**Genetic variability**

Aligned sequences (obtained in present study and downloaded from GenBank) were trimmed to 591, 498 and 700 bp for COI (four sequences; two species), 18S rRNA (eight sequences; four species and two sequences of *Bryodelphax* sp.) and 28S rRNA (14 sequences; seven species and two sequences of *Bryodelphax* sp.) molecular markers, respectively. Only sequences of *Bryodelphax* downloaded from GenBank that coincided with our four aforementioned molecular markers were selected.

In the case of the COI molecular marker, only three sequences of *Bry. parvulus* [8] were available in GenBank. Analysis of the p-distances between our sequences (GenBank accession numbers: MW655785-87) and three sequences of *Bry. parvulus* was from 16% (GenBank accession numbers: JX683827, JX683826, unpublished) to 18% (GenBank accession number: HM193405 [61]). In the case of the 18S rRNA molecular marker (GenBank accession numbers of our sequences: MW680639-40), no genetic differences were observed when compared with *Bry. parvulus* (GenBank accession numbers: HM193371 [61]; JX676189, [62]) and p-distance between other sequences, i.e., *Bryodelphax* sp. (GenBank accession numbers: EU266963 and EF632433 [63]) and *Bry. tatrensis* [64] (GenBank accession numbers: JX676188 and JX676190 [62]) was 0.01%. The ranges of uncorrected genetic p-distances between our 28S rRNA sequences (GenBank accession numbers: MW680637-38) and the most similar *Bry*. cf. *parvulus* (GenBank accession number: MT333466 [30]) was 0.01% and the least similar *Bryodelphax* sp. (GenBank accession numbers: MH414964 [65]) was 0.16% (see Supplementary Materials—SM1).

**Class**: Eutardigrada Richters, 1926 [57] **Order**: Parachela Schuster, Nelson, Grigarick and Christenberry, 1980 [66] **Superfamily**: Macrobiotoidea Thulin, 1928 [8] **Family**: Macrobiotidae Thulin, 1928 [8] **Genus**: *Macrobiotus* C.A.S. Schultze, 1834 [11] *Macrobiotus birendrai* **sp. nov.** (Tables 4 and 5, Figures 5–8) LSID http://zoobank.org/urn:lsid:zoobank.org:act:5617E45B-865A-42DA-A70A-DB6 FE1A52163




**Table 4.** *Cont*.

**Table 5.** Measurements (in μm) of selected morphological structures of eggs of *Macrobiotus birendrai* **sp. nov**. mounted in Hoyer's medium (N—number of specimens/structures measured; RANGE refers to the smallest and the largest structure among all measured eggs; SD—standard deviation).


**Figure 5.** *Macrobiotus birendrai* sp. nov.: (**A**) dorso-ventral projection (holotype); (**B**) cuticular pores on dorsal side of the body (paratype). All PCM. Scale bars in μm.

**Figure 6.** *Macrobiotus birendrai* sp. nov.: bucco-pharyngeal apparatus (dorso–ventral projection): (**A**) general view (paratype); (**B**) oral cavity armature with filled arrowhead indicating teeth of the first band (paratype); (**C**) oral cavity armature with arrow indicating teeth of the second band and indented filled arrowhead indicating dorsal teeth of the third band (paratype); (**D**) oral cavity armature with empty arrowhead indicating ventral teeth of the third band (paratype); (**E**) ventral placoids; the filled arrowhead indicates a first macroplacoid with central constriction (holotype). All PCM. Scale bars in μm.

**Figure 7.** *Macrobiotus birendrai* sp. nov.: (**A**) claws III (paratype); (**B**) claws IV with dentate lunulas (arrowhead); arrow indicates granulation on legs IV (paratype); (**C**) lunulas under claws III with small teeth; indented arrowhead indicates cuticular bar under claws (paratype); (**D**) granulation on leg III (arrow) (holotype). All PCM. Scale bars in μm.

**Figure 8.** *Macrobiotus birendrai* sp. nov.: eggs: (**A**,**B**) egg chorion (PCM and SEM, respectively); (**C**) the surface between egg processes visible in PCM; (**D**) egg processes visible in PCM; (**E**,**F**) egg surface and processes visible in SEM; (**G**,**H**) egg processes visible in PCM. Scale bars in μm.

