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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."]}

Animals that colonize soil show specific adaptations to soil. Compared to closely related species living on the surface, the limbs of soil-dwelling animals are often shortened, reduced, or absent to allow a less restricted passage through cavities between soil particles. This pattern of limb reduction has also been observed in tardigrades, where multiple lineages that colonized the below-ground habitat show independent reduction and/or loss of legs and claws. In the tardigrade superfamily Macrobiotoidea, leg and claw reductions are a common trait found in the Macrobiotus pseudohufelandi complex. This rarely found species complex currently contains four nominal taxa. Here we describe, with the use of integrative taxonomy, Macrobiotus naginae sp. nov., a new species in the Macrobiotus pseudohufelandi complex from inland sand dunes in Finland. We also provide a dichotomous key to the Macrobiotus pseudohufelandi complex to assist with their identification in future studies. peerReviewed

Macrobiotus sottilei Pilato, Kiosya, Lisi & Sabella, 2012 Material examined. 102 animals, and 47 eggs. Specimens mounted on microscope slides in Hoyer’s medium (89 animals + 37 eggs), fixed on SEM stubs (10+10), and processed for DNA sequencing (3+0). Population locality. 54°4’55.37”N, 15°0’53.78”E, 15 asl: Poland, Rewal; mixed sample of lichen and moss collected from bark on a cherry tree in an orchard on the Polish coast; coll. Daniel Stec and Krzysztof Miler. Slides and SEM stub depository. 89 animals (slides: PL.352.*, where the asterisk can be substituted by any of the following numbers 03–08) and 37 eggs (slides: PL.352.*: 01–02, 09–10) as well as the SEM stub (code 18.13) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30- 387, Kraków, Poland. Morphology. Animals (measurements and statistics in Table 3). Body transparent in juveniles and creamywhite to slightly yellowish in adults but transparent after fixation in Hoyer’s medium (Fig. 1A). Eyes present in live animals and visible also after fixation. Round to subrounded pores (0.6–1.2 μm in diameter), clearly visible under both PCM and SEM, scattered randomly over the entire body cuticle (Fig. 1 B–С). Granulation on all legs present (Fig. 2 A–F). A patch of clearly visible granulation is present only on the external surface of legs I–III (Fig. 2 A–B). Granulation on legs IV is always clearly visible and consists of a single large, granulated patch on each leg (Fig. 2 E–F). A cuticular bulge/fold (pulvinus) is present on the internal surface of legs I–III (Fig. 2 C–D). Claws Y-shaped, of the hufelandi type. Primary branches with distinct accessory points, a common tract, and an evident stalk connecting the claw to the lunula (Fig. 3 A–F). Lunulae I–III are smooth (Fig. 3A, E), whereas lunulae IV are dentate (Fig. 3 C–D, F) although sometimes the dentation is only faintly visible (Fig. 3B). A divided cuticular bar is situated between the claws and the muscle attachments on legs I–III and is only faintly visible under PCM (Fig. 3A; see also Discussion section for more information on this character). A horseshoe-shaped structure connects anterior and posterior lunules (Fig. 3B). Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Fig. 4A), with ventral lamina and ten small peribuccal lamellae (Fig. 5 A–B). Under PCM, the oral cavity armature is of the patagonicus type, i.e., with only the second and third bands of teeth visible (Fig. 4 B–C). