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 mileri sp. nov. urn:lsid:zoobank.org:act: 99372E2F-595A-4AB2-8398-21198A2CBD7E Figs 9–20 , Tables 4–5 Etymology The species is named in honour of Krzysztof Miler, who has developed an impressive tolerance for the daily tardigrade madness that surrounds him. Fig. 9. Macrobiotus mileri sp. nov. , PCM images of habitus and body granulation. A . Dorso-ventral projection (holotype, IL.001.11, ISEA PAS, forma aporata). B . Granulation in the dorsal cuticle (paratype, ISEA PAS, forma aporata). C . Granulation in the dorsal body cuticle (paratype, ISEA PAS, forma porata). Scale bars in μm. Material examined 39 animals, 7 eggs mounted on microscope slides in Hoyer’s medium, 7 animals and 3 eggs examined under SEM , and 4 animals processed for DNA sequencing. Type material Holotype ISRAEL • Tel-Aviv ; 32°2′42.82″ N , 34°46′14.88″ E ; 19 m a.s.l. ; Nov. 2019 ; K. Miler leg.; moss growing on a stone wall in urban park ; ISEA PAS, slide IL.001.11. Paratypes ISRAEL • 45 animals; same collection data as for the holotype; ISEA PAS , slides IL.001.08 to IL.001.12, SEM stub TAR.014 • 10 eggs ; same collection data as for the holotype; ISEA PAS , slides IL.001.06 to IL.001.07, SEM stub TAR.014 . Description Animals Body transparent in juveniles and white in adults, after fixation in Hoyer’s medium transparent ( Fig. 9A ). Eyes present. Granulation is present on the entire body cuticle and is visible under PCM and SEM, but granulation on the ventral side of the body is less dense ( Figs 9B–C , 10A–F ). In terms of cuticular pores, two morphological forms of animals are present in this species. One form (forma porata) with large, evident pores arranged specifically in five patches ( Figs 11A–B , 12A–D , 13 ) and second form (forma aporata) with only small, single pores randomly distributed on the body (almost indetectable under PCM and hardly detectable also under SEM; 0.2–0.4 μm in diameter; Figs 10E–F , 14A , 16C , 18A–B ). In forma porata, the round and oval pores (0.4–0.7 μm in diameter) are arranged into five distinct patches: (I) a sparse patch of pores on the external surface of the distal portion of leg I ( Figs 13 , 15A ); (II) a dense patch of pores on the external surface of the proximal portion of leg II extending also towards the lateral body cuticle ( Figs 11A , 13 ); (III) a dense patch of pores on the lateral body surface between legs II and II ( Figs 11A , 12A, C , 13 ); (IV) a dense large patch of pores covering the whole external surface of leg III, extending also towards the lateral body cuticle ( Figs 11A – B , 12A, C , 13 ); and (V) the largest patch of pores that extends from the left caudo-lateral surface, through the dorsal caudal surface to the right caudo-lateral surface, extending also towards lateral and dorsal surfaces of legs IV ( Figs 11B , 12A–D , 13 , 15D ). Only the V patch is single and continuous, while patches I–IV are doubled and present symmetrically on each side of the body. In both forms, some evident dense granulation patches are visible on the external and internal surfaces of all legs I–III, as well as on the lateral and dorsal surfaces of legs IV under PCM and SEM ( Figs 14A–D , 15A–D ). Small pores, visible only under SEM, can be seen in between the granulation on the hind legs ( Fig. 15D ). A pulvinus-shaped cuticular bulge is not visible on the internal surface of legs I–III, but there is a garter-shaped structure on the external surface of all legs I–III ( Figs 13 , 14A–B , 15A–B ) above which there is a small cuticular bulge / fold (visible only under SEM; Fig. 15A–B ). Small and robust hufelandi - type claws ( Fig. 16A–E ). Primary branches with distinct accessory points, a moderately long common tract, and an evident stalk