Integrative taxonomy identifies two new tardigrade species (Eutardigrada: Macrobiotidae) from Greenland

. In this paper we describe Macrobiotus engbergi sp. nov. and Tenuibiotus zandrae sp. nov. from Greenland. Our study has involved both classical taxonomic methods, which include morphological and morphometric analyses conducted with the use of light and scanning electron microscopy, and genetic analysis based on four molecular markers (three nuclear: 18S rRNA, 28S rRNA, ITS-2, and one mitochondrial: COI). Moreover, we re-examined the type series of Tenuibiotus voronkovi (Tumanov, 2007) as well as the original sample where the species was found and we provide new morphological data from light and scanning electron microscopy which enabled us to amend its description. Finally, we also analysed slides with animals and egg of two populations from Nordaustlandet and Edgeøya (archipelago of Svalbard, Norway) designated as T. voronkovi within its recent redescription. The results and comparisons presented in our study question the validity of this designation. tardigrade


Microscopy and imaging
Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's medium and secured with a cover slip, following the protocol by Morek et al. (2016). Slides were examined under an Olympus BX53 light microscope with phase and Nomarski contrasts (together termed later as light contrast microscopy), associated with an Olympus DP74 digital camera. Subsequently, after mounting, the specimens in the medium slides were also checked under phase contrast microscopy for the presence of males and females in the studied populations, as the spermatozoa in testis and spermathecae are visible for several hours after mounting . In order to obtain clean and extended specimens for scanning electron microscopy, tardigrades were processed according to the protocol by Stec et al. (2015). In short, specimens were first subjected to a 60°C water bath for 30 mn to obtain fully extended animals, next to a water/ethanol and an ethanol/acetone series, then to CO 2 critical point drying and finally sputter coated with a thin layer of gold. Bucco-pharyngeal apparatuses were extracted following the protocol of Eibye-Jacobsen (2001) as modified by . Specimens were examined under high vacuum in a Versa 3D DualBeam scanning electron microscope at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. Moreover, one egg of Tenuibiotus voronkovi was examined under high vacuum in a MIRA3 LMU scanning electron microscope at the Centre for Molecular and Cell Technologies, St Petersburg State University (for details please see Comparative material section below). All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For structures that could not be satisfactorily focused in a single light microscope photograph, a stack of 2-6 images was taken with an equidistance of ca 0.2 μm and assembled manually into a single deep-focus image in Corel Photo-Paint X6.

Morphometrics and morphological nomenclature
All measurements are given in micrometres (μm). Sample size was adjusted following recommendations by Stec et al. (2016). Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The terminology used to describe oral cavity armature and egg shell morphology follows Michalczyk & Kaczmarek (2003) and Kaczmarek & Michalczyk (2017). Macroplacoid length sequence is given according to Kaczmarek et al. (2014). Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). All other measurements and nomenclature follow Kaczmarek & Michalczyk (2017). Specifically, buccal tube width was measured as the external and internal diameter at the level of the stylet support insertion point. Heights of the claw branches were measured from the base of the claw (i.e., excluding the lunula) to the top of the branch, including accessory points. Distance between egg processes was measured as the shortest line connecting base edges of the two randomly chosen closest processes. Morphometric data were handled using the "Parachela" ver. 1.6 template available from the Tardigrada Register . Raw morphometric data for each analysed species are provided as supplementary files (SM.01 and SM.02). Tardigrade taxonomy follows Guil et al. (2019).

Additional comparative material
For morphological comparison of our new Tenuibiotus species we used slides with animals and eggs from the type series of Tenuibiotus voronkovi. Moreover, the re-examination of the original sample in which this species was found resulted in the discovery of one more egg which has been observed in a scanning electron microscope. Additionally, we re-examined slides with animals and eggs attributed to T. voronkovi by Zawierucha et al. (2016a) of populations from two islands in the archipelago of Svalbard, Norway: Nordaustlandet and Edgeøya. For more collection details please see Zawierucha et al. (2013Zawierucha et al. ( , 2016aZawierucha et al. ( , 2016b.

