Integrative taxonomy supports two new species of Macrobiotus (Tardigrada: Eutardigrada: Macrobiotidae) allowing further discussion on the genus phylogeny Author Stec, Daniel 13C435F8-25AB-47DE-B5BB-8CE788E92CF6 Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31 - 016 Kraków, Poland daniel.stec@isez.pan.krakow.pl text European Journal of Taxonomy 2024 2024-03-26 930 79 123 https://europeanjournaloftaxonomy.eu/index.php/ejt/article/download/2481/11065 journal article 292132 10.5852/ejt.2024.930.2481 7dcacff1-4cea-4dc9-8606-064d4308c9c5 2118-9773 10893800 A09EB44B-286F-439A-A970-48F09416584A Macrobiotus ovovittatus sp. nov. urn:lsid:zoobank.org:act: 9C2931D3-8EBE-47AB-A3B0-991AB28F03A2 Figs 1–8 , Tables 2–3 Etymology The species name refers to the terminal discs of the egg processes which resemble crocheted napkins. From the Latin “egg” = “ovo” and “chaplet” = “vittatus”. Material examined 32 animals, 57 eggs mounted on microscope slides in Hoyer’s medium, 15 animals and 15 eggs examined under SEM , 15 animals stained with orcein and two animals processed for DNA sequencing. Type material Holotype GREENLAND • near Zackenberg Valley ; 74°29′0.766″ N , 20°32′18.308″ W ; 77 m a.s.l. ; Jul. 2021 ; M. Kolasa leg.; mixed sample of moss and lichen collected from the rock in arctic tundra ; ISEA PAS, slide GL.001.01. Paratypes GREENLAND • 46 animals; same collection data as for the holotype; ISEA PAS , slides GL.001.01 to GL.001.03, SEM stub TAR.015 • 72 eggs ; same collection data as for the holotype; ISEA PAS , slides GL.001.04 to GL.001.09, SEM stub TAR.015 . Description Animals Body transparent in juveniles and white in adults, after fixation in Hoyer’s medium transparent ( Fig. 1A ). Eyes present. Round and oval pores (0.4–0.6 μm in diameter), scattered randomly throughout the cuticle (distributed more sparsely on the ventral side of the body) ( Figs 1B–E , 2A–B ), including the external and internal surface of all legs ( Fig. 3A–F ). Granulation is present on the entire body cuticle and clearly visible under PCM and SEM, with granulation on the ventral side of the body being less dense ( Figs 1B– E , 2A–B ). Moreover, evident dense granulation patches on the external and internal surface of all legs I–III are visible under PCM and SEM ( Fig. 3A–D ). This dense granulation is also present on the lateral and dorsal surfaces of legs IV ( Fig. 3E–F ). A pulvinus-shaped cuticular bulge is centrally present on the internal surface of all legs I–III ( Fig. 3C–D ). This structure is visible only if the legs are fully extended and well oriented. Claws small and slender, of the hufelandi type ( Fig. 4A–F ). Primary branches with distinct accessory points, a long common tract, and an evident stalk connecting the claw to the lunula ( Fig. 4A–F ). The lunulae on legs I–III are smooth ( Figs 4A, D, E ), while there is a dentation in the lunulae on legs IV ( Fig. 4B, C, F ). A single continuous cuticular bar and double muscle attachments are present above claws I–III ( Figs 3C–D , 4A, D, E ). Shadowed extensions that extend from the lunulae of the claws on legs I–III are present and visible only under PCM ( Figs 3C , 4A ). A horseshoe-shaped structure connects the anterior and posterior lunules on leg IV ( Fig. 4B ). Mouth antero-ventral. Bucco-pharyngeal apparatus of Macrobiotus type , with ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes ( Figs 5A , 6A–B ). Under PCM, the oral cavity armature is of hufelandi type – three bands of teeth are always visible ( Fig. 5B–C ). The first band of teeth is composed of numerous extremely small cones arranged in four to six rows located anteriorly in the oral cavity, at the bases of the peribuccal lamellae and just behind them ( Figs 5B–C , 6A–B ). The second band of teeth is located between the ring fold and the third band of teeth and comprises 4–6 rows of small cones, larger than those of the first band ( Figs 5B–C , 6A–B ). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the opening of the buccal tube ( Figs 5B–C , 6A–B ). The third band of teeth is discontinuous and divided into the dorsal and ventral portions. 