connecting the claw to the lunula ( Fig. 16A–F ). The lunulae on legs I–III are smooth ( Fig. 16A, D ), while there is dentation in the lunulae on legs IV ( Fig. 16B, C, E ). The cuticular bars are absent, but double muscle attachments are present above the claws I–III ( Fig. 16A, D ). Shadowed extensions extending from lunulae on legs I–III are present and faintly visible only under PCM ( Fig. 16A ). A horseshoe-shaped structure connects the anterior and posterior lunules on leg IV ( Fig. 16C ). Fig. 10. Macrobiotus mileri sp. nov. , SEM images of body granulation and cuticular pores (paratype, ISEA PAS, forma aporata). A . Body granulation in the dorsal head region. B . Body granulation in the dorsal central body region. C . Body granulation in the dorsal caudal body region. D . General view of body granulation in the lateral caudal body region. E–F . Singular pores in the body cuticle. Scale bars in μm. Mouth antero-ventral. Bucco-pharyngeal apparatus of Macrobiotus type , with ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes ( Figs 17A , 18A–D ). Under PCM, the oral cavity armature is of hufelandi type – three bands of teeth are always visible ( Fig. 17B–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, just behind the bases of the peribuccal lamellae ( Figs 17B–C , 18C–D ). The second band of teeth is located between the ring fold and the third band of teeth and comprises about four rows of small cones, larger than those of the first band ( Figs 17B–C , 18C–D ). 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 17B–C , 18C–D ). The third band of teeth is discontinuous and divided into the dorsal and ventral portions. Under PCM, dorsal teeth are seen as three distinct transverse ridges, and the medio-dorsal tooth is evidently longer than the latero-dorsal teeth ( Fig. 17B ). The ventral teeth appear as two separate lateral transverse ridges between which a median tooth is visible and rarely divided into two teeth ( Fig. 17C ). Under SEM, the dorsal and ventral teeth are also clearly distinct ( Fig. 18C–D ). Under SEM, the margins of the dorsal teeth are serrated and the medio-dorsal tooth is clearly longer than latero-dorsal teeth ( Fig. 18C ) whereas the ventral teeth are smaller and their margins are less serrated ( Fig. 18D ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a large triangular microplacoid ( Fig. 17A ). The macroplacoid length sequence being 2<1. The first and the second macroplacoid are constricted centrally and subterminally, respectively ( Fig. 17D–E ). The animals’ measurements and statistics are given in Table 4 . Fig. 11. Macrobiotus mileri sp. nov. , PCM images of cuticular pores patches in forma porata (paratype, ISEA PAS). A . Patches II, III and IV. B . Patches IV and V. Scale bars in μm. Fig. 12. Macrobiotus mileri sp. nov. , SEM images of cuticular pores patches in two paratypes of forma porata (ISEA PAS). A , C . Patches II, III and IV. B , D . Dorsal view on the caudal body region with continuous patch V of pores. Scale bars in μm. Fig. 13. Macrobiotus mileri sp. nov. , a schematic drawing of a specimen belonging to forma porata, showing the distribution of patches of cuticular pores, body and leg granulation as well as the gartershaped structures on legs I–III. Fig. 14. Macrobiotus mileri sp. nov. , PCM images of dense granulation patches and cuticular structures on legs (paratypes, ISEA PAS). A . Granulation on the external, proximal and internal surface of leg II. B . Garter-shaped structure and granulation on the external surface of leg II. C . Granulation on the internal surface of leg III. D . Granulation on the hind leg. The empty arrow indicates a cuticular pore, filled arrows indicate the garter-shaped structure. All photographs taken from specimens