Genotyping
The DNA was extracted from individual animals following a Chelex® 100 resin (Bio-Rad) extraction method by Casquet et al. (2012) with modifications described in detail in Stec et al. (2015). We sequenced four DNA fragments: the small ribosome subunit (18S rRNA, nDNA), the large ribosome subunit (28S rRNA, nDNA), the internal transcribed spacer (ITS-2, nDNA), and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015); primers and original references for specific PCR programs are listed in Table 1. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of Jagiellonian University, Kraków, Poland. Sequences were processed in BioEdit ver. 7.2.5 (Hall 1999) and submitted to GenBank.

Comparative molecular analysis
For molecular comparisons, all published sequences of the four above-mentioned markers for species of the hufelandi complex were downloaded from GenBank (Appendix 1). To compare sequences of the new Tenuibiotus species we used all sequences of T. voronkovi published by Zawierucha et al. (2016a). The sequences for the M. hufelandi complex and two Tenuibiotus species were aligned separately using the default settings (in the case of ITS-2 and COI) and the Q-INS-I method (in the case of ribosomal markers: 18S rRNA, 28S rRNA) of MAFFT ver. 7 (Katoh et al. 2002;Katoh & Toh 2008) and manually checked against non-conservative alignments in BioEdit. Then, the aligned sequences were trimmed to: 763 (18S rRNA), 714 (28S rRNA), 395 (ITS-2) and 618 (COI) bp for the M. hufelandi complex and 999 (18S rRNA), 737 (28S rRNA), 376 (ITS-2) and 584 (COI) bp for two Tenuibiotus species. All COI sequences were translated into protein sequences in MEGA7 version 7.0 (Kumar et al. 2016) to check against pseudogenes. Uncorrected pairwise distances were calculated using MEGA7 and are provided as a supplementary file (SM.03).

Description
Animals (measurements and statistics in Table 2) Body transparent in juveniles and whitish in adults, after fixation in Hoyer's medium transparent (Fig. 1A). Eyes present, visible also in specimens mounted in Hoyer's medium. Cuticle porous with two types of pores: large (up to 5.0 μm in diameter) lenticular pores of shape resembling paper wrapped candy, with transversal wrinkles in extremities distributed randomly on entire body cuticle and being the biggest on anterior and posterior dorsal region (Figs 1B-C, 2); and small round cuticular pores (0.3-0.7 μm in diameter) scattered in between lenticular pores (Figs 1C, 2B). Patches of granulation on all legs present (Fig. 3). A patch of clearly visible granulation is present on the external surface of legs I-III ( Fig. 3A-B). A pulvinus present on internal surface of legs I-III, together with a faint cuticular fold covered with faint granulation and paired muscles attachments which are present just below claws (  Claws stout, of the hufelandi type (Fig. 4). Primary branches with distinct accessory points, a common tract and with an evident stalk connecting the claw to the lunula (Fig. 4). Lunulae on all legs smooth (Fig. 4). Cuticular bars under claws are absent. Double muscle attachments are faintly marked under LCM but clearly visible under SEM (Fig. 4A, C). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM (Fig. 4B) and sometimes also on legs I-III, but in this case inverted and not connecting the external and the internal claw (Fig. 4A).
Mouth antero-ventral, followed by ten peribuccal lamellae and a circular sensory lobe, surrounded by the ring of large pores ( ( Fig. 5A). Under LCM, the oral cavity armature is of the patagonicus type, i.e., with only the 2 nd and 3 rd bands of teeth visible ( Fig. 5B-C). However, in SEM all three bands of teeth are visible, with the first band situated at the base of peribuccal lamellae and composed of a 1-2 rows of small, cone-shaped teeth arranged around the oral cavity (Fig. 6). The second band of teeth is situated between the ring fold and the third band of teeth, and comprises 3-6 rows of small cone-shaped teeth (Figs 5B-C, 6). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 5B-C, 6). The third band of teeth is discontinuous and divided into dorsal and ventral portions. Under LCM, the dorsal teeth are seen as three distinct transversal ridges, whereas the ventral teeth appear as two separate lateral transverse ridges and a median roundish tooth ( Fig. 5B-C). In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 6). Under SEM, the margins of the dorsal teeth slightly serrated (Fig. 6A). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small, triangular microplacoid ( Fig. 5A, D-E). The macroplacoid length sequence 2<1. The first macroplacoid has a central constriction, whereas the second macroplacoid is sub-terminally constricted ( Fig. 5D-E). Table 3) Laid freely, yellowish, spherical (Figs 7A, 8A). The surface between processes is of the persimilis type, i.e., with the continuous smooth chorion, never with pores or reticulum (Figs 7F-G, 8). Under PCM labyrinthine layer is visible as dark dots/thickenings on the surface between processes, whereas under SEM the surface is smooth (Figs 7F-G and 8, respectively). Processes are of the inverted goblet shape, with slightly concave trunks and concave terminal discs (

