Macrobiotus rybaki Stec & Vecchi, 2021

Vecchi, Matteo & Stec, Daniel, 2021, Integrative descriptions of two new Macrobiotus species (Tardigrada, Eutardigrada, Macrobiotidae) from Mississippi (USA) and Crete (Greece), Zoosystematics and Evolution 97 (1), pp. 281-306 : 281

Macrobiotus rybaki Stec & Vecchi sp. nov. Tables 5 , 6, Figures 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16, SM.02

Etymology.

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

Material examined.

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).

Type locality.

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.

Type depositories.

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).

Description of the new species.

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 ).

Claws Y-shaped, of the Macrobiotus 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 ).

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 ).

Eggs (measurements and statistics in Table 6 ):

The surface between processes is of the Macrobiotus 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 Macrobiotus 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 ).

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 ).

DNA sequences.

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 2nd and 3rd bands of teeth visible under light microscopy), egg chorion of the Macrobiotus 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:

• 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).

• 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).

• 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).

• 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).

Phylogenetic analysis.

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 ).

Figures

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.

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.

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.

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.

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.

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.

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).