*Type Locality*: 51◦24 21" N, 116◦14 27" W, 1900 m asl, Canada, Alberta, Banff National Park, near east end of the Louise Lake, moss on stone, May 2019, leg. Milena Roszkowska and Łukasz Kaczmarek.

*Material examined*: The 57 specimens, i.e., holotype (slide: CN8.43) + 56 paratypes (adults: 44 and eggs: 12) were mounted on microscope slides in Hoyer's medium, four eggs prepared for SEM and five animals prepared for molecular analyses (not included in type series). However, DNA sequences were obtained from one female specimen (exoskeleton) which was later mounted on microscope slide in Hoyer's medium and included into type series.

*Type depositories*: Holotype (CN8.43) and 57 paratypes (slides: CN8.\*, where the asterisk can be substituted by any of the following numbers: 3, 5–7, 15–17, 21, 29–33, 39, 40, 42–45, 101/S) are deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Pozna ´n, Uniwersytetu Pozna ´nskiego 6, 61-614 Pozna ´n, Poland. Six paratypes (five adults and one egg; slides CN8.41 and CN8.28) are deposited in the Natural History Museum of Denmark, University of Copenhagen.

*Etymology*: The first author would like to dedicate this species to her father—Birendra Prasad Lal Karna.

*Description of the new species.*

Adults (measurements and statistics in Table 4). Body transparent after fixation in Hoyer's medium, eyes present in all fixed specimens (Figure 5A). Entire cuticle covered with conspicuous round and lenticular pores (0.6–1.8 μm in diameter) distributed randomly (Figure 5B). However, larger pores present, on dorsal side, at the anterior and posterior part of the body. Bucco–pharyngeal apparatus of the *Macrobiotus* type, with ventral lamina and 10 peribuccal lamellae (Figure 6A). Mouth antero-ventral. Oral cavity armature of the *hufelandi* type, with first and the second band composed of numerous minute teeth (visible as granules in PCM) and third composed of three dorsal and three ventral transverse ridges (Figure 6B–D). Pharyngeal bulb spherical with triangular apophyses, two rod–shaped macroplacoids and a triangular microplacoid. Macroplacoid length sequence 2<1 (Figure 6A,E). The first macroplacoid with central constriction (Figure 6E, arrowhead), second with sub-terminal constriction. Claws of the *hufelandi* type (Figure 7A,B). Primary branches with distinct accessory points. Lunules under claws I–III with hardly visible teeth (visible only in bigger specimens) (Figure 7C, arrowhead) and dentate under claws IV (Figure 7B, arrowhead). Thin single continuous cuticular bars under claws I–III present (Figure 7C, indented arrowhead). Easily visible granulation present on legs I–IV (Figure 7B,D, arrows).

Eggs (measurements and statistics in Table 5). Eggs spherical, ornamented and laid freely with egg chorion of the *hufelandi* type (Figure 8A,B). Pores of egg surface mesh circular, similar in size and rather small, i.e., 0.2–0.8 μm in diameter (Figure 8C,E,F). Processes in the shape of inverted concave cups with terminal discs (Figure 8D–H). Terminal discs concave with serrated margins or with small irregular teeth (Figure 8D–H).

#### **DNA sequences**

We obtained good quality sequences for the applied molecular markers:

**COI**: single sequence; 609 bp long;

**18S rRNA**: single sequence; 553 bp long;

**28S rRNA**: single sequence; 721 bp long;

**ITS-2**: single sequence; 350 bp long.

**Differential diagnosis.** Based on egg processes morphology, the new species is most similar to *Mac. canaricus* Stec et al. [67], *Mac. hannae* Nowak and Stec [68], *Mac. crustulus* Stec et al. [12], *Mac. joannae* Pilato and Binda [69], *Mac. kamilae* Coughlan and Stec [70], *Mac. madegassus* Maucci [71], *Mac. noemiae* Roszkowska and Kaczmarek [72], *Mac. noongaris* Coughlan and Stec [70], *Mac. papei* Stec et al. [73], *Mac. paulinae* Stec et al. [16], *Mac. polypiformis* Roszkowska et al. [38] and *Mac. porifini* Kuzdrowska et al. [39], but differs specifically from:

*Mac. canaricus* **Stec, Krzywa ´nski and Michalczyk, 2018**, known only from the type locality on Canary Islands [67], by a different oral cavity armature (*hufelandi* type in the new species vs. *maculatus* type in *Mac. canaricus*), lunules I-III with hardly visible teeth, larger buccal tube internal width (3.4–5.6 μm in the new species vs. 2.1–3.3 μm in *Mac. canaricus*), higher *pt* of placoid row (*57.9–67.9* in the new species vs. *42.6–55.3* in *Mac. canaricus*), larger cuticular pores (0.7–1.8 μm in the new species vs. 0.4–0.7 μm in *Mac. canaricus*), different egg shell surface (porous shell in the new species vs. mesh shell in *Mac. canaricus*) and absence of granulation on terminal discs of the egg process.