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 a round median tooth is visible ( Fig. 5B–C ). Under SEM, both dorsal and ventral teeth are also clearly distinct ( Fig. 6A– B ). Under SEM, the margins of the dorsal teeth are clearly serrated ( Fig. 6A ) whereas the margins of the ventral teeth are less serrated ( Fig. 6B ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a large triangular microplacoid ( Fig. 5A ). The macroplacoid length sequence being 2<1. The first and the second macroplacoid are constricted centrally and subterminally, respectively ( Fig. 5D–E ). The animals’ measurements and statistics are given in Table 2 . Eggs Laid freely, white, spherical and ornamented ( Figs 7A–H , 8A–F ). The surface between processes is of intermediate state between the maculatus and the persimilis type, that is, the surface is continuous/solid and clearly wrinkled with sparse, very small and irregularly spaced pores ( Figs 7B, D, F , 8C–D ). These pores are faintly visible under PCM but clearly visible under SEM (0.3–0.5 μm in diameter; Figs 7B, D, F , 8C–D ). Under PCM the wrinkles in egg surface are visible as dark dots/comas/bars making the impression of incomplete reticulation ( Fig. 7B, D, F ). The processes are in the shape of inverted goblets with concave conical trunks and well-defined terminal discs ( Figs 7A–H , 8A–F ). Faint annulations are visible on the trunk of the process, especially in its distal portion, which is also covered by fine granulation (characters visible only under SEM; Fig. 8C–D ). A crown of gently marked thickenings is visible around the bases of the processes as darker dots under PCM ( Fig. 7B, D, F ) and as thicker wrinkles at the processes bases under SEM ( Fig. 8C–D ). The terminal discs are cog-shaped, with a concave central area and 10–18 distinct teeth ( Figs 7A–H , 8A–F ). Terminal discs under PCM are covered by multiple light-refracting dots, and as such resemble crocheted napkins ( Fig. 7A, C, E ). These light refracting dots, when viewing the egg process laterally, give the impression that the terminal discs are rough and ragged (visible under PCM; Fig. 7 B, D, F–H ). However, the terminal discs under SEM are solid without any pores or light refracting dots, their teeth, are covered by small granules (visible only under SEM) that probably serve to improve the adhesive properties of the egg processes ( Fig. 8C–F ). The measurements and statistics of eggs are given in Table 3 . Fig. 1. Macrobiotus ovovittatus sp. nov. , PCM images of habitus, body granulation and cuticular pores of the holotype (GL.001.01, ISEA PAS). A . Habitus, dorso-ventral projection. B . Granulation in the dorsal cuticle. C . Granulation in the ventral body cuticle. D . Pores in the dorsal cuticle. E . Pores in the ventral cuticle. Scale bars in μm. Fig. 2. Macrobiotus ovovittatus sp. nov. , SEM images of body granulation and cuticular pores of a paratype (ISEA PAS). A . Body granulation and pores in the dorsal cuticle. B . Body granulation and pores in the ventral cuticle. Scale bars in μm. Fig. 3. Macrobiotus ovovittatus sp. nov. , PCM ( A , C , E ) and SEM ( B , D , F ) images of dense granulation patches on legs of paratypes (ISEA PAS). A–B . Granulation on the external surface of leg III. C–D . Granulation on the internal surface of leg III and II, respectively. E–F . Granulation on the hind legs. The empty flat arrowheads indicate a single continuous cuticular bar above the claws, the filled flat arrowheads indicate a pulvinus-shaped cuticular bulge, and the filled indented arrowheads indicate shadowed extensions extending from the lunulae. Scale bars in μm. Figure 4. Macrobiotus ovovittatus sp. nov. , images of claws. A–C . PCM, holotype (GL.001.01, ISEA PAS). D–F . SEM, paratype (ISEA PAS). A . Claws II with smooth lunulae. B . Claws IV with dentate lunulae. C . Dentate lunulae. D–E . Claws II and III with smooth lunulae, respectively. F . Claws IV with dentate lunulae. The empty flat arrowheads indicate a single continuous cuticular bar above the claws, the filled indented arrowheads indicate shadowed extensions extending from the lunulae (under PCM) or the places where they should be expected (SEM), the filled flat arrowheads indicate paired muscles attachments, and the empty indented arrowheads indicate the horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm. Fig. 5. Macrobiotus ovovittatus sp. nov. , PCM images of the buccal apparatus, A–D . Holotype (GL.001.01, ISEA PAS). E . Paratype (ISEA PAS). A . An entire buccal apparatus. B–C . The oral cavity armature, dorsal and ventral teeth, respectively. D–E . Placoid morphology, dorsal and ventral placoids respectively. The filled flat arrowheads indicate the first band of teeth, the empty flat arrowheads indicate the second band of teeth, the filled indented arrowheads indicate the third band of teeth, and the empty indented arrowheads indicate central and subterminal constrictions in the first and second macroplacoid, respectively. Scale bars in μm. Table 2. Measurements [in μm] and pt values of selected morphological structures of animals of Macrobiotus ovovittatus sp. nov. ; specimens mounted in Hoyer’s medium; N: number of specimen/ structures measured; range: refers to the smallest and the largest structure among all measured specimens; SD: standard deviation.

