We dedicate this species to MV friend and colleague Dr. Anne Winters, evolutionary ecologist, who collected the sample in which the new species was found.

146 animals and 56 eggs. Specimens mounted on microscope slides in Hoyer’s medium (93 animals + 38 eggs), fixed on SEM stubs (51+18), and processed for DNA sequencing (2+0).

32°21'05"N, 89°56'30"W; 106 m asl: suburban area of Jackson, Mississippi, USA; mixed leaf litter on ground; coll. December 2019 by Anne Winters.

Holotype ♀ (slide US.084.01 with 10 paratypes) and 63 paratypes (slides: US.084.*, where the asterisk can be substituted by any of the following numbers: 02–05) and 20 eggs (slides US.084.*: 06–08) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University (Gronostajowa 9, 30-387, Kraków, Poland). Additional paratypes (71 animals + 29 eggs) (slides: S207_SL*: 1–15; SEM stubs: S207_Stub*:1–4) are deposited at the Department of Biological and Environmental Sciences, University of Jyväskylä (Survontie 9C, 40500, Jyväskylä, Finland).

Animals (measurements and statistics in Table 3):

In live animals, body translucent in smaller specimens and opaque whitish in larger animals; transparent after fixation in Hoyer’s medium (Figure 1). Eyes present in live animals and after fixation in Hoyer’s medium. Small roundish cuticular pores on the dorsal and lateral cuticle, as well as on the external cuticle of all legs (0.2–0.6 μm in diameter), visible under both PCM and SEM (Figures 1B, C, 2D). On the dorsal surface, pores are absent between cuticle folds and arranged in loose belts (Figure 1C). Pores sparse on the ventral surface and visible only under SEM (Figure 8C). Patches of fine granulation, on the external surface of legs I–III as well as on the dorsal and dorso-lateral sides of legs IV, visible in PCM (Figure 2A, C) and SEM (Figure 2D). A pulvinus is present on the internal surface of legs I–III (Figure 2B, E).

Figure 1. 

Macrobiotus annewintersae sp. nov. – habitus and cuticular pores: A. Dorso-ventral view of the body (Holotype ♀;, PCM); B, C. Cuticular pores on the dorsal part of the body under PCM and under SEM, respectively. Arrowheads indicate pores and empty arrows indicate places on dorsal cuticle without pores. Scale bars in μm.

Figure 2. 

Macrobiotus annewintersae sp. nov. – cuticular structures on legs: A. External granulation on leg III under PCM; B. A cuticular bulge (pulvinus) on the internal surface of leg III under PCM; C. Granulation on leg IV under PCM; D. External granulation on leg III under SEM; E. A cuticular bulge (pulvinus) on the internal surface of leg III under SEM. Filled flat arrowheads indicate the granulation patch, empty flat arrowheads indicate pulvinus and filled indented arrowheads indicate muscle attachments. C assembled from several photos. Scale bars in μm.

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 (Figure 3). The lunulae I–III are smooth (Figure 3A, C), whereas lunulae IV are dentate (Figure 3B, D). A divided cuticular bar with double muscle attachments are poorly visible under PCM (Figure 3A).

Figure 3. 

Macrobiotus annewintersae sp. nov. – claws: A, B. Claws III and IV, respectively, under PCM; C, D. Claws III and IV, respectively, under SEM. Filled indented arrowheads indicate double muscle attachments under the claws, empty indented arrowheads indicate a faintly visible divided cuticular bar. A and B assembled from several photos. Scale bars in μm.

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Figure 4) with ventral lamina and ten peribuccal lamellae. The stylet furcae typically-shaped, the basal portion is enlarged and has two caudal branches with thickened, swollen, rounded apices. Under PCM, the oral cavity armature is of the patagonicus type, i.e., with only the second and third bands of teeth visible (Figure 4B, C). However, under SEM the first band of teeth is visible and composed of one row of very small cones situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae (Figure 5). The second band of teeth is situated between the ring fold and the third band of teeth and composed of 3–4 rows of teeth visible in PCM as granules (Figure 4B, C). The third band of teeth is divided into a dorsal (Figure 4B) and a ventral portion (Figure 4C). Under PCM, the dorsal teeth are seen as three distinct transverse ridges whereas the ventral teeth appear as two separate lateral transverse ridges between which one big tooth (sometimes circular in PCM) is visible (Figure 4B, C).

Figure 4. 