belonging to forma aporata; scale bars in μm. Fig. 15. Macrobiotus mileri sp. nov. , SEM images of dense granulation patches and cuticular structures on legs (paratypes, ISEA PAS). A . Granulation and garter-shaped structure on the external surface of leg I (forma porata). B . Granulation and garter-shaped structure on the external surface of leg II (forma aporata). C . Granulation on the internal surface of leg II (forma porata). D . Granulation on the hind leg (forma porata). Empty arrows indicate cuticular pores, filled arrows indicate the garter-shaped structure, empty flat arrowheads indicate the small cuticular bulge / fold, filled flat arrowheads indicate small pores in between granulation that are visible only under SEM. Scale bars in μm. Fig. 16. Macrobiotus mileri sp. nov. , images of claws (paratypes, ISEA PAS). A . Claws II with smooth lunulae (PCM, forma aporata). B . Claws IV with dentate lunulae (PCM, forma aporata). C . Dentate lunulae (PCM, forma aporata). D . Claws II with smooth lunulae, respectively (SEM, forma porata). E . Claws IV with dentate lunulae (SEM, forma porata). The empty arrow indicates a singular cuticular pore, filled indented arrowheads indicate shadowed extensions extending from the lunulae (under PCM), filled flat arrowheads indicate paired muscles attachments, and empty indented arrowheads indicate the horseshoe structure connecting the anterior and the posterior claw. Scale bars in μm. Fig. 17. Macrobiotus mileri sp. nov. , PCM images of the buccal apparatus (all from holotype, IL.001.11, ISEA PAS, forma aporata). 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 4. Measurements [in μm] and pt values of selected morphological structures of animals of Macrobiotus mileri 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	326–523	1101–1392	449	1251	54	71	488	1217
Buccal tube
Buccal tube length	20	29.5–40.8	–	35.8	–	3.4	–	40.1	–
Stylet support insertion point	20	23.3–32.8	78.9–81.3	28.5	79.7	2.7	0.6	32.0	79.8
Buccal tube external width	20	4.3–6.7	14.4–17.3	5.8	16.1	0.7	0.8	6.4	16.0
Buccal tube internal width	20	3.0–5.2	9.7–13.1	4.2	11.7	0.6	0.9	5.1	12.7
Ventral lamina length	19	17.2–24.2	53.5–60.4	20.8	57.6	1.9	1.9	23.4	58.4
Placoid lengths
Macroplacoid 1	20	7.0–12.2	23.5–30.2	9.7	26.9	1.6	2.2	12.1	30.2
Macroplacoid 2	20	4.6–7.5	12.3–20.8	6.1	17.1	0.9	1.8	7.3	18.2
Microplacoid	20	2.5–4.9	8.4–13.3	3.8	10.5	0.6	1.2	4.1	10.2
Macroplacoid row	20	12.9–20.9	42.7–52.2	17.2	47.9	2.4	2.9	20.9	52.1
Placoid row	20	16.3–26.6	54.3–67.7	22.1	61.6	3.0	3.8	26.6	66.3
Claw I heights
External primary branch	18	7.2–12.9	24.4–34.1	10.5	29.0	1.4	2.3	11.0	27.4
External secondary branch	11	7.1–10.0	22.8–30.2	9.1	25.2	0.9	2.1	10.0	24.9
Internal primary branch	18	7.1–11.5	24.1–31.1	9.9	27.3	1.1	1.6	10.7	26.7
Internal secondary branch	12	7.0–10.1	21.4–27.1	9.0	24.3	1.0	1.6	9.1	22.7
Claw II heights
External primary branch	18	8.3–13.0	26.8–38.1	11.3	31.3	1.5	2.8	12.5	31.2
External secondary branch	10	7.7–12.0	23.1–32.3	10.0	27.2	1.4	3.0	10.9	27.2
Internal primary branch	15	7.4–11.0	24.8–29.5	9.7	26.9	1.1	1.4	10.1	25.2
Internal secondary branch	10	6.2–10.2	20.8–27.3	8.9	23.9	1.2	2.1	9.8	24.4
Claw III heights
External primary branch	16	8.1–12.9	27.2–34.6	11.2	30.9	1.2	1.9	12.3	30.7
External secondary branch	11	7.5–11.9	22.8–31.9	10.0	27.1	1.3	2.6	10.6	26.4
Internal primary branch	13	7.3–11.2	24.5–30.0	9.9	27.2	1.2	1.5	10.8	26.9
Internal secondary branch	10	6.0–10.4	20.1–27.9	9.0	24.0	1.5	2.6	10.0	24.9
Claw IV heights
Anterior primary branch	18	7.4–12.1	24.8–31.4	10.4	28.8	1.4	2.0	12.1	30.2
Anterior secondary branch	13	8.2–10.7	20.1–27.8	9.1	24.5	0.7	2.1	10.7	26.7
Posterior primary branch	20	8.2–13.1	23.8–38.4	11.1	31.1	1.5	3.2	12.2	30.4
Posterior secondary branch	9	6.9–10.9	20.8–27.9	9.3	25.0	1.3	2.2	?	?