Reproduction
The new species is dioecious. Spermathecae in females as well as testis in males have been found to be filled with spermatozoa, clearly visible under LCM up to 24 hours after mounting in Hoyer's medium. The new species exhibits a male secondary sexual dimorphism trait in the form of evident lateral gibbosities on legs IV (Fig. 9).

DNA sequences
We obtained sequences for all four of the above mentioned DNA markers. Sequences of 18S rRNA and 28S rRNA were represented by single haplotypes, whereas sequences of ITS-2 and COI were represented by two (distance: 0.5%) and three (distance: 1.3-1.8%) haplotypes, respectively:

Etymology
We take great pleasure in dedicating this new species to the friend of the first and third authors. Zandra Maria Skandrup Sigvardt, who recently completed her PhD studies working on crustaceans (Section of Biosystematics) at the Natural History Museum of Denmark in Copenhagen.

Description
Animals (measurements and statistics in Table 4) Body transparent in juveniles and whitish in adults, after fixation in Hoyer's medium transparent (Fig. 10). Eyes present in specimens mounted in Hoyer's medium. Body cuticle without pores but covered with fine granulation including ventral side of the body and all legs (Figs 11-13). Granulation is distributed uniformly on the body (Fig. 11A-B, E) but sometimes, especially in larger specimens, random patches without granulation are present on the body cuticle ( Fig. 11C-D, F). Patches of dense granulation composed of cushions with aggregated granules present on all legs (Figs 12-13). A patch of clearly visible granulation, is present on the external surface of legs I-III just below the claws (Figs 12A,  13A). A pulvinus is absent on the internal surface of legs I-III, whereas a patch of dense granulation is present and wider than the patch on the external leg surface (Figs 12B, 13B). A patch of dense granulation on legs IV is always visible and covers the dorsal and the lateral sides of hind legs (Figs 12C, 13C-D). Claws slender, of the Tenuibiotus type (Fig. 14). Primary branches with distinct accessory points, a long common tract, and with an evident stalk connecting the claw to the very wide lunula (Fig. 14). Lunulae I-III smooth (Fig. 14A, C), whereas lunulae IV exhibit clear dentation (Fig. 14B, D). The horseshoe structure connecting the anterior and the posterior claw is present and is visible only in PCM (Fig. 14B).
Mouth antero-ventral, followed by ten peribuccal lamellae (Figs 15A, 16). Bucco-pharyngeal apparatus of the Macrobiotus type (Figs 15A, 17A). Under LCM, only the second and third bands of teeth visible, with the second band being faintly marked (Fig. 15B-C). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of 1-2 rows of small, cone-shaped teeth arranged around the oral cavity (Figs 16,  17B). The second band of teeth is situated between the ring fold and the third band of teeth and comprises 3-6 rows of small, cone-shaped teeth (Figs 15B-C, 16). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 15B-C,16). The third band of teeth is discontinuous and divided into dorsal and ventral portions. Under LCM, the dorsal teeth are seen as three distinct transversal ridges of which the median tooth is triangular and is wedged between the lateral teeth (Fig. 15B). The ventral teeth under LCM appear as three to four separate roundish teeth, largest than those of the second band (Fig.  15C), only sometimes they can be seen as one faintly marked, elongated tooth. In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 16). Under SEM, the medio-dorsal tooth is the largest within the third band and is positioned anteriorly with respect to the lateral teeth (Fig. 16A), whereas the ventral portion consist of cone-shaped teeth of which the lateral ones are larger than the medial ones (Fig. 16B). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small triangular microplacoid (Fig. 15A, D-E). The macroplacoid length sequence is 2<1. The first macroplacoid exhibits central constriction whereas the second macroplacoid is sub-terminally constricted (Figs 15D-E, 17C). Table 5) Laid freely, whitish, spherical or ovoid (Figs 18A-B, 19A). The surface between processes is smooth, with thickenings/striae often radiating from the processes bases (Figs 18B-D, 19B-C, E-F). Under PCM, these thickenings together with labyrinthine layer within chorion are visible as dark dots and lines on the surface between processes, whereas under SEM they are smooth striae coming out of the process bases (Figs 18B-D and 19B-C, E-F, respectively). Under SEM, the surface between processes and between the peribasal striae is covered with micropores ( Fig. 19E-F). Processes are of conical shape, with elongated apices which are sometimes bi-or trifurcated (Figs 18E-H, 19A-D). The labyrinthine layer between the process walls is clearly visible under LCM as a reticular pattern  with sinuous margins (Fig. 18C-D). The elongated meshes decrease in size from the base to the top of the processes (Fig. 18C-D). Under SEM, the surface of the processes is covered with small tubercles, whereas the surface of the elongated apices is smooth (Fig. 18B-E).