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of 1–2 rows of small, conical teeth (Fig. 5 A–B). The second band of teeth is situated between the ring fold and third band of teeth and comprises 2–3 rows of small cones, slightly larger than those of the first band (Fig. 5 A–B), which under PCM are visible as dark dots (Fig. 4 B–C). The third band of teeth is discontinuous and divided into a dorsal and a ventral portion. Under both PCM and SEM, the dorsal teeth of the third band are fused and form a single transverse ridge (Fig. 4B, 5A), whereas the ventral teeth appear as three separate transverse ridges with the median tooth being positioned more anteriorly compared to the lateral teeth (Fig. 4C, 5B). In SEM the margins of these ridges are serrated (Fig. 5 A–B). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a drop-shaped microplac-oid (Fig. 4A). The macroplacoid length sequence is 2 Eggs (measurements and statistics in Table 4). Laid freely, creamy-white to yellowish and spherical (Figs 6 A–D and 7A). The surface between processes is of the hufelandi type, i.e., chorion surface covered by an evident reticulum with several rows of pores between processes (Figs 6 A–B and 7A–F). Under PCM the reticulation appears uniform over the entire surface (Fig. 6 A–B,); however, in SEM it is evident that the shape and size of the mesh is highly variable, with the mesh diameter ranging from 0.2 to 0.8μm (Fig. 7 B–F). The pillars connecting the reticulum with the chorion surface are visible only in SEM (Fig. 7 C–D). The bases of the processes are surrounded by cuticular thickenings that merge into the bars and nodes of the reticulum (Fig. 7B). These basal thickenings appear in PCM as short dark projections around the process bases (Fig. 6 A–B). Processes are in the shape of inverted goblets with slightly concave conical trunks and well-defined terminal discs (Figs 6 E–G and 7C–D). Margins of the terminal discs are strongly serrated to dentated, with a concave central area (Figs 6 C–G and 7B–F). Under SEM, the teeth of the terminal discs appear to be covered by microgranulation (Fig.7 C–F). Reproduction / Sexual dimorphism. This species is dioecious: both males with testes and females with ovaries were recorded within the same population. Males exhibited a secondary sexual dimorphic trait in the form of poorly developed lateral gibbosities on legs IV (Fig. 8 A–B). DNA sequences obtained in this study. We obtained sequences for all four of the above-mentioned molecular markers from each of the six individuals destined for DNA extraction and sequencing in this study. Sequences of each marker were represented by a single haplotype in each species as follows: Macrobiotus sottilei: 18S rRNA (GenBank: MW 247178), 1011 bp long; 28S rRNA (MW 247175), 785 bp long; ITS-2 (MW 247179), 379 bp long; COI (MW 246133), 658 bp long. Macrobiotus glebkai: 18S rRNA (GenBank: MW 247177), 1012 bp long; 28S rRNA (MW 247176), 719 bp long; ITS-2 (MW 247180), 400 bp long; COI (MW 246134), 620 bp long. Phylogenetic position of the studied species. The bayesian reconstruction produced a well-supported tree (Fig. 9). The same three Macrobiotus clades (A, B and C) from Stec et al. (2020e) were recovered with high support. Macrobiotus glebkai belongs to clade A, without any clear close affinity to any of the other species in the clade, whereas Macrobiotus sottilei instead belongs to clade C and is closely related to Macrobiotus sapiens Binda & Pilato, 1984.

The honeybee continues to be developed as a model species in many research areas, including studies related to the effects of alcohol. Here, we investigate whether workers display one of the key features of alcoholism, namely withdrawal symptoms. We show that workers fed for a prolonged time on food spiked with ethanol, after discontinuation of access to such food, exhibited a marked increase in the consumption of ethanol and a slight increase in mortality. We additionally show that withdrawal symptoms do not include an increase in appetitiveness of ethanol diluted in water. Our results demonstrate that workers can develop alcohol dependence, which might be especially important in the natural setting of repeated exposure to ethanol in floral nectar and for their potential as a model of alcohol addiction.

