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This work presents two new Mesobiotus species from the Republic of South Africa, formally described using integrative analyses. Specimens of the new species are examined in terms of morphology and morphometry under a contrast phase light microscope (PCM) and scanning electron microscope (SEM). For both new species, genetic data in the form of DNA sequences of commonly used molecular markers are also provided (18S rRNA, 28S rRNA, COI, ITS-2). Furthermore, such genetic data are also provided for the first time for Mesobiotus peterseni (Maucci, 1991) from Greenland. The study also presents a multilocus molecular phylogeny of the genus and an elaborated discussion on the taxa groupings and species composition. This results in the ratification of three informal morpho-groups in order to ease and improve communication in further taxonomic studies on the genus. Finally, an updated key to all valid nominal Mesobiotus taxa (71 species) is provided to enhance species identification in this morphologically diverse group of limno-terrestrial tardigrades.

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."]}