Reproduction
The examination of specimens freshly mounted in Hoyer's medium did not revealed any spermathecae or testes filled with spermatozoa. Also, male secondary sexual dimorphism traits such as lateral gibbosities on legs IV were absent. Thus, reproductive mode could not be unambiguously determined.

Amended description of Tenuibiotus voronkovi
The examination of the holotype and paratype of T. voronkovi under LCM revealed the presence of granulation on the body cuticle. Faint granulation with small and uniformly sized granules is regularly arranged and covers the dorso-medial region of the body, from its cephalic to the caudal end (Fig. 20A). On the dorso-lateral surface of the body, granulation is unevenly distributed and resembles patches of granules, within which granule size gradually increases in the dorsal to lateral direction (Fig. 20B). The granulation is absent or not visible in LCM on the ventral side of the body. Similarly, the granulation is absent on the legs except a typical dense granulation patch on the external and internal surface of the legs near the claws. However, note that this observation was made on two different specimens (not very well oriented/positioned and stretched on the slide) thus, the certain distribution pattern of body granulation requires a further examination when a new population of T. voronkovi becomes available. As in the original description, the eggs of T. voronkovi have small conical processes with elongated and flexible apices which are often folded and not clearly visible or even broken. (Figs 21A,22). The process walls are smooth, without any obvious thickenings or tubercles (Fig. 22) but with obvious annulation and with the labyrinthine layer within process walls, visible under LCM as roundish polygonal reticulation (Fig. 21C, E-F), on one egg being abnormally developed and visible more like pores than true reticulation (Fig. 21D). Under SEM, the surface between processes is covered with short irregular striae/ridges/wrinkles which often radiate from the process bases, with small micropores randomly scattered in between them (Fig. 22). However, under PCM the surface is visible as being covered with dark dots which are probably the thickenings of the labyrinthine layer within the chorion (Fig. 21B).
The morphological analysis conducted on two populations designated as "T. voronkovi" by Zawierucha et al. (2016a), from Edgeøya and Nordaustlandet (islands within the Svalbard archipelago, Norway), showed distinct differences in cuticle morphology in comparison to the T. voronkovi type series. Specifically, animals of the Edgeøya population exhibit faint, dense and uniformly distributed granulation on the whole dorso-lateral cuticle from its cephalic to the caudal end (excluding ventral and leg cuticle) (Fig. 23A), whereas this granulation is absent or not visible under LCM in animals of the Nordaustlandet population. The morphology of egg processes in both these populations is very similar: specifically, processes are of a conical shape with elongated apices, with the labyrinthine layer between the process walls clearly visible under LCM as a reticular pattern with sinuous margins and elongated meshes decreasing in size from the base to the processes top in most cases (Figs 23B-D, 24). Other traits are as described by Zawierucha et. al. (2016a) however, it should be noted that as for the similarly to T. voronkovi, no more conclusions can be made based on this material due to the bad condition of the slides, with bubbles of air and crystalized Hoyer which prevent further investigation and limit the number of specimens suitable for imaging.