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

Tenuibiotus zandrae sp. nov. urn:lsid:zoobank.org:act: 99D6163D-E8E6-4223-B975-F68AA2268304 Figs 10���19 Etymology We take great pleasure in dedicating this new species to the friend of the first and third authors. Zandra Maria Skandrup Sigvardt, who recently completed her PhD studies working on crustaceans (Section of Biosystematics) at the Natural History Museum of Denmark in Copenhagen. Material examined 62 animals (including 18 simplex) and 171 eggs. Specimens mounted on microscope slides in Hoyer���s medium (49 animals + 161 eggs), fixed on SEM stubs (including two extracted buccal apparatuses) (10 +10), and processed for DNA sequencing (3 +0). Holotype GREENLAND ��� Disko Island, ��sterlien; 69��15���17��� N, 53��30���46��� W; 30 m a.s.l.; sample of moss collected from the rock in arctic tundra; IZiBB, slide GL.011.17. Paratypes GREENLAND ��� 58 paratypes; same collection data as for holotype; IZiBB, slides GL.011.08 to 011.09, 011.16 to 011.19, 011.22 to 011.23, 011.27 to 011.28, SEM stubs GL.012.06 to 012.07, 012.13 ��� 171 eggs; same collection data as for holotype; IZiBB, slides GL.011.10 to 011.15, 011.20 to 011.21, 011.24 to 011.26, 011.29, SEM stub GL.12.13. Description Animals (measurements and statistics in Table 4) Body transparent in juveniles and whitish in adults, after fixation in Hoyer���s medium transparent (Fig. 10). Eyes present in specimens mounted in Hoyer���s medium. Body cuticle without pores but covered with fine granulation including ventral side of the body and all legs (Figs 11���13). Granulation is distributed uniformly on the body (Fig. 11 A���B, E) but sometimes, especially in larger specimens, random patches without granulation are present on the body cuticle (Fig. 11 C���D, F). Patches of dense granulation composed of cushions with aggregated granules present on all legs (Figs 12���13). A patch of clearly visible granulation, is present on the external surface of legs I���III just below the claws (Figs 12A, 13A). A pulvinus is absent on the internal surface of legs I���III, whereas a patch of dense granulation is present and wider than the patch on the external leg surface (Figs 12B, 13B). A patch of dense granulation on legs IV is always visible and covers the dorsal and the lateral sides of hind legs (Figs 12C, 13 C���D). Claws slender, of the Tenuibiotus type (Fig. 14). Primary branches with distinct accessory points, a long common tract, and with an evident stalk connecting the claw to the very wide lunula (Fig. 14). Lunulae I���III smooth (Fig. 14A, C), whereas lunulae IV exhibit clear dentation (Fig. 14B, D). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM (Fig. 14B). Mouth antero-ventral, followed by ten peribuccal lamellae (Figs 15A, 16). Bucco-pharyngeal apparatus of the Macrobiotus type (Figs 15A, 17A). Under LCM, only the second and third bands of teeth visible, with the second band being faintly marked (Fig. 15 B���C). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of 1���2 rows of small, cone-shaped teeth arranged around the oral cavity (Figs 16, 17B). The second band of teeth is situated between the ring fold and the third band of teeth and comprises 3���6 rows of small, cone-shaped teeth (Figs 15 B���C, 16). 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 15 B���C,16). The third band of teeth is discontinuous and divided into dorsal and ventral portions. Under LCM, the dorsal teeth are seen as three distinct transversal ridges of which the median tooth is triangular and is wedged between the lateral teeth (Fig. 15B). The ventral teeth under LCM appear as three to four separate roundish teeth, largest than those of the second band (Fig. 15C), only sometimes they can be seen as one faintly marked, elongated tooth. In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 16). Under SEM, the medio-dorsal tooth is the largest within the third band and is positioned anteriorly with respect to the lateral teeth (Fig. 16A), whereas the ventral portion consist of cone-shaped teeth of which the lateral ones are larger than the medial ones (Fig. 16B). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small triangular microplacoid (Fig. 15A, D���E). The macroplacoid length sequence is 2Eggs (measurements and statistics in Table 5) Laid freely, whitish, spherical or ovoid (Figs 18 A���B, 19A). The surface between processes is smooth, with thickenings/striae often radiating from the processes bases (Figs 18 B���D, 19B���C, E���F). Under PCM, these thickenings together with labyrinthine layer within chorion are visible as dark dots and lines on the surface between processes, whereas under SEM they are smooth striae coming out of the process bases (Figs 18 B���D and 19B���C, E���F, respectively). Under SEM, the surface between processes and between the peribasal striae is covered with micropores (Fig. 19 E���F). Processes are of conical shape, with elongated apices which are sometimes bi- or trifurcated (Figs 18 E���H, 19A���D). The labyrinthine layer between the process walls is clearly visible under LCM as a reticular pattern with sinuous margins (Fig. 18 C���D). The elongated meshes decrease in size from the base to the top of the processes (Fig. 18 C���D). Under SEM, the surface of the processes is covered with small tubercles, whereas the surface of the elongated apices is smooth (Fig. 18 B���E). Reproduction The examination of specimens freshly mounted in Hoyer���s medium did not revealed any spermathecae or testes filled with spermatozoa. Also, male secondary sexual dimorphism traits such as lateral gibbosities on legs IV were absent. Thus, reproductive mode could not be unambiguously determined. DNA sequences We obtained sequences of good quality for all four of the above-mentioned DNA markers. Sequences of each marker were represented by single haplotypes: 18S rRNA sequence (GenBank: MN443040), 1035 bp long; 28S rRNA sequence (GenBank: MN443035), 780 bp long; ITS-2 sequence (GenBank: MN443038), 439 bp long; COI sequence (GenBank: MN444827), 658 bp long. Morphological observations of comparative material Amended description of Tenuibiotus voronkovi The examination of the holotype and paratype of T. voronkovi under LCM revealed the presence of granulation on the body cuticle. Faint granulation with small and uniformly sized granules is regularly arranged and covers the dorso-medial region of the body, from its cephalic to the caudal end (Fig. 20A). On the dorso-lateral surface of the body, granulation is unevenly distributed and resembles patches of granules, within which granule size gradually increases in the dorsal to lateral direction (Fig. 20B). The granulation is absent or not visible in LCM on the ventral side of the body. Similarly, the granulation is absent on the legs except a typical dense granulation patch on the external and internal surface of the legs near the claws. However, note that this observation was made on two different specimens (not very well oriented/positioned and stretched on the slide) thus, the certain distribution pattern of body granulation requires a further examination when a new population of T. voronkovi becomes available. As in the original description, the eggs of T. voronkovi have small conical processes with elongated and flexible apices which are often folded and not clearly visible or even broken. (Figs 21A, C���J, 22). The process walls are smooth, without any obvious thickenings or tubercles (Fig. 22) but with obvious annulation and with the labyrinthine layer within process walls, visible under LCM as roundish polygonal reticulation (Fig. 21C, E���F), on one egg being abnormally developed and visible more like pores than true reticulation (Fig. 21D). Under SEM, the surface between processes is covered with short irregular striae/ridges/wrinkles which often radiate from the process bases, with small micropores randomly scattered in between them (Fig. 22). However, under PCM the surface is visible as being covered with dark dots which are probably the thickenings of the labyrinthine layer within the chorion (Fig. 21B). The morphological analysis conducted on two populations designated as ��� T. voronkovi ��� by Zawierucha et al. (2016a), from Edge��ya and Nordaustlandet (islands within the Svalbard archipelago, Norway), showed distinct differences in cuticle morphology in comparison to the T. voronkovi type series. Specifically, animals of the Edge��ya population exhibit faint, dense and uniformly distributed granulation on the whole dorso-lateral cuticle from its cephalic to the caudal end (excluding ventral and leg cuticle) (Fig. 23A), whereas this granulation is absent or not visible under LCM in animals of the Nordaustlandet population. The morphology of egg processes in both these populations is very similar: specifically, processes are of a conical shape with elongated apices, with the labyrinthine layer between the process walls clearly visible under LCM as a reticular pattern with sinuous margins and elongated meshes decreasing in size from the base to the processes top in most cases (Figs 23 B���D, 