Macrobiotus annewintersae sp. nov. – buccal apparatus and the oral cavity armature under PCM: A. Dorso-ventral view of the entire buccal apparatus; B, C. Oral cavity armature in dorsal and ventral view, respectively; D, E. Placoid morphology in dorsal and ventral view, respectively. Empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity, and empty indented arrowheads indicate central constriction in the first macroplacoid and subterminal constriction in the second macroplacoid. A, D and E assembled from several photos. Scale bars in μm.

Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a drop-shaped microplacoid (Figure 4A, D, E). The macroplacoid length sequence is 2<1. The first and the second macroplacoid have a central and a subterminal constriction, respectively (Figure 4D, E).

Figure 5. 

Macrobiotus annewintersae sp. nov. – anterior view of the mouth opening under SEM. Filled flat arrowhead indicates the first band of teeth. Scale bar in μm.

Eggs (measurements and statistics in Table 4):

The surface between processes is of the persimilis type, i.e., with a continuous smooth chorion, never with pores or reticulum (Figures 6, 7). Under PCM the surface between the processes is covered with wrinkles that appear as dark thickenings/striae, whereas under SEM the surface appears clearly wrinkled (Figures 6, 7). Processes are of a modified hufelandi type (Figures 6, 7). The proper terminal disc is absent and instead 2–8 thick tentacular arms (typically 5–6) are present in the distal part of the process (Figures 6, 7). The tentacular arms present bubble-like structures (visible in PCM). Under SEM, each tentacular arm is distally divided into many irregular digitations that are sometime covered with micro-granulation (Figure 7C–F). Also, under SEM micro-pores can be seen on the egg surface between the processes and around the process bases (Figure 7C, E).

Figure 6. 

Macrobiotus annewintersae sp. nov. – egg chorion morphology under PCM: A, B. Egg surface; C, D. Midsection of the processes. Filled flat arrowheads indicate bubble-like structures within tentacular arms in the distal portion of the egg processes and empty flat arrowheads indicate dark thickenings/striae on the egg surface between processes. Scale bars in μm.

Figure 7. 

Macrobiotus annewintersae sp. nov. – egg chorion morphology under SEM: A, B. Entire egg; C–E. Details of the egg processes and egg surface between them; F. Details of the tentacular arms in the distal portion of each egg process. Filled indented arrowheads indicate micropores and empty indented arrowheads indicate lobes in tentacular arms covered by micro-granulation. Scale bars in μm.

Reproduction / Sexual dimorphism. The species is dioecious. Spermathecae in females as well as testis in males, clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium, have been found to be filled with spermatozoa (Figure 8A, B). The species exhibits secondary sexual dimorphism in the form of clearly visible lateral gibbosities on the hind legs in males (Figure 8B, C).

Figure 8. 

Macrobiotus annewintersae sp. nov. – reproduction: A. Female under PCM; B. Male under PCM; C. Male under SEM. Filled indented arrowhead indicates spermathecae filled with spermatozoa, empty indented arrowhead indicates male’s testis, arrows indicate lateral gibbosities on legs IV and filled flat arrowhead indicates cuticular pore on the ventral side of the body. Scale bars in μm.

18S rRNA: GenBank: MW588024–MW588025; 659 and 664 bp long.

28S rRNA: GenBank: MW588030–MW588031; 679 and 703 bp long.

ITS-2: GenBank: MW588018–MW588019; 298 bp long.

COI: GenBank: MW593927–MW593928; 532 and 535 bp long.

Phenotypic differential diagnosis. By having an egg chorion of the persimilis type (smooth or wrinkled chorion) and by having thick tentacular arms instead of a proper terminal disc on the distal part of egg processes, M. annewintersae sp. nov. resembles only one species: Macrobiotus anemone Meyer, Domingue & Hinton, 2014 from USA. However, the new species differs specifically from:

1. • M. anemone by having eyes (absent in M. anemone), by the presence of granulation on all legs (absent in M. anemone), by having the oral cavity armature (OCA) of the patagonicus type (maculatus type – only the third band of teeth visible under light microscope – in M. anemone), by the presence of dentate lunulae in legs IV (smooth lunulae in legs IV in M. anemone), by having the thick tentacular arms in the distal part of the processes filled with bubble-like structures (tentacular arms solid in M. anemone, Figure 17) and by lacking a cavity between the process trunk and tentacular arms that appears in PCM as a clearly refracting dot (the cavity present in M. anemone, Figure 17).

