)]. • Barbaria jenningsi, by the cirrus A length (B. paucigranulata vs.> 50% of the body length in B. jenningsi), the type of perforation in the dorsal plates [pores in B. paucigranulata (Fig. 7I) vs. pseudopores in B. jenningsi (Fig. 7E, F)] and by the primary spur morphology [slightly heteronych, needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. fully heteronych, robust, hook-shaped and divergent from the claw branch in B. jenningsi (Fig. 11E, F)]. • Barbaria madonnae, by striae between pillars in the scapular and the caudal plate [present in B. paucigranulata (Figs 7I, 10I) vs. absent in B. madonnae (Figs 7G, 10G)], the shape of papilla IV [tubby in B. paucigranulata (Fig. 11L) vs. elongated in B. madonnae (Fig. 11H)] and the primary spur morphology [needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. robust, hook-shaped and divergent from the claw branch in B. madonnae (Fig. 11G, H)]. • Barbaria ollantaytamboensis, by the distribution of pores in plates [absent in the anterior portion of median plate 2 (Fig. 8I), paired segmental plates (Fig. 9I) and the entirety of median plate 3 in B. paucigranulata vs. present in the anterior portion of m2 (Fig. 8H), paired segmental plates (Fig. 9H) and the entirety of m 3 in B. ollantaytamboensis] and claw isomorphy [slightly heteronych (heteromorphic) in B. paucigranulata (Fig. 11K, L) vs. isonych (homomorphic) in B. ollantaytamboensis (Fig. 11I, J)]. • Barbaria quitensis, by the type of perforation in the dorsal plates [pores in B. paucigranulata (Figs 7I, 8I, 9I, 10I) vs. pseudopores in B. quitensis (Figs 7J, 8J, 9J, 10J)] and the distribution of pores/pseudopores [scarcer on lateralmost portions of the caudal plate (Fig. 10I) in B. paucigranulata vs. roughly equally distributed in all portions of the caudal plate (Fig. 10J) in B. quitensis]. • Barbaria ranzii, by the cirrus A length (B. paucigranulata vs.> 50% of the body length in B. ranzii), the distribution of pores in plates [absent in the anterior portion of median plate 2 (Fig. 8I), paired segmental plates (Fig. 9I) and the entirety of median plate 3 in B. paucigranulata vs. present in the anterior portion of m2 (Fig. 8K), paired segmental plates (Fig. 9K) and the entirety of m 3 in B. ranzii] and the by secondary spurs directed upwards on external claws IV [absent in B. paucigranulata (Fig. 11L) vs. present in B. ranzii, Fig. 11R]. • Barbaria weglarskae, by the cirrus A length (B. paucigranulata vs.> 50% of the body length in B. weglarskae) and by the primary spur morphology [needle-like and adjacent to the claw branch in B. paucigranulata (Fig. 11K, L) vs. robust, hook-shaped and divergent from the claw branch in B. weglarskae (Fig. 11M, N)].

BARBARIA WEGLARSKAE GĄSIOREK, WILAMOWSKI, VONČINA & MICHALCZYK, SP. NOV. (FIGS 4, 7L, 8L, 9L, 10L, 11M, N; TABLE 5) Zoobank registration: u r n: l s i d: z o o b a n k. org:act: F083907B-741F-48C0-A127-2FC28482C9AA Morphometric data: w w w. t a r d i g r a d a. n e t / register/0110.htm Ty p e m a t e r i a l: Holotype (adult female, slide AR.059.04) and two paratypes (adult females, slides AR.059.01, 05). Type locality: 48°25′42″S, 71°44′48″W, 803 m asl: Argentina, Patagonia, Santa Cruz Province, Río Chico Department, vicinity of La Florida; lichen from on in the Andean Patagonian forest (see also Table 1). Etymology: A patronym honouring Professor Barbara Węglarska, 20.02.1922 – 02.10.2020, whose death left a void in the community of tardigradologists. A noun in the genitive case. Description: Adult females (i.e. from the third instar onwards, measurements in Table 5). Orange body with large, red eyes; the entire pigment and eyes dissolve quickly after mounting in Hoyer’s medium. Body massive (Fig. 4A). Cylindrical, Echiniscus - type cephalic papillae (secondary clavae) and (primary) clavae; cirri embedded in bulbous cirrophores. Cirrus A is long (> 50% of the body length) and with evident, conical cirrophore (Fig. 4A, B). Dorsal plate sculpturing of the bigranulata - type, composed of pillars present in all plate portions (pseudogranulation) and pores and pseudopores present in different elements of armour (Figs 4A, B, 7L, 8L, 9L, 10L). Pseudopores can be present or absent exclusively in the anterior portion of