Eggs Laid freely, white, spherical and ornamented ( Figs 19A–E , 20A–F ). The surface between processes is of intermediate state between the maculatus and the persimilis types , that is, the surface is solid and wrinkled with very small pores, which are present mainly around the bases of the egg processes, and only some are sparse and irregularly distributed in the egg surface between processes ( Figs 19A–C , 20C–F ). These pores are visible under PCM, but better visible under SEM (0.1–0.3) μm in diameter; Figs19A–C , 20C–F ). The processes are not in the shape of inverted goblets with mostly sigmoidal (sometimes concave) conical trunks and weakly defined convex terminal discs with smooth edges ( Figs 19A–C , 20C–F ). Very faint annulations are visible on the process trunk, especially on the distal portion of the process (character visible only under SEM; Fig. 20D ). A crown of gently marked thickenings is visible around the bases of the processes as darker dots under PCM ( Fig. 19A–C ) and as thicker wrinkles at the processes bases under SEM ( Fig. 20D–F ). In some processes under SEM the terminal discs have pores in the center ( Fig. 20D–F ), which under PCM are visible as large light-refracting dot in the disc center ( Fig. 19A–C ). However, it cannot be excluded that the actual pores in the terminal discs are preparation artefacts, while light refracting dots visible under PCM are caused by thinner chorion layers in this place. The measurements and statistics of eggs are given in Table 5 . Fig. 18. Macrobiotus mileri sp. nov. , mouth opening and the oral cavity armature seen under SEM (paratypes, ISEA PAS). A–B . The mouth opening of a single paratype visible from lateral and frontal view respectively. C–D . The oral cavity armature of a single paratype seen under SEM from different angles, dorsal (C) and ventral (D) view, respectively. Empty arrows indicate cuticular pores, filled flat arrowheads indicate the first band of tenth, empty flat arrowheads indicate the second band of teeth, and filled indented arrowheads indicate the third band of teeth. Scale bars in μm. Table 5. Measurements [in μm] of the eggs of Macrobiotus mileri sp. nov. ; eggs mounted in Hoyer’s medium; process base/height ratio is expressed as percentage; N: number of eggs/structures measured; range: refers to the smallest and the largest structure among all measured specimens; SD: standard deviation.

Character	N	Range	Mean	SD
Egg bare diameter	7	76.5– 92.6	82.5	6.2
Egg full diameter	7	93.0– 107.6	98.2	6.3
Process height	21	6.2– 8.7	7.6	0.8
Process base width	21	7.3– 10.3	8.5	0.6
Process base/height ratio	21	93%– 140%	113%	14%
Terminal disc width	21	2.2– 4.2	3.2	0.4
Inter-process distance	21	1.2– 3.3	2.4	0.5
Number of processes on the egg circumference	7	23– 25	24.0	0.8

Fig. 19. Macrobiotus mileri sp. nov. , PCM images of the eggs under ×1000 magnification (ISEA PAS). A–C . Egg surface. D–E . Midsections of egg processes. Empty indented arrowheads indicate pores/ light-refracting dots in the center of the terminal discs, filled flat arrowheads indicate small pores around bases of the egg processes, filled indented arrowheads indicate dark thickenings around bases of the egg processes. Scale bars in μm. Fig. 20. Macrobiotus mileri sp. nov. , SEM images of eggs (ISEA PAS). A–B . Entire egg. C–F . Details of egg processes and the surface between them. Empty indented arrowheads indicate pores in center of terminal discs, filled flat arrowheads indicate small pores around bases of egg processes, filled indented arrowheads indicate thickenings around bases of egg processes. Scale bars in μm. Reproduction The type population of M. mileri sp. nov. is dioecious. Both males with testes filled with sperm and females with ovaries containing oocytes were observed in specimens freshly mounted in Hoyer’s medium in both specimens ascribed to each of the two morphological forms. Differential diagnosis By having (i) three bands of teeth in the oral cavity armature that are well visible under light microscope, (ii) the entire body cuticle covered by granulation (sometimes visible only under SEM), the new species is the most similar