We dedicate this species to the singer, composer, musician, actor and the 2009 Eurovision Song Contest winner, Alexander Rybak.

173 animals and 37 eggs. Specimens mounted on microscope slides in Hoyer’s medium (156 animals + 32 eggs), fixed on SEM stubs (15+5), and processed for DNA sequencing (2+0).

35°15'00"N, 23°49'28"E; 30 m asl: Omalos, Crete, Greece; moss on rock in a xeric shrubland; coll. June 2015 by Małgorzata Mitan and Małgorzata Osielczak.

Holotype ♂ (slide GR.011.11 with 11 paratypes) and 160 paratypes (slides: GR.011.*, where the asterisk can be substituted by any of the following numbers: 02–08, 10–13, 15–16; SEM stub: 18.10) and 37 eggs (slides GR.011.*: 01, 09, 14; SEM stub: 18.10) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University (Gronostajowa 9, 30-387, Kraków, Poland).

Animals (measurements and statistics in Table 5):

In live animals, body translucent in smaller specimens and opaque whitish in larger animals; transparent after fixation in Hoyer’s medium (Figure 9A). Eyes present in live animals and after fixation in Hoyer’s medium. Elliptical cuticular pores (0.6–1.5 μm in length) present all over the body and clearly visible under both PCM and SEM (Figures 9B–D, 10). Patches of fine granulation on the external surface of legs I–III as well as on the dorsal and dorso-lateral sides of legs IV clearly visible under both PCM and SEM (Figure 10A, B, E, F). A pulvinus is present on the internal surface of legs I–III (Figure 10C, D).

Figure 9. 

Macrobiotus rybaki sp. nov. – habitus and cuticular pores: A. Dorso-ventral view of the body (Holotype ♂; Hoyer’s medium, PCM); B. Cuticular pores on the dorsal part of the body under SEM; C, D. Cuticular pores on the dorsal and ventral part of the body under PCM, respectively. Filled arrows indicate lateral gibbosities. Arrowheads indicate elliptical pores. Scale bars in μm.

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 (Figure 11). The lunulae I–III are smooth (Figure 11A, D, E), whereas lunulae IV are dentate (Figure 11B, C, F). A divided cuticular bar and doubled muscle attachments are visible under PCM (Figures 10C, D, 11A, D, E).

Figure 10. 

Macrobiotus rybaki sp. nov. – cuticular structures on legs: A, B. External granulation on leg III and II under PCM and SEM, respectively; C, D. A cuticular bulge (pulvinus) on the internal surface of legs III under PCM and SEM, respectively; E, F. Granulation on legs IV under PCM and SEM, respectively. Filled flat arrowheads indicate the granulation patch, empty flat arrowheads indicate pulvinus and filled indented arrowheads indicate muscle attachments. A and E assembled from several photos. Scale bars in μm.

Figure 11. 

Macrobiotus rybaki sp. nov. – claws: A, B. Claws III and IV, respectively, under PCM; C. Magnification of lunulae IV of a different specimen; D–F. Claws II, III and IV respectively, under SEM. Filled indented arrowheads indicate double muscle attachments under the claws, empty indented arrowheads indicate a divided cuticular bar. A and B assembled from several photos. Scale bars in μm.

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Figure 12), with ventral lamina and ten peribuccal lamellae (Figure 13A). The stylet furcae typically-shaped, the basal portion is enlarged and has two caudal branches with thickened, swollen, rounded apices. Under PCM, the oral cavity armature is of the patagonicus type, i.e., with only the second and third bands of teeth visible (Figure 12B, C). However, under SEM the first band of teeth is visible as a row of irregularly distributed small teeth situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae (Figure 13A, B). The second band of teeth is situated between the ring fold and the third band of teeth and comprised of 3–4 rows of teeth faintly visible in PCM (Figure 12B, C) and visible as cones in SEM (Figure 13A). Teeth of the second band are larger than those in the first band. 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 (Figures 12B, C, 13A, B). The third band of teeth is divided into a dorsal and the ventral portion. Under both PCM and SEM, the dorsal teeth are seen as three distinct transverse ridges (Figures 12B, 13A). The ventral teeth appear as two separate lateral transverse ridges between which one conical medial tooth (roundish in PCM) is visible (Figures 12C, 13B). Lateral cribrose area present in the buccal tube behind the third band of teeth (Figure 13B). Pharyngeal bulb spherical, with triangular apophyses, three anterior cuticular spikes (typically only two are visible in any given plane), two rod-shaped macroplacoids and a drop-shaped microplacoid (Figures 12A, D, E). The macroplacoid length sequence is 2<1. The first macroplacoid has a weak central constriction, whereas the second is weakly constricted only subterminally (Figures 12D, E).

