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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.

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 2hufelandi 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., Published as part of Kiosya, Yevgen, Pogwizd, Justyna, Matsko, Yelyzaveta, Vecchi, Matteo & Stec, Daniel, 2021, Phylogenetic position of two Macrobiotus species with a revisional note on Macrobiotus sottilei Pilato, Kiosya, Lisi & Sabella, 2012 (Tardigrada: Eutardigrada Macrobiotidae), pp. 113-135 in Zootaxa 4933 (1) on pages 118-127, DOI: 10.11646/zootaxa.4933.1.5, http://zenodo.org/record/4548040, {"references":["Pilato, G., Kiosya, Y., Lisi, O. & Sabella, G. (2012) New records of Eutardigrada from Belarus with the description of three new species. Zootaxa, 3179, 39 - 60. https: // doi. org / 10.11646 / zootaxa. 3179.1.2","Stec, D., Vecchi, M., Calhim, S. & Michalczyk, L. (2020 e) New multilocus phylogeny reorganises the family Macrobiotidae (Eutardigrada) and unveils complex morphological evolution of the Macrobiotus hufelandi group. Molecular Phylogenetics & Evolution. https: // doi. org / 10.1016 / j. ympev. 2020.106987","Binda, M. G. & Pilato, G. (1984) Macrobiotus sapiens, nuova specie di Eutardigrado di Sicilia. Animalia, 11, 85 - 90."]}