Discussion
Phenotypic differential diagnosis of Macrobiotus engbergi sp. nov.
Macrobiotus engbergi sp. nov., by having the patagonicus type of oral cavity armature (i.e., with only the 2 nd and 3 rd bands of teeth in the oral cavity visible under LCM), the persimilis type of egg shell ornamentation (i.e., with a continuous smooth chorion, never with pores or reticulum) and large lenticular pores within the body cuticle is most similar to a species recently discovered in Ecuador,  (Tumanov, 2007). Body granulation seen in PCM. A. Uniformly distributed granulation of uniform size on the dorso-medial part of the body (cephalic region, paratype). B. Patch of dorso-lateral granulation composed of granules of different size (holotype). Scale bars in μm.

Macrobiotus dulciporus
processes (microgranulation absent in M. dulciporus; this character is visible only in SEM), the presence of a smooth chorion surface between the processes under SEM (the surface under SEM slightly wrinkled with 1-2 obvious rows of peribasal thickenings/tubercles in M. dulciporus). Remarks: The new species has a secondary sexual dimorphic trait in the form of gibbosities on legs IV in males, and this trait has not been seen in the population of M. dulciporus, since males were not observed. Thus, this character cannot be used for differentiation until the reproduction mode of M. dulciporus is ascertained.
The ranges of uncorrected genetic p-distances between the new species and species of the Macrobiotus hufelandi complex, for which sequences are available from GenBank, are as follows (from the most to the least conservative):   (Tumanov, 2007), egg chorion morphology seen in SEM. A. Entire egg. B-D. Details of the egg processes and surface between them. Filled flat arrowheads indicate thickenings/ striae on the surface between processes, filled indented arrowheads indicate elongated and flexible apices of egg processes which are often folded, whereas empty indented arrowheads indicate micro pores on the chorion surface between processes. Scale bars in μm.