Character	N	Range		Mean		SD		Holotype
µm	pt	µm	pt	µm	pt	µm	pt
Body length	20	570–879	981–1249	726	1120	95	67	757	1083
Buccal tube
Buccal tube length	20	54.5–75.3	–	64.6	–	5.6	–	69.9	–
Stylet support insertion point	20	44.7–60.8	80.1–82.4	52.4	81.1	4.5	0.6	56.4	80.7
Buccal tube external width	20	7.9–13.4	14.5–18.5	10.6	16.4	1.4	1.0	11.6	16.6
Buccal tube internal width	20	5.9–10.5	10.8–13.9	8.1	12.5	1.1	0.8	8.5	12.2
Ventral lamina length	20	33.1–48.2	60.0–68.7	41.5	64.2	4.4	2.9	46.6	66.7
Placoid lengths
Macroplacoid 1	20	15.5–24.3	28.4–34.4	20.3	31.3	2.6	1.8	22.8	32.6
Macroplacoid 2	20	10.1–17.0	18.2–22.6	13.1	20.2	1.9	1.4	13.4	19.2
Microplacoid	20	6.3–9.6	9.3–13.5	7.5	11.6	1.0	1.0	8.4	12.0
Macroplacoid row	20	27.1–43.3	48.5–59.0	35.3	54.4	4.8	3.0	38.9	55.7
Placoid row	20	35.2–55.1	62.7–75.2	44.5	68.6	5.7	3.2	49.6	71.0
Claw I heights
External primary branch	19	13.7–21.7	24.6–29.3	17.4	26.8	2.2	1.5	17.5	25.0
External secondary branch	18	10.5–15.5	18.7–23.5	13.7	21.1	1.4	1.1	14.7	21.0
Internal primary branch	19	12.4–19.8	22.1–26.3	15.9	24.7	1.8	1.1	16.9	24.2
Internal secondary branch	18	10.5–14.5	17.5–21.6	12.5	19.6	1.2	1.0	14.1	20.2
Claw II heights
External primary branch	20	13.6–21.4	25.0–30.7	18.0	27.8	2.2	1.5	19.9	28.5
External secondary branch	15	10.9–16.9	19.7–24.1	14.1	22.1	1.8	1.4	16.7	23.9
Internal primary branch	20	11.9–20.9	21.8–27.8	16.3	25.2	2.1	1.5	17.3	24.7
Internal secondary branch	19	9.3–17.2	17.1–22.8	13.2	20.3	1.8	1.6	14.3	20.5
Claw III heights
External primary branch	20	14.5–23.2	26.4–30.8	18.3	28.2	2.2	1.3	19.5	27.9
External secondary branch	18	12.3–18.4	21.5–25.0	15.0	23.1	1.6	1.0	16.2	23.2
Internal primary branch	20	13.2–19.9	23.5–27.0	16.5	25.5	1.8	1.0	17.7	25.3
Internal secondary branch	17	11.2–16.5	18.4–22.1	13.4	20.8	1.5	1.1	15.2	21.7
Claw IV heights
Anterior primary branch	20	16.1–25.1	28.0–33.6	19.6	30.2	2.4	1.6	19.6	28.0
Anterior secondary branch	15	12.5–17.5	20.9–24.9	15.0	23.0	1.6	1.1	15.6	22.3
Posterior primary branch	20	17.1–27.0	30.7–35.9	20.9	32.3	2.5	1.5	21.7	31.0
Posterior secondary branch	10	13.5–18.9	21.9–25.3	15.6	23.9	1.6	1.2	16.2	23.2

Reproduction The reproduction mode of M. ovovittatus sp. nov. is unknown. Examination of orcein-stained specimens revealed no spermatozoa, but only developing oocytes. The same was true for the observation of freshly mounted individuals in Hoyer’s medium that did not reveal any sperm either but only developing oocytes. Therefore, this population could be parthenogenetic or hermaphroditic (considering its close relationship with hermaphroditic taxa in Fig. 21 ). In the second case, it might have been possible that there were no specimens in the development stage when the sperm could be detectable. Differential diagnosis By having (i) three bands of teeth in the oral cavity armature that are well visible under light microscope, (ii) entire body cuticle covered by granulation (sometimes visible only under SEM), (iii) eggs with inverted goblet shaped processes, the new species is the most similar to four other taxa of Macrobiotus , namely Macrobiotus joannae Pilato & Binda, 1983 reported from its type locality in Australia ( Pilato & Binda 1983 ), and several uncertain localities in central, eastern, and southeastern Russia ( Biserov 1990 ) and from Italy ( Bertolani et al. 2014 ), Macrobiotus hannae Nowak & Stec, 2018 known only from its type locality in Poland ( Nowak & Stec 2018 ), Macrobiotus punctillus Pilato, Binda & Azzaro, 1990 known only from its type locality in Chile ( Pilato et al. 1990 ) and Macrobiotus rebecchii Stec, 2022 known only from its type locality in Kyrgyzstan ( Stec 2022b ). However, it can be easily distinguished from all of them by having a different morphology of the egg surface (the surface is continuous/solid and clearly wrinkled with sparse, very small and irregularly spaced pores in the new species vs chorion surface covered by evident reticulum in the other species), a different appearance of the terminal discs under PCM (the terminal discs are covered by multiple light-refracting dots, and as such resemble crocheted napkins vs terminal discs without light-refracting dots in other species).