the median plate 2 and paired segmental plates and the entirety of median plate 3 (Fig. 4A, B). Minute pores of equal size are regularly distributed in the remaining plate portions (Figs 4A, B, 7L, 8L, 9L, 10L). The cephalic plate large, with a pronounced chalice-shaped anterior incision and lateral sutures demarcating roughly triangular lateralmost portions of the plate (Fig. 4A, B). Thin cervical plate with developed pillars and pseudopores. Lateral sutures in the scapular plate demarcate lateralmost, rectangular portions with identical sculpturing as on the rest of the plate (Fig. 4A, B). Median plates 1 and 3 unipartite (the latter strongly reduced and partially covered by the caudal plate), median plate 2 bipartite (Figs 4A, B, 8L). Paired segmental plates with broad, transverse, unsculptured bands (Figs 4A, B, 9L). The caudal plate with short, poorly sclerotized incisions clearly joined by a transversal suture (Figs 4A, B, 10L). Ventral cuticle with minute endocuticular pillars distributed evenly throughout the entire venter; a pair of small, subcephalic plates present (Fig. 4C–D). Sexpartite gonopore (Fig. 4E) placed anteriorly to a trilobed anus between legs IV. Pedal plates I–IV with evident pillars and pseudopores (Figs 4A, 11M, N). Evident pulvini on outer sides of all legs. Dentate collar IV with numerous irregular short teeth (Fig. 11N). A tiny spine I and a tubby papilla IV (Fig. 11N). Claws slightly heteronych, because primary spurs on internal claws IV are positioned higher than those on claws I–III (Fig. 11M, N). The shape and angle at which spurs diverge from branches are almost identical on all limbs. Cuticular bars below claw bases on the inner side of legs present. Buccal apparatus with a rigid tube and round pharynx containing placoids. Lacking stylet supports. Adult males, juveniles, larvae or eggs not found. Differential diagnosis: There are only two other species of Barbaria with a cirrus A /body length ratio> 50% [the titles and values in the last five rows of table 1 in Michalczyk & Kaczmarek (2007) are mismatched in the case of sexes treated separately, but the ratio statistics for all measured specimens of B. ganczareki stands valid: min = 15%, max = 23%, mean = 19%]: B. jenningsi and B. ranzii, but B. weglarskae can be distinguished from: • Barbaria jenningsi, by the type of perforation in the dorsal plates [dominant pores in B. weglarskae (Fig. 7L) vs. pseudopores in B. jenningsi (Figs 7E, F, 8F, 9F, 10F)] and claw isomorphy [anisonych/ slightly heteronych in B. weglarskae (Fig. 11M, N) vs. strongly heteronych in B. jenningsi (Fig. 11E, F)]. • Barbaria ranzii, by the pedal plate sculpturing [with evident pillars in B. weglarskae (Fig. 11M, N) vs. without pillars in B. ranzii (Fig. 11R)], the shape of papilla IV [tubby in B. weglarskae (Fig. 11N) vs. elongated in B. ranzii] and by the presence of secondary spurs directed upwards on external claws IV [absent in B. weglarskae (Fig. 11N) vs. present in B. ranzii (Fig. 11R)]. MOLECULAR PHYLOGENY Phylogeny based on the five concatenated markers brought fully resolved relationships between all eight analysed species of Barbaria, which form clades characterized by low intraspecific and large interspecific genetic variability (Fig. 5). The topology is as follows: B. madonnae is a sisterspecies to all other sequenced Barbaria spp., which are clustered in two clades: the first comprising (B. paucigranulata (B. danieli + B. charrua)) and the second grouping ((B. weglarskae + B. jenningsi) + (B. ollantaytamboensis + B. bigranulata)). INTRA- AND INTERSPECIFIC GENETIC VARIABILITY Regarding COI sequences deposited in GenBank, the data are available only for B. bigranulata and B. jenningsi. COI p -distances between populations of B. bigranulata and the previously published data for a population from Chile (HM193406; Jørgensen et al., 2011) ranged between 2.6 and 2.9% (alignment length = 585 bp). Analogous index for the pair B. weglarskae – B. jenningsi (KP013596; Velasco-Castrillón et al., 2015) was 18.9% (alignment length = 472 bp). More than one haplotype per marker has been found for all markers, but only in a few species. The intraspecific p -distances are as follows: 18S rRNA: 0.1% (in B. bigranulata