Phenotypic differential diagnosis of Tenuibiotus zandrae sp. nov.
Tenuibiotus zandrae sp. nov., by having two macroplacoids as well as elongated and sharp apices of the egg processes, is most similar to seven Tenuibiotus species, T. bondavallii (Manicardi, 1989), T. ciprianoi (Guil et al., 2007), T. danilovi (Tumanov, 2007), T. hyperonyx (Maucci, 1983), T. kozharai (Biserov, 1999), T. mongolicus (Maucci, 1988) and T. voronkovi (Tumanov, 2007), but it differs specifically from: Tenuibiotus bondavallii, known only from a few Arctic and Sub-arctic localities in Canada (including type locality) and Russia (Manicardi 1989;Biserov 1996;Dudichev & Biserov 2000;Sutcliffe & Blake 2000;Kaczmarek et al. 2016), by the absence of small areoles on the chorion surface between the processes (one row of small areoles surrounding the egg processes present in T. bondavallii); Tenuibiotus ciprianoi, known only from its type locality in Spain (Guil et al. 2007), by: the presence of dense granulation patches on the internal surface on legs I-III (internal leg surface smooth in T. ciprianoi), the processes distant from each other enabling the observation of the chorion surface covered Fig. 23. Tenuibiotus cf. voronkovi (Tumanov, 2007) from the Edgeøya population, body cuticle and eggs seen in PCM. A. Uniformly distributed granulation on the dorsal side of the body at the level between leg pairs II and III. B-D. Three different eggs under 1000× magnification. Scale bars in μm.
with thickenings between processes, visible under LCM as dots and lines (chorion surface between processes invisible under LCM due to small distance between neighbouring processes, which are almost always in contact to each other in T. ciprianoi), the absence of a bubble-like structure/reticulation system in the elongated apices of the egg processes (the bubble like structure/reticulation system present in T. ciprianoi), and by wider egg process bases (13.6-29.6 µm in the new species vs 6.9-9.9 µm in T. ciprianoi); Tenuibiotus danilovi, known only from its type locality in Kyrgyzstan (Tumanov 2007 (Tumanov, 2007) from the Nordaustlandet population, eggs seen in PCM. Four different eggs under 1000× magnification. Scale bars in μm.
T. danilovi), higher egg processes (16.8-38.9 µm in the new species vs 12.0-13.5 µm in T. danilovi) and by wider egg process bases (13.6-29.6 µm in the new species vs 8.0-8.5 µm in T. danilovi); Tenuibiotus hyperonyx, known only from its type locality in Italy (Maucci 1983), by: the absence of pores in the body cuticle (the pores are present in T. hyperonyx), the presence of a microplacoid in the pharynx (the microplacoid absent in T. hyperonyx), the presence of reticulation on the egg processes caused by the labyrinthine layer (the reticulation absent in T. hyperonyx), higher egg processes (16.8-38.9 µm in the new species vs 10.0-11.0 µm in T. hyperonyx) and by wider egg process bases (13.6-29.6 µm in the new species vs 5.0-6.0 µm in T. hyperonyx); Tenuibiotus kozharai, known only from its type locality in Turkmenistan (Biserov 1999), by: the presence of well-developed and distinct accessory points on the primary branches of all claws (the accessory points weakly developed, short and connected with primary branches over almost their entire length in T. kozharai), higher egg processes (16.8-38.9 µm in the new species vs 5.0-9.0 µm in T. kozharai), and by wider egg process bases (13.6-29.6 µm in the new species vs 6.0-6.5 µm in T. kozharai); T. mongolicus, known only from its type locality in Mongolia (Maucci 1988), by: the presence of eyes (eyes absent in T. mongolicus), the presence of continuous granulation on the body cuticle (granulation on body cuticle absent in T. mongolicus; note: similarly to the new species, specimens of T. mongolicus were also observed under SEM by Maucci (1988)), different morphology of claws (not very elongated primary branches of all claws which after furcation with secondary branches at a right angle from the common tract extend immediately horizontally in the opposite direction as the secondary branch in the new species vs. primary braches clearly elongated which after furcation with secondary branches at a right angle from the common tract extend immediately almost vertically in T. mongolicus), the presence of faintly marked tubercles on the surface of the egg processes (the surface of process walls smooth, without tubercles in T. mongolicus; this character is visible only in SEM) and slightly higher egg processes (16.8-38.9 µm (average 28.3 ± 3.9 µm) in the new species vs 15.0-17.0 µm in T. mongolicus); Tenuibiotus voronkovi, known only from its type locality in Spitsbergen (Norway) (Tumanov 2007, please see also notes on this species below), by: the presence of granulation uniform in size on the entire body cuticle (the granulation present only in dorsal and dorso-lateral regions, in the latter being composed of variable-sized granules in T. voronkovi), the presence of inflexible, stout, not very elongated apices of the egg processes (the apices are very flexible, narrow and more elongated in T. voronkovi), the presence of reticulation caused by the labyrinthine layer within process walls with elongated meshes decreasing in size from the base to the process top (roundish polygonal reticulation with uniform size of meshes present in T. voronkovi), the presence of faintly marked tubercles on the process wall surface and egg processes without annulation (the surface of process walls smooth, without tubercles, but with obvious annulations in T. voronkovi; this character is visible only in SEM), by a denser distribution of micropores between the striae/ridges on the chorion surface between processes (micropores with lower density scattered randomly on the chorion surface between processes in T. voronkovi; this character is visible only in SEM), higher processes (16.8-38.9 µm in the new species vs. 9.6-14.8 µm in T. voronkovi) and a lower number of processes on the egg circumference (13-15 in the new species vs. 20-22 in T. voronkovi). Remarks: for this comparison, we only used the original description of T. voronkovi, type specimens and a newly found egg from the original sample, as the designation of two additional populations in the redescription by Zawierucha et al. (2016a) as "T. voronkovi" is uncertain. See also specific notes below.
To date, DNA sequences of only one Tenuibiotus species, namely of a population identified by Zawierucha et al. (2016a) as "T. voronkovi", have been published and we used all of them in this comparison: 18S rRNA: no differences in the analysed fragment between the new species and T. voronkovi (KX810045); 28S rRNA: 0.3% and 0.4% between the new species and two haplotypes of T. voronkovi (KX810049-50, respectively); ITS-2: 2.4% between the new species and all three haplotypes of T. voronkovi (KX810046-48); COI: 14.4%, 15.3% and 13.7% between the new species and all three haplotypes of T. voronkovi (KX810042-44, respectively).

