Macrobiotus ariekammensis, GROENLANDICUS, 2022

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, Zoological Journal of the Linnean Society 195, pp. 1067-1099 : 1072-1076

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 <1) and a triangular small microplacoid ( Figs 8A, E , 9A, C, D ). The first macroplacoid exhibits strong central constriction, whereas the second macroplacoid is subterminally constricted ( Figs 8E , 9C, D ). Two globular protuberances are present on the ventral side of the buccal apparatus, one on the buccal crown anteriorly to the beginning of ventral lamina, second posteriorly to the ventral lamina at the level of stylet support insertion point (character visible only under SEM; Fig. 9A, B, E ).

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: MZ463662 , MZ463663 , MZ463664 . 28S rRNA: MZ463677 , MZ463678 , MZ463679 . ITS2: MZ463653 , MZ463654 , MZ463655 .

COI: MZ461005 , MZ461006 , MZ461007 .

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 ).

Figures

Figure 5. Macrobiotus ariekammensis groenlandicus subsp. nov. A, habitus, dorsoventral projection (holotype, Hoyer’s medium, PCM); B, C, well-visible granulation on the dorsal (B) and ventral (C) parts of the body seen in PCM; D, less-visible granulation on the dorsal part of the body seen in PCM; E, F, granulation on the dorsal part of the body seen in SEM; G, magnification on the cuticular pore and granulation on the dorsal part of the body in SEM. Filled flat arrowheads indicate the granules of granulation seen in SEM. Scale bars in µm.

Figure 6. Macrobiotus ariekammensis groenlandicus subsp. nov. – cuticular structures on legs: A–C, granulation on the external surface of legs III seen in PCM (A) and SEM (B, C), respectively; C, shows a magnification of leg granulation above the dense granulation patch; D–F, granulation on the internal surface of legs III seen in PCM (D) and SEM (E, F), respectively; F, shows a magnification of leg granulation above the dense granulation patch; G–I, granulation on the dorsal and dorsolateral surface of leg IV seen in PCM (G) and SEM (H, I); I, shows a magnification of leg granulation above the dense granulation patch. Filled flat arrowheads indicate the dense granulation patch on the external leg surface, empty indented arrowhead indicates the cuticular bulge (pulvini), empty flat arrowheads indicate the dense granulation patch on the internal leg surface, filled indented arrowhead indicates cuticular bar under the claws. Scale bars in µm.

Figure 7. Macrobiotus ariekammensis groenlandicus subsp. nov. – claws: A, B, claws III and IV seen in PCM, respectively; C–E, claws I, III and IV seen in SEM, respectively. Filled flat arrowheads indicate double muscles attachments under the claws, filled indented arrowhead indicates cuticular bar under the claws. Scale bars in µm.

Figure 8. Macrobiotus ariekammensis groenlandicus subsp. nov. – buccal apparatus and the oral cavity armature seen in PCM:A, dorsoventral projection of the entire buccal apparatus; B, C, oral cavity armature visible from dorsal (B) and ventral (C) view, respectively; D, oral cavity armature visible from lateral view; E, macroplacoid morphology. Filled flat arrowheads indicate a single tooth in dorsal portion of the third band of teeth in the oral cavity, empty arrows indicate dorsal cuticular spikes, empty indented arrowheads indicate central constrictions in first macroplacoids and subterminal constriction in second macroplacoid. Scale bars in µm.

Figure 9. Macrobiotus ariekammensis groenlandicus subsp. nov. – buccal apparatus and oral cavity armature seen in SEM: A, entire buccal apparatus; B, ventral view of the buccal crown; C, D, placoids morphology; E, magnification of the middle part of the buccal apparatus with stylet support insertion points; F, the oral cavity armature. Filled arrows indicate two globular protuberances on the ventral side of the buccal crown and buccal tube, empty arrows indicate dorsal cuticular spikes, empty indented arrowheads indicate central constrictions in first macroplacoid and subterminal constriction in second macroplacoid, filled flat arrowhead indicates a single tooth in dorsal portion of the third band of teeth in the oral cavity. Scale bars in µm.

Figure 10. Macrobiotus ariekammensis groenlandicus subsp. nov. – egg chorion morphology seen in PCM:A–D, egg surface under 1000× magnification; E–P, egg processes midsections under 1000× magnification. Filled flat arrowheads indicate a crown of dark thickenings and pores arranged alternately around egg process bases, filled indented arrowheads indicate light-refracting dots in the egg surface between the processes, empty flat arrowheads indicate septum between the basal and distal portion of egg process. Scale bars in µm.

Figure 11. Macrobiotus ariekammensis groenlandicus subsp. nov. – egg chorion morphology seen in SEM: A, entire egg; B, magnification of the egg surface; C, D, details of the egg processes; E, F, details of the terminal portion of egg processes. Filled flat arrowheads indicate pores within the basal portion of the process wall. Scale bars in µm.