*Mac. crustulus* **Stec, Dudziak and Michalczyk, 2020**, known only from the type locality in French Guiana [12], by a different oral cavity armature (*hufelandi* type in the new species vs. *lissostomus* type in *Mac. crustulus*), lunules I-III with hardly visible teeth, higher *pt* of stylet support insertion points (*78.0–82.8* in the new species vs. *69.3–72.7* in *Mac. crustulus*), larger buccal tube internal width (3.4–5.6 μm in the new species vs. 1.6–2.7 μm in *Mac. crustulus*), higher *pt* of buccal tube external and internal width (*13.5–17.0* and *8.7–11.9*, respectively, in the new species vs. *9.3–12.2* and *5.1–6.5*, respectively, in *Mac. crustulus*), higher *pt* of ventral lamina (*56.0–66.2* in the new species vs. *46.9–55.8* in *Mac. crustulus*), different egg shell surface (porous shell in the new species vs. mesh shell in *Mac. crustulus*), absence of granulation on convex central area of terminal discs and lower number of processes on the egg circumference (22–26 in the new species vs. 26–34 in *Mac. crustulus*).

*Mac. hannae* **Nowak and Stec, 2018**, known only from the type locality in Poland [68], by lunules I-III with hardly visible teeth, larger cuticular pores (0.6–1.8 μm in the new species vs. up to 0.55 μm in *Mac. hannae*), smaller distance between egg processes (1.0–3.3 μm in the new species vs. 4.0–8.1 μm in *Mac. hannae*), smaller egg bare diameter (74.9–86.9 μm in the new species vs. 88.6–109.2 μm in *Mac. hannae*) and absence of granules inside pores around egg processes.

*Mac. joannae* **Pilato and Binda, 1983**, known only from the type locality in Australia (Pilato and Binda [69]; see also comments in Nowak and Stec [68]), by lower average *pt* of stylet support insertion points (*80.2* in the new species vs. *81.9* in *Mac. joannae*), smaller size of macroplacoid 1 and microplacoid (8.2–16.0 and 2.4–4.9 μm, respectively, in the new species vs. 19.3 and 6.4 μm, respectively, in *Mac. joannae* in specimen of body length 400 μm), smaller size of claws I and II external and internal primary branch (I. 8.5–13.2 and 8.2–12.7 μm, respectively; II. 8.1–14.0 and 8.6–13.6 μm, respectively, in the new species vs. I. 25.0 and 23.5 μm, respectively; II. 27.0 and 24.5 μm, respectively in *Mac. joannae* in specimen of body length 400 μm) and smaller size of claw IV anterior and posterior primary branch (9.5–15.8 and 8.4–14.2 μm, respectively, in the new species vs. 28.0 and 27.0 μm, respectively, in *Mac. joannae* in specimen of body length 400 μm).

*Mac. kamilae* **Coughlan and Stec, 2019**, known only from the type locality in India [70], by a different oral cavity armature (*hufelandi* type in the new species vs. *patagonicus* type in *Mac. kamilae*), lunules I-III with hardly visible teeth, higher *pt* of stylet support insertion points (*78.0–82.8* in the new species vs. *71.6–75.9* in *Mac. kamilae*), shorter claws (for details see Table 4 in this paper and Table 4 in Coughlan and Stec [65]), absence of body granulation and absence of scattered granules on the terminal discs of egg process.