Notes on Tenuibiotus voronkovi (Tumanov, 2007)
Tenuibiotus voronkovi was described from Ny Ålesund (Konigsfjorden, Spitsbergen, Norway) based on two animals and five eggs. Since the animals from the original description have destroyed claws on the hind legs and there was a lack of complete morphometric data for the buccal tube and claws, Zawierucha et al. (2016a) aimed to redescribe this species. They supplemented the original description with a reexamination of the paratype and one egg from the type series alongside with additional animals and eggs from three new populations from Spitsbergen, Nordaustlandet and Edgeøya (all within Svalbard Archipelago, Norway). Together with a morphological redescription, they also provided DNA sequences of four molecular markers (18S rRNA, 28S rRNA, ITS-2 and COI) which were obtained from specimens of the Nordaustlandet population. In this study, we once again re-examined the type series of T. voronkovi together with one additional egg (SEM observation) from the original sample in which this species was discovered (Tumanov 2007). We also examined the slides with animals and eggs of the Nordaustlandet and the Edgeøya populations studied by Zawierucha et al. (2016a). Although the holotype and the paratype are partially destroyed and not optimally oriented on the slides, and even though the slides with specimens from Nordaustlandet and Edgeøya are in bad condition, we were able to note some important morphological differences between the type series and the other two populations. Specifically, the holotype and the paratype of T. voronkovi exhibit fine granulation on the dorso-medial and dorso-lateral cuticle that seems to be unevenly distributed from the cephalic to the caudal end of the body (Fig. 20A-B). Fine granulation is also present on the dorsal and dorso-lateral body cuticle in specimens from Edgeøya, but here its distribution and size are uniform (Fig. 23A). However, granulation on the body cuticle is absent or invisible in specimens of the Nordaustlandet population. The examination of eggs of T. voronkovi showed that the processes exhibit very narrow, elongated and flexible apices (Figs 21A-J, 22A-D), which is in contrast to the eggs from Nordaustlandet and Edgeøya, which are more similar to the eggs of T. zandrae sp. nov. described herein (Figs 18A-H, 19A-F, 23B-D, 24A-D). Nevertheless, the processes presented in figures 17 and 18 in Zawierucha et al. (2016a) are more similar to those of T. voronkovi, with strongly elongated and flexible apices. Moreover, the SEM images of the Tenuibiotus eggs presented by Zawierucha et al. (2013), who examined both populations from Nordaustlandet and Edgeøya, are indeed very similar to those of T. voronkovi in process proportions as well as by having annulation on the processes. These new findings question the designation of additional populations by Zawierucha et al. (2016a) as T. voronkovi. All these observations suggest that the most parsimonious scenario is that the additional material used within the mentioned redescription comprises three separate species: T. voronkovi mixed with T. zandrae sp. nov. (the Edgeøya population), and a new Tenuibiotus species (the Nordaustlandet population). However, since the condition of slides and specimens is bad and only one population (Nordaustlandet) has been genetically characterised, it cannot be excluded that even more species are present there. Thus, also the designation of DNA sequences presented by Zawierucha et al. (2016a) as sequences of T. voronkovi should be treated as invalid. The comparison of these sequences with sequences of T. zandrae sp. nov. revealed a close relationship between these species by a high similarity of the three nuclear markers typically used in tardigrade taxonomy. This pattern has already been noted in some tardigrade species within the genera Richtersius Pilato & Binda, 1989, Paramacrobiotus Guidetti et al., 2009and Mesobiotus Vecchi et al., 2016(Guidetti et al. 2016Stec 2019;Stec et al. 2020). Given that specimens of the Nordaustlandet population are morphologically more similar to T. zandrae sp. nov. than to specimens from the type series of T. voronkovi, it is even more probable that they represent a new distinct species. To summarise, T. voronkovi still urgently needs a detailed redescription based on a new population collected exactly from the locus typicus in Ny Ålesund. Therefore, until new detailed morphological and genetic data for this species are available, the two populations (Nordaustlandet and Edgeøya, respectively) studied by Zawierucha et al. (2016a) and the associated DNA sequences (KX810042-50) should be referred as T. cf. voronkovi.

Conclusions
Thanks to detailed morphological and morphometric analysis as well as integration of these data with DNA sequences of the studied tardigrade populations, we described two tardigrade species of the family Macrobiotidae new for science. Moreover, the re-examination of the type series of T. voronkovi, but also slides with specimens of two Tenuibiotus populations studied by Zawierucha et al. (2016a), enabled us to amend the description of this species. At the same time, our results question the designation of these two populations and the DNA sequences presented in the redescription as representing T. voronkovi.