Figure 12. 

Macrobiotus rybaki sp. nov. – buccal apparatus and the oral cavity armature under PCM: A. Dorso-ventral view of the entire buccal apparatus; B, C. Oral cavity armature in dorsal and ventral view, respectively; D, E. Placoid morphology in dorsal and ventral view, respectively. Empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity, empty indented arrowheads indicate central constriction in the first macroplacoid and subterminal constriction in the second macroplacoid and arrows indicate cuticular spikes between end of the buccal tube and anterior portion of the bulbus. A, D, E assembled from several photos. Scale bars in μm.

Eggs (measurements and statistics in Table 6):

Figure 13. 

Macrobiotus rybaki sp. nov. – anterior view of the oral cavity armature under SEM: A, B. Dorsal and ventral view, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowhead indicates the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity. Scale bars in μm.

The surface between processes is of the hufelandi type, i.e., covered with a reticulum (Figures 14A, B, 15A–E). Peribasal meshes of slightly larger diameter compared to interbasal meshes (Figures 14A, B, 15A–D). Typically, the reticulation between neighbouring processes is composed of two rows of peribasal meshes and with a third row of smaller mashes interposed (the third row sometimes missing) (Figures 14A, B, 15A–D). Mesh diameter is usually larger than the mesh walls and nodes (Figures 14A, B, 15A–D). The meshes are 0.4–1.4 μm in diameter, with roundish irregular shape. The pillars connecting the reticulum with the chorion surface are visible only under SEM (Figure 15C). The bases of the processes are surrounded by cuticular thickenings that merge into the bars and nodes of the reticulum (Figure 15C, D). These basal thickenings appear under PCM as short dark projections around the process bases (Figure 14A, B). Processes are of the hufelandi type with very elongated concave trunk and extremely reduced (narrow), round and convex terminal discs with irregularly jagged edges (Figures 14C–F, 15). Under SEM the surface of the convex terminal discs is covered by small irregular granules and tubercles (Figures 15C–F).

Figure 14. 

Macrobiotus rybaki sp. nov. – egg chorion morphology under PCM: A, B. Egg surface; C–F. Midsection of the processes. Filled flat arrowheads indicate cuticular thickenings around the processes base that merge into the bars and nodes of the reticulum. Scale bars in μm.

Figure 15. 

Macrobiotus rybaki sp. nov. – egg chorion morphology under SEM: A, B. Entire egg; C–E. Details of the egg processes and egg surface between them; F. Details of the reduced terminal disc. Filled flat arrowheads indicate cuticular thickenings around the processes base that merge into the bars and nodes of the reticulum. Scale bars in μm.

Reproduction / Sexual dimorphism. The species is dioecious. Testis in males, which were clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium, have been found to be filled with spermatozoa, (Figure 16). In females spermathecae filled with spermatozoa were not observed. The species exhibits secondary sexual dimorphism in the form of small lateral gibbosities on the hind legs of males (Figure 16).

Figure 16. 

Macrobiotus rybaki sp. nov. – reproduction: male under PCM. Empty indented arrowhead indicates male’s testis and arrows indicate lateral gibbosities on legs IV. Scale bar in μm.

18S rRNA: GenBank: MW588028–MW588029; 1018 bp long.

28S rRNA: GenBank: MW588034–MW588035; 783 bp long.

ITS-2: GenBank: MW588022–MW588023; 391 bp long.

COI: GenBank: MW593931–MW593932; 658 bp long.

Phenotypic differential diagnosis. By having the OCA of the patagonicus type (only the 2^nd and 3^rd bands of teeth visible under light microscopy), egg chorion of the hufelandi type (covered with a reticulum), and egg processes with reduced (narrow) terminal disc, Macrobiotus rybaki sp. nov. is most similar to four species: Macrobiotus dariae Pilato & Bertolani, 2004, Macrobiotus noemiae Roszkowska & Kaczmarek, 2019, Macrobiotus santoroi Pilato & D’Urso, 1976 and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996. The new species differs specifically from:

Figure 17. 