*Mac. madegassus* **Maucci, 1993**, known only from the type locality in Madagascar [71], by presence of eyes, different oral cavity armature (*hufelandi* type in the new species vs. *maculatus* type in *Mac. madegassus*), presence of cuticular pores, lunules I-III with hardly visible teeth, longer buccal tube (33.2–52.6 μm in the new species vs. up to 30.0 μm in *Mac. madegassus*), larger buccal tube external width (5.2–8.4 μm in the new species vs. up to 2.1 μm in *Mac. madegassus*), higher *pt* of buccal tube external width (*13.5–17.0* in the new species vs. 7.0 in *Mac. madegassus* in specimen of body length 316 μm), longer macroplacoids (I: 8.2–16.0 μm and II: 6.0–11.2 μm in the new species vs. I: up to 6.4 μm and II: up to 3.6 μm in *Mac. madegassus* in specimen of body length 316 μm), stylet supports inserted in more caudal position (26.2–43.0 μm in the new species vs. up to 20.4 μm in *Mac. madegassus* in specimen of body length 316 μm), higher *pt* of stylet support insertion

points (*78.0–82.8* in the new species vs. up to *68.0* in *Mac. madegassus* in specimen of body length 316 μm) and lower number of processes on the egg circumference (20–26 in the new species vs. 30–34 in *Mac. madegassus*).

*Mac. noemiae* **Roszkowska and Kaczmarek, 2019**, known only from the type locality in Spain [72], by a different oral cavity armature (*hufelandi* type in the new species vs. *patagonicus* type in *Mac. noemiae*), lunules I-III with hardly visible teeth, higher *pt* of ventral lamina (*56.0–66.2* in the new species vs. *48.4–55.7* in *Mac. noemiae*), higher *pt* of macroplacoid row and placoid row (*49.1–57.9* and *57.9–67.9*, respectively, in the new species vs. *39.2–47.1* and *47.1–57.9*, respectively, in *Mac. noemiae*), smaller egg full and bare diameter (87.8–103.9 and 74.9–86.9 μm, respectively, in the new species vs. 118.5–123.5 100.6–105.7 μm, respectively, in *Mac. noemiae*), lower number of processes on the egg circumference (20–26 in the new species vs. 30–34 in *Mac. noemiae*), presence of discs on egg processes and absence of filaments on apisal part of egg processes.

*Mac. noongaris* **Coughlan and Stec, 2019**, known only from the type locality in Australia [70], by a different oral cavity armature (*hufelandi* type in the new species vs. *patagonicus* type in *Mac. noongaris*), lunules I-III with hardly visible teeth, larger maximum size of the cuticular pores (up to 1.8 μm in the new species vs. up to 0.8 μm in *Mac. noongaris*), absence of scattered granulation on the terminal discs of the egg processes, higher mean of egg bare diameter and egg full diameter (82.9 and 96.3 μm, respectively, in the new species vs. 70.7 and 82.1 μm, respectively, in *Mac. noongaris*), larger mean process height, process base width and terminal disc width (7.9, 6.6 and 4.1 μm, respectively, in the new species vs. 6.2, 5.0 and 3.3 μm, respectively, in *Mac. noongaris*), and smaller mean inter processes distance (2.1 μm in the new species vs. 3.4 μm in *Mac. noongaris*).

*Mac. papei* **Stec, Kristensen and Michalczyk, 2018** known only from type locality in Tanzania [73], by a different oral cavity armature (*hufelandi* type in the new species vs. *patagonicus* type in *Mac. papei*), lunules I-III with hardly visible teeth, absence of patches of cuticular granulation on the internal surface of legs I–III, higher *pt* of macroplacoid II (*17.2–22.0* in the new species vs. *10.3–16.2* in *Mac. papei*), absence of flexible filaments on the terminal disc of the egg processes, lower mean of egg bare diameter and egg full diameter (82.9 and 96.3 μm, respectively, in the new species vs. 95.0 and 109.7 μm, respectively, in *Mac. papei*), smaller mean terminal disc width (4.1 μm in the new species vs. 4.3 μm in *Mac. papei*), and smaller mean inter processes distance (2.1 μm in the new species vs. 4.3 μm in *Mac. papei*).