to five 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 south-eastern 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 ), Macrobiotus rebecchii Stec, 2022 known only from its type locality in Kyrgyzstan ( Stec 2022b ) and M. ovovittatus sp. nov. described above. However, it can be easily distinguished from all of them by having different pores arrangements on the body cuticles (two forms: porata with pores arranged in five distinct patches; and aporata with singular, small, almost undetectable pores vs typical, more or less evenly distributed cuticular pores in the other species) and a different morphology of the terminal discs (weakly defined convex terminal discs with smooth edges in the new species vs cog-shaped terminal discs, with a concave central area and 10–18 distinct teeth in the other species). Phylogenetic and delimitation results Both phylogenetic analyses resulted in trees of similar topology, and most of the nodes well and moderately supported, in which three distinct monophyletic Macrobiotus lineages ( Macrobiotus clades A, B, and C) were confidently recovered ( Fig. 21 , Supp. file 3). The analyses confirmed also monophyly for the M. ariekammensis , M. pallarii , and M. pseudohufelandi complexes ( Fig. 21 ). At first, it seems that the Macrobiotus polonicus - persimilis complex as defined by Bertolani et al. (2023) has also been recovered to be monophyletic. However, the position of M. cf. polonicus 1 and 2 from Sweden ( Vecchi & Stec 2021 ), whose morphology also fits this definition, makes this species complex paraphyletic. Also, the M. polonicus species complex as defined by Stec et al. (2021a) or the M. persimilis morpho-group as defined by Bertolani et al. (2023) is paraphyletic, since M. cf. polonicus from Sweden and Macrobiotus annewintersae Vecchi & Stec, 2021 cluster together with species of the M. pallarii complex. Macrobiotus mileri sp. nov. belongs to the Macrobiotus clade B staying in sister relationship with the clade containing Macrobiotus caelestis Coughlan, Michalczyk & Stec, 2019 and nominal taxa of the M. polonicus - persimilis complex ( Fig. 21 ). The second new species, M. ovovittatus sp. nov. , belongs to the Macrobiotus clade A, as the closest relative of Macrobiotus hupingensis Yuan, Wang, Liu, Liu & Li, 2022 and together they cluster with Macrobiotus birendrai Kayastha, Roszkowska, Mioduchowska, Gawlak & Kaczmarek, 2021 , M. hannae and M. rebecchii ( Fig. 21 ). Importantly, M. hupingensis is a species of the M. pallarii complex, but the DNA sequences associated with this description belong to different unspecified Macrobiotus and the authors are working on correcting this mistake (Z. Yuan, Shaanxi Normal University, pers. com.). Therefore, the species name is given within quotation marks in the phylogenetic tree ( Fig. 21 ). The delimitation results of both ASAP analyses were congruent (Supp. file 4). The number of delimited species from the COI data set representing Macrobiotidae superclade I, and for the data set comprising only taxa of Macrobiotus , were 73 and 47, respectively. The number of taxa of Macrobiotus delimited in the larger data set was the same as for the smaller data set (47; Supp. file 4). Two new species described in this study have always been distinguished as two distinct entities (Supp. file 4). Both morphological forms within M. mileri sp. nov. have also been recognized as a single species (Supp. file 4). Interestingly, there were several cases where taxa that were potentially thought as being distinct have been lumped together into singular putative species. These were: (i) Macrobiotus sandrae Bertolani & Rebecchi, 1993 and Macrobiotus azzunae Ben Marnissi, Cesari, Rebecchi & Bertolani, 2021 , (see Ben Marnissi et al. 2021 ), (ii) Macrobiotus hufelandi Schultze, 1834 and M. cf. hufelandi (see Bertolani et al. 2011 ), (iii) Macrobiotus fontourai Bertolani, Cesari, Giovannini, Rebecchi, Guidetti, Kaczmarek & Pilato, 2022 and M. cf. muralis (see Bertolani et al. 2023 ), (iv) Macrobiotus kosmali Kayastha, Mioduchowska, Gawlak, Sługocki, Gonçalves Silva & Kaczmarek, 2023 and M. cf. recens (see Kayastha et al. 2023 ).