and B. charrua); 28S rRNA: 0.1–0.4% (B. bigranulata, B. ollantaytamboensis); ITS 1: 0.5 % (B. bigranulata), 0.1 – 1.0 % (B. charrua); ITS2: 0.2% (B. bigranulata), 0.2–0.4% (B.ollantaytamboensis); COI: 0.1–2.3% (B. bigranulata) and 0.7% (B. charrua). Interspecific p -distances in the analysed dataset are as follows: • 18S rRNA: 0.0–2.5% (1.2% on average), with the most similar being B. charrua (MZ820796) and B. danieli (MZ820800); and the least similar being B. madonnae (MZ820803) and B. ollantaytamboensis (MZ820804). • 28S rRNA: 0.0–4.3% (2.4% on average), with the most similar being B. charrua (MZ820814) and B. danieli (MZ820818); and the least similar being B. madonnae (MZ820821) and B. ollantaytamboensis (MZ820823). • ITS1: 0.3–10.3% (5.5% on average), with the most similar being B. charrua (MZ820833) and B. danieli (MZ820836); and the least similar being B. madonnae (MZ820839) and B. bigranulata (MZ820828). • ITS2: 1.6–10.8% (8.1% on average), with the most similar being B. charrua (MZ822380) and B. danieli (MZ822384); and the least similar being B. danieli + B. madonnae (MZ822384, MZ822387) and B. ollantaytamboensis (MZ822388 –91). • COI: 9.1–20.4% (16.0% on average), with the most similar being B. charrua (MZ820850) and B. danieli (MZ820853); and the least similar being B. ollantaytamboensis (MZ820855) and B. weglarskae (MZ820860). MORPHOLOGICAL EVOLUTION Mapping morphological traits on to the phylogeny suggests that the ancestor of Barbaria was most probably covered with uniform dorsal sculpturing comprising both pillars and pores (Fig. 6A) and welldelimited pedal plates on legs I–III with densely packed pillars and pseudopores (Fig. 6B). Its papillae on legs IV were elongated (Fig. 6C), meaning that they were much longer than wide. Internal claws were exhibiting heteronychy or lacking primary spurs (Fig. 6D). In other words, among the extant species, B. bigranulata is morphologically overall the most similar to the last common ancestor of the analysed species., Published as part of Gąsiorek, Piotr, Wilamowski, Andrzej, Vončina, Katarzyna & Michalczyk, Łukasz, 2022, Neotropical jewels in the moss: biodiversity, distribution and evolution of the genus Barbaria (Heterotardigrada: Echiniscidae), pp. 1037-1066 in Zoological Journal of the Linnean Society 195 on pages 1048-1054, DOI: 10.1093/zoolinnean/zlab087, http://zenodo.org/record/6993173, {"references":["Michalczyk L, Kaczmarek L. 2007. Echiniscus ganczareki, a new species of Tardigrada (Heterotardigrada: Echiniscidae, bigranulatus group) from Costa Rica. Zootaxa 1471: 15 - 25.","Jorgensen A, Mobjerg N, Kristensen RM. 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.","Velasco-Castrillon A, McInnes SJ, Schultz MB, Arroniz- Crespo M, D'Haese CA, Gibson JAE, Adams BJ, Page TJ, Austin AD, Cooper SJB, Stevens MI. 2015. Mitochondrial DNA analyses reveal widespread tardigrade diversity in Antarctica. Invertebrate Systematics 29: 578 - 590."]}

Additional file 1: SM.01. Saturation plots for COI and ITS markers.

Mesobiotus dilimanensis, new species (Tables 3, 4, Figs. 1–5) Material examined: 65 animals (including eight simplex), 47 eggs, and 7 empty chorions mounted on microscope slides in Hoyer's medium, 5 eggs fixed on SEM stubs, and 8 specimens processed for DNA sequencing. Type locality: 14°39′40″N, 121°04′07″E; 76 m asl: Philippines, Quezon City, Diliman, University of the Philippines, A. Roces St.; moss on a rock; September 2015; coll. Lowelyn Itang. Etymology: The species is named after Diliman, the district in Quezon City, Philippines, where it was discovered. Type depositories: Holotype: slide PH.006.10 with six paratypes; 58 paratypes (slides: PH.006. *, where the asterisk can be substituted by any of the following numbers: 02–03, 11–16), 47 eggs (slides: PH.006. *: 5–9, 17); seven empty chorions (slides: PH.006. *: 01, 04) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387, Kraków, Poland.

Itang, Lowelyn A. M., Stec, Daniel, Mapalo, Marc A., Mirano-Bascos, Denise, Michalczyk, Łukasz (2020): An integrative description of Mesobiotus dilimanensis, a new tardigrade species from the Philippines (Eutardigrada: Macrobiotidae: furciger group). Raffles Bulletin of Zoology 68: 19-31, DOI: 10.26107/RBZ-2020-0003