*Mac. paulinae* **Stec, Smolak, Kaczmarek and Michalczyk, 2015** known only from type locality in Kenya [16], by lack of dorso-lateral patches of granulation, smaller maximum size of cuticular pores (up to 1.8 μm in the new species vs. up to 0.5 μm in *Mac. paulinae*), different oral cavity armature (*hufelandi* type in the new species vs. *maculatus* type in *Mac. paulinae*), different third band of teeth (three dorsal and three ventral teeth in the new species vs. single dorsal and single ventral tooth in *Mac. paulinae*), lunules I-III with hardly visible teeth, larger buccal tube external and internal width with higher *pt* (5.2–8.4 [*13.5–17.0*] and 3.4–5.6 μm [*8.7–11.9*], respectively, in the new species vs. 2.2–4.6 μm [*8.8–12.6*] and vs. 1.0–2.9 μm [*3.5–8.1*], respectively, in *Mac. paulinae*), longer ventral lamina (19.9–32.9 μm in the new species vs. 15.0–19.6 μm in *Mac. paulinae*), longer macroplacoid row with higher *pt* (16.3–30.2 μm [*49.1–57.9*] in the new species vs. 9.2–14.4 μm [*32.5–40.4*] in *Mac. paulinae*), longer placoid row with higher *pt* (19.2–35.2 μm [*57.9–67.9*] in new species vs. 10.8–17.4 μm [*37.8–48.9*] in *Mac. paulinae*), larger egg bare and full diameter (74.9–86.9 and 87.8–103.9 μm, respectively, in the new species vs. 57.0–70.5 and 66.3–85.6 μm, respectively, in *Mac. paulinae*) and absence of filaments on egg processes discs.

*Mac. polypiformis* **Roszkowska, Ostrowska, Stec, Janko and Kaczmarek, 2017** known only from type locality in Ecuador [38], by different oral cavity armature (*hufelandi* type in the new species vs. *maculatus* type in *Mac. polypiformis*), lunules I-III with hardly visible teeth, longer buccal tube (33.2–52.6 μm in the new species vs. 24.4–32.5 μm in *Mac. polypiformis*), stylet supports inserted in more caudal position with higher *pt* (26.2–43.0 μm

[*78.0–82.8*] in the new species vs. 17.1–23.5 μm [*70.1–72.9*] in *Mac. polypiformis*), larger buccal tube external and internal width and with higher *pt* (5.2–8.4 [*13.5–17.0*] and 3.4–5.6 μm [*8.7–11.9*], respectively, in the new species vs. 2.8–4.0 [*11.0–13.0*] and 1.6–2.4 μm [*6.1–8.6*], respectively, in *Mac. polypiformis*), longer ventral lamina with larger *pt* (19.9–32.9 μm [*56.0–66.2*] in the new species vs. 13.5–17.3 μm [*52.1–55.1*] in *Mac. polypiformis*), longer macroplacoid 1 (8.2–16.0 μm in the new species vs. 5.2–6.8 μm in *Mac. polypiformis*), longer macroplacoid 2 with higher *pt* (6.0–11.2 μm [*17.2–22.0*] in the new species vs. 2.8–4.1 μm [*11.4–14.5*] in *Mac. polypiformis*), longer microplacoid (2.4–4.9 μm in the new species vs. 1.5–2.3 μm in *Mac. polypiformis*), longer macroplacoid row with higher *pt* (16.3–30.2 μm [*49.1–57.9*] in the new species vs. 9.0–11.8 μm [*34.3–39.9*] in *Mac. polypiformis*) and longer placoid row with higher *pt* (19.2–35.2 μm [*57.9–67.9*] in the new species vs. 11.1–14.5 μm [*41.4–49.0*] in *Mac. polypiformis*), larger egg bare and full diameter (74.9–86.9 and 87.8–103.9 μm, respectively, in the new species vs. 61.9–70.5 and 70.4–81.2 μm, respectively, in *Mac. polypiformis*) and absence of filaments on egg processes discs.

*Mac. porifini* **Kuzdrowska, Mioduchowska, Gawalak, Bartylak, Kepel, Kepel and Kaczmarek 2021** known only from the type locality in Madagascar [39] by presence of eyes, different oral cavity armature (*hufelandi* type in the new species vs. *patagonicus* type in *Mac. porifini*), lunules I-III with hardly visible teeth, presence of dentate lunules on legs IV, higher *pt* of macroplacoid row (*49.1–57.9* in the new species vs. *36.2–47.7* in *Mac. porifini*), higher *pt* of placoid row (*57.9–67.9* in the new species vs. *43.5–57.7* in *Mac. porifini*), larger egg bare diameter (74.9–86.9 μm in the new species vs. 72.2–74.0 μm in *Mac. porifini*), absence of very small irregular granules on the surface of the discs and absence of micro granulation on the teeth of the terminal discs of egg process.