Macrobiotus anemone Meyer, Domingue & Hinton, 2014 (type series) – egg chorion morphology under PCM: A, B. Egg surface (slides 9551 and 9552 respectively). Filled flat arrowheads indicate a cavity between the process trunk and tentacular arms that appears in PCM as a clearly refracted dot. Scale bars in μm.

1. • M. dariae by having a more anteriorly placed stylet support insertion point (pt 73–75.5 in the new species vs. 77.2–77.9 in M. dariae), a narrower buccal tube external diameter (pt 12.3–15.6 in the new species vs. 15.6–25.7 in M. dariae), a smaller number of processes on the egg circumference (25–34 in the new species vs. 34–38 in M. dariae), a different egg process morphology (processes with very elongated concave trunks and extremely reduced – narrow – convex terminal discs in the new species vs. conical processes with flexible distal portion without terminal discs in M. dariae; Figure 18A–C).
2. • M. noemiae by having a more anterior stylet support insertion point (pt 73.0–75.5 in the new species vs. 78.3–81.8 in M. noemiae), by a smaller number of processes on the egg circumference (25–34 in the new species vs. 35–36 in M. noemiae), by well-defined reticulation on the chorion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than the walls and nodes of the reticulum (very delicate and faint reticulation with mesh of uniform size distributed randomly on the egg surface between the processes in M. noemiae), a different egg processes morphology (processes with very elongated concave trunks and extremely reduced – narrow – convex terminal discs without flexible filaments in the new species vs. conical processes without terminal discs but with hair-like, and flexible filaments in M. noemiae).
3. • M. santoroi by having taller egg processes (6.7–13.4 µm in the new species vs. 4 µm or less in M. santoroi), by a smaller number of processes on the egg circumference (25–34 in the new species vs. 37–40 in M. santoroi), by processes with very elongated concave trunks (processes peg-shaped in M. santoroi), by well-defined reticulation on the chorion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than walls and nodes of the reticulum (very fine mesh with evident and wide walls and nodes, giving the false impression of a granulated surface in M. santoroi).
4. • M. serratus by having a more anterior stylet support insertion (pt 73.0–75.5 in the new species vs. 75.6–77.7 in M. serratus), by a taller egg process height (6.7–13.4 µm in the new species vs. 5.5–6.0 µm in M. serratus) and by well-defined reticulation on the chorion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than walls and nodes of the reticulum (very delicate and faint reticulation with mesh of similar sizes distributed uniformly on the egg surface between processes in M. serratus; Figure 18D, E).

The phylogenetic reconstruction (Figure 19) recovered the genus Macrobiotus as well as the three clades found by Stec et al. (2021) and by Kiosya et al. (2021) to be monophyletic. All three clades have high support values (pp=1). The new species Macrobiotus annewintersae sp. nov. belongs to subclade B, within the Macrobiotus persimilis complex, even though the monophyly of this complex was not strongly supported (pp=0.73). Macrobiotus engbergi Stec, Tumanov & Kristensen, 2020 was recovered as the closest relative of M. annewintersae sp. nov. (Figure 19). The second species analysed in this study, Macrobiotus rybaki sp. nov., belongs to subclade A with its closest relatives being Macrobiotus wandae Kayastha, Berdi, Miaduchowska, Gawlak, Łukasiewicz, Gołdyn & Kaczmarek, 2020 and Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011 (Figure 19). The newly found Swedish population identified in this study as Macrobiotus aff. polonicus, as could have been predicted from its morphological similarity with that species, clusters together with two populations of Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003 from Austria and Slovakia (Figure 19).

Figure 18. 

Macrobiotus dariae Pilato & Bertolani, 2004 and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996 (type series) – egg chorion morphology under PCM: A–C. Egg surface (A) and midsections of the processes (B, C) of M. dariae (slides PC45s1 and PC45s3 respectively); D, E. Egg surface of M. serratus (slides C1907s17 and C1907s30 respectively). Scale bars in μm.

Figure 19. 

Phylogenetic reconstruction of the genus Macrobiotus, topology of BI analysis. Nodes with pp<70 were collapsed. Clades A–C from Stec et al. (2021) are indicated. * indicates nodes with support pp=1. Numbers after species names (when present) indicate different haplotypes or individuals from the same population. Outgroups not shown. Country abbreviations after species names (when present) indicate different populations (AT: Austria; SE: Sweden; SK: Slovakia).