24). Other traits are as described by Zawierucha et. al. (2016a) however, it should be noted that as for the similarly to T. voronkovi, no more conclusions can be made based on this material due to the bad condition of the slides, with bubbles of air and crystalized Hoyer which prevent further investigation and limit the number of specimens suitable for imaging., Published as part of Stec, Daniel, Tumanov, Denis V. & Kristensen, Reinhardt M��bjerg, 2020, Integrative taxonomy identifies two new tardigrade species (Eutardigrada: Macrobiotidae) from Greenland, pp. 1-40 in European Journal of Taxonomy 614 on pages 15-26, DOI: 10.5852/ejt.2020.614, http://zenodo.org/record/3710893, {"references":["Zawierucha K., Kolicka M. & Kaczmarek L. 2016 a. Re-description of the Arctic tardigrade Tenuibiotus voronkovi (Tumanov, 2007) (Eutardigrada; Macrobiotidea), with the first molecular data for the genus. Zootaxa 4196 (4): 498 - 510. https: // doi. org / 10.11646 / zootaxa. 4196.4.2"]}

Macrobiotus engbergi sp. nov. urn:lsid:zoobank.org:act: C592B357-37F6-4C92-B16A-C28EDB17A231 Figs 1���9 Etymology We take great pleasure in dedicating this new species to the friend of the third author, Lars Engberg Hansen, who is a teacher emeritus from Qeqertarsuaq and Alluitsup Paa in Greenland and is always happy to help with collecting samples of mosses and lichens for us. Material examined 112 animals (including 9 simplex) and 108 eggs. Specimens mounted on microscope slides in Hoyer���s medium (98 animals + 103 eggs), fixed on SEM stubs (10+ 5) and processed for DNA sequencing (4+ 0). Holotype GREENLAND ��� ♀; Alluitsup Paa; 60��28���1.5���N, 45��34���27.8���W; 25 m a.s.l.; mixed sample of moss and lichen collected from rock in arctic tundra; IZiBB, slide GL.052.22. Paratypes GREENLAND ��� 107 paratypes; same collection data as for holotype; IZiBB, slides GL.052.17 to 052.24, SEM stub 17.08 ��� 108 eggs; same collection data as for holotype; IZiBB, slides GL.052.09 to 052.16, SEM stub 17.08. Description Animals (measurements and statistics in Table 2) Body transparent in juveniles and whitish in adults, after fixation in Hoyer���s medium transparent (Fig. 1A). Eyes present, visible also in specimens mounted in Hoyer���s medium. Cuticle porous with two types of pores: large (up to 5.0 ��m in diameter) lenticular pores of shape resembling paper wrapped candy, with transversal wrinkles in extremities distributed randomly on entire body cuticle and being the biggest on anterior and posterior dorsal region (Figs 1 B���C, 2); and small round cuticular pores (0.3���0.7 ��m in diameter) scattered in between lenticular pores (Figs 1C, 2B). Patches of granulation on all legs present (Fig. 3). A patch of clearly visible granulation is present on the external surface of legs I���III (Fig. 3 A��� B). A pulvinus present on internal surface of legs I���III, together with a faint cuticular fold covered with faint granulation and paired muscles attachments which are present just below claws (Fig. 3 C���D). Both structures are visible only if the legs are fully extended and well oriented on slide. Granulation on legs IV always visible and consists of a single large granulation patch on each leg (Fig. 3 E���F). Claws stout, of the hufelandi type (Fig. 4). Primary branches with distinct accessory points, a common tract and with an evident stalk connecting the claw to the lunula (Fig. 4). Lunulae on all legs smooth (Fig. 4). Cuticular bars under claws are absent. Double muscle attachments are faintly marked under LCM but clearly visible under SEM (Fig. 4A, C). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM (Fig. 4B) and sometimes also on legs I���III, but in this case inverted and not connecting the external and the internal claw (Fig. 4A). Mouth antero-ventral, followed by ten peribuccal lamellae and a circular sensory lobe, surrounded by the ring of large pores (Figs 2A, 5A, 6). Bucco-pharyngeal apparatus of the Macrobiotus type (Fig. 5A). Under LCM, the oral cavity armature is of the patagonicus type, i.e., with only the 2 nd and 3 rd bands of teeth visible (Fig. 5 B���C). However, in SEM all three bands of teeth are visible, with the first band situated at the base of peribuccal lamellae and composed of a 1���2 rows of small, cone-shaped teeth arranged around the oral cavity (Fig. 6). The second band of teeth is situated between the ring fold and the third band of teeth, and comprises 3���6 rows of small cone-shaped teeth (Figs 5 B���C, 6). 