Macrobiotus mileri Stec 2024, sp. nov.
Creators: name: Stec, Daniel
Publisher: name: Zenodo
Year: 2024
Subjects: Biodiversity
Taxonomy
Animalia
Tardigrada
Eutardigrada
Parachela
Macrobiotidae
Macrobiotus
Macrobiotus mileri

description: 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.
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).
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.
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.
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).
description: Published as part of Stec, Daniel, 2024, Integrative taxonomy supports two new species of Macrobiotus (Tardigrada: Eutardigrada: Macrobiotidae) allowing further discussion on the genus phylogeny, pp. 79-123 in European Journal of Taxonomy 930 on pages 93-106, DOI: 10.5852/ejt.2024.930.2481, http://zenodo.org/record/10893800