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 5 B���C, 6). The third band of teeth is discontinuous and divided into dorsal and ventral portions. Under LCM, the dorsal teeth are seen as three distinct transversal ridges, whereas the ventral teeth appear as two separate lateral transverse ridges and a median roundish tooth (Fig. 5 B���C). In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 6). Under SEM, the margins of the dorsal teeth slightly serrated (Fig. 6A). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small, triangular microplacoid (Fig. 5A, D���E). The macroplacoid length sequence 2Eggs (measurements and statistics in Table 3) Laid freely, yellowish, spherical (Figs 7A, 8A). The surface between processes is of the persimilis type, i.e., with the continuous smooth chorion, never with pores or reticulum (Figs 7 F���G, 8). Under PCM labyrinthine layer is visible as dark dots/thickenings on the surface between processes, whereas under SEM the surface is smooth (Figs 7 F���G and 8, respectively). Processes are of the inverted goblet shape, with slightly concave trunks and concave terminal discs (Figs 7 B���C, 8A���C). Terminal discs are round with margins ranging from only serrated to clearly indented (Figs 7 D���E, 8). Each terminal disc has a distinct concave central area, which may contain some scattered granulation within, and micro-granulations which are always present on the margins (visible only under SEM; Fig. 8 C���D). Reproduction The new species is dioecious. Spermathecae in females as well as testis in males have been found to be filled with spermatozoa, clearly visible under LCM up to 24 hours after mounting in Hoyer���s medium. The new species exhibits a male secondary sexual dimorphism trait in the form of evident lateral gibbosities on legs IV (Fig. 9). DNA sequences We obtained sequences for all four of the above mentioned DNA markers. Sequences of 18S rRNA and 28S rRNA were represented by single haplotypes, whereas sequences of ITS-2 and COI were represented by two (distance: 0.5%) and three (distance: 1.3���1.8%) haplotypes, respectively: 18S rRNA sequence (GenBank: MN443039), 1017 bp long; 28S rRNA sequence (GenBank: MN443034), 783 bp long; ITS-2 haplotype 1 sequence (GenBank: MN443036), 374 bp long; ITS-2 haplotype 2 sequence (GenBank: MN443037), 374 bp long; COI haplotype 1 sequence (GenBank: MN444824), 638 bp long; COI haplotype 2 sequence (GenBank: MN444825), 638 bp long; COI haplotype 3 sequence (GenBank: MN444826), 638 bp long. Genus Tenuibiotus Pilato & Lisi, 2011, Published as part of Stec, Daniel, Tumanov, Denis V. & Kristensen, Reinhardt M��bjerg, 2020, Integrative taxonomy identifies two new tardigrade species (Eutardigrada: Macrobiotidae) from Greenland, pp. 1-40 in European Journal of Taxonomy 614 on pages 4-15, DOI: 10.5852/ejt.2020.614, http://zenodo.org/record/3710893, {"references":["Stec D., Zawierucha K. & Michalczyk L. 2017 a. An integrative description of Ramazzottius subanomalus (Biserov, 1985) (Tardigrada) from Poland. Zootaxa 4300 (3): 403 - 420. https: // doi. org / 10.11646 / zootaxa. 4300.3.4","Zeller C. 2010. Untersuchung der Phylogenie von Tardigraden anhand der Genabschnitte 18 S rDNA und Cytochrom c Oxidase Untereinheit 1 (COX I). MSc Thesis, Technische Hochschule Wildau, Germany.","Gasiorek P., Stec D., Zawierucha Z., Kristensen R. M. & Michalczyk L. 2018. Revision of Testechiniscus Kristensen, 1987 (Heterotardigrada: Echiniscidae) refutes the polar-temperate distribution of the genus. Zootaxa 4472 (2): 261 - 297. https: // doi. org / 10.11646 / zootaxa. 4472.2.3","Mironov S. V., Dabert J., Dabert M. 2012. A new feather mite species of the genus Proctophyllodes Robin, 1877 (Astigmata: Proctophyllodidae) from the Long-tailed Tit Aegithalos caudatus (Passeriformes: Aegithalidae): morphological description with DNA barcode data. Zootaxa 3253: 54 - 61. https: // doi. org / 10.11646 / zootaxa. 3253.1.2","Stec D., Morek W., Gasiorek P. & Michalczyk L. 2018 a. Unmasking hidden species diversity within the Ramazzottius oberhaeuseri complex, with an integrative redescription of the nominal species for the family Ramazzottiidae (Tardigrada: Eutardigrada: Parachela). Systematics and Biodiversity 16 (4): 357 - 376. https: // doi. org / 10.1080 / 14772000.2018.1424267","Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294 - 299.","Michalczyk L., Welnicz W., Frohme M. & Kaczmarek L. 2012. Redescriptions of three Milnesium Doyere, 1840 taxa (Tardigrada: Eutardigrada: Milnesiidae), including the nominal species for the genus. Zootaxa 3154: 1 - 20. https: // doi. org / 10.11646 / zootaxa. 3154.1.1"]}