Redescription of Milnesium alpigenum Ehrenberg, 1853 (Tardigrada: Apochela) and a description of Milnesium inceptum sp. nov., a tardigrade laboratory model Author Morek, Witold Author Suzuki, Atsushi C. Author Schill, Ralph O. Author Georgiev, Dilian Author Yankova, Maria Author Marley, Nigel J. Author Michalczyk, Łukasz text Zootaxa 2019 2019-04-16 4586 1 35 64 journal article 27131 10.11646/zootaxa.4586.1.2 6c27fa6f-8be8-4b9a-8f3f-f6581007b008 1175-5326 2642667 2D01FAB0-53DF-4B89-A891-A51C548D4B72 Milnesium inceptum sp. nov. Figure 2 , Tables 4–5 M. tardigradum : Suzuki 2003 , Schill at al . 2004, Suzuki 2006 , Pfannkuchen et al. 2007 , Schill 2007, Schill & Steinbruck 2007 , Hengherr et al. 2008a , Hengherr et al. 2008b , Jönsson et al. 2008 , Schill & Fritz 2008 , Suzuki 2008 , Takahashi et al . 2008 , Förster et al. 2009 , Hengherr et al. 2009a , Hengherr et al. 2009b , Neumann et al. 2009 , Hengherr et al. 2010 , Mali et al. 2010 , Reuner et al. 2010a , Reuner et al. 2010b , Schökraie et al. 2010 , Shcherbakov et al. 2010 , Grohme et al. 2011 , Förster et al. 2011 , Schökraie et al. 2011 , Wełnicz, et al. 2011 , Beisser, et al. 2012 , Förster et al. 2012 , Schökraie et al. 2012 , Grohme et al. 2013 , Wang et al. 2014 , Jönsson et al. 2016 . M. cf. alpigenum strain Mil.alp_DE.001: Kosztyła et al . (2016) , Morek et al . (2016ab), Stec et al . (2016) . FIGURE 2. Milnesium inceptum sp. nov. A —habitus, ventral view (holotype, PCM). B— dorsal cuticle (holotype, German population, PCM). C— dorsal cuticle with faint pseudopores (specimen from Bulgaria, the white arrowhead indicates the area where the pseudopores are more densely arranged, PCM). D —dorsal cuticle with the barely visible outline of a pseudoplate (paratype, SEM). E —pseudoplate surface (paratype, SEM). F —buccal apparatus (holotype, PCM). G —six peribuccal lamellae with the 4+2 configuration. H —claws I with the cuticular bar below (paratype, PCM). I —claws IV (paratype, PCM). All scale bars in µm. TABLE 4. Measurements (in μm) and the pt values of selected morphological structures of 30 specimens (holotype and paratypes) of Milnesium inceptum sp. nov. from the laboratory culture derived from the type locality, from Tübingen, Germany, mounted in Hoyer’s medium. Individuals were chosen to represent the entire body length range, with as equal representation of all available life stages as possible.

CHARACTER	N	RANGE		MEAN		SD		Holotype
ΜM			pt		ΜM	pt	ΜM	pt	ΜM	pt
Body length	30	326	–	848	1136	–	1588	583	1381	164	122	743	1561
Peribuccal papillae length	13	4.5	–	11.1	17.2	–	21.6	8.6	19.1	2.0	1.4	10.3	21.6
Lateral papillae length	27	3.7	–	10.6	13.4	–	19.8	6.8	15.5	1.9	1.6	8.0	16.8
Buccal tube
Length	30	25.8	–	53.5	–	41.8	–	9.7	–	47.6	–
Stylet support insertion point	30	17.1	–	34.3	61.4	–	69.4	27.1	65.0	5.9	1.8	30.9	64.9
Anterior width	30	8.4	–	20.9	30.8	–	42.9	15.4	36.5	4.4	3.1	20.4	42.9
Standard width	30	7.1	–	19.6	23.1	–	37.8	13.8	32.5	4.2	3.7	18.0	37.8
Posterior width	30	7.4	–	20.1	25.2	–	39.9	14.1	33.0	4.3	3.7	19.0	39.9
Standard width/length ratio	30	23%	–	38%	–	32%	–	4%	–	38%	–
Posterior/anterior width ratio	30	81%	–	99%	–	91%	–	4%	–	93%	–
Claw 1 lengths
External primary branch	27	11.0	–	22.4	35.1	–	45.6	17.7	40.8	3.7	2.9	20.9	43.9
External base + secondary branch	28	8.3	–	17.1	26.8	–	35.2	12.6	30.6	2.9	2.4	15.6	32.8
External spur	16	2.1	–	6.6	7.6	–	14.8	4.6	11.0	1.4	1.9	5.0	10.5
External branches length ratio	25	67%	–	79%	–	74%	–	4%	–	75%	–
Internal primary branch	28	10.0	–	21.4	33.3	–	43.8	16.6	39.6	3.7	2.6	20.7	43.5
Internal base + secondary branch	21	8.2	–	17.4	24.4	–	32.7	11.9	29.7	3.0	2.6	14.7	30.9
Internal spur	19	3.4	–	8.4	11.5	–	15.7	5.8	13.6	1.6	1.4	6.7	14.1
Internal branches length ratio	19	68%	–	86%	–	74%	–	4%	–	71%	–
Claw 2 lengths
External primary branch	29	10.8	–	25.0	37.5	–	50.0	18.3	43.3	4.2	3.1	21.6	45.4
External base + secondary branch	21	8.8	–	17.2	27.4	–	36.7	13.2	31.6	3.1	2.4	?	?
External spur	15	2.9	–	7.1	9.2	–	15.2	4.8	11.8	1.2	1.5	?	?

……continued on the next page TABLE 4. (Continued)

CHARACTER	N	RANGE		MEAN		SD		Holotype
ΜM			pt		ΜM	pt	ΜM	pt	ΜM	pt
External branches length ratio	20	67%	–	81%	–	73%	–	4%	–	?	–
Internal primary branch	28	10.6	–	22.9	38.8	–	46.3	17.4	42.4	4.0	2.3	20.0	42.0
Internal base + secondary branch	16	8.3	–	17.7	27.3	–	33.6	12.3	30.7	3.3	2.1	15.3	32.1
Internal spur	16	3.6	–	9.1	12.1	–	18.0	6.3	14.8	1.8	1.7	6.2	13.0
Internal branches length ratio	14	68%	–	77%	–	71%	–	2%	–	77%	–
Claw 3 lengths
External primary branch	30	11.7	–	24.0	38.8	–	51.2	18.4	44.2	4.1	2.8	21.7	45.6
External base + secondary branch	26	8.5	–	18.2	27.6	–	36.3	13.5	32.0	3.2	2.2	15.7	33.0
External spur	15	3.3	–	7.9	8.9	–	14.8	5.0	12.0	1.5	1.9	?	?
External branches length ratio	26	67%	–	80%	–	72%	–	3%	–	72%	–
Internal primary branch	28	10.6	–	22.2	37.3	–	47.7	17.4	42.5	4.0	2.6	20.0	42.0
Internal base + secondary branch	11	8.3	–	14.9	25.9	–	33.8	10.6	30.4	2.4	2.3	?	?
Internal spur	14	3.7	–	9.0	13.1	–	17.8	5.9	15.1	1.7	1.5	6.5	13.7
Internal branches length ratio	11	62%	–	78%	–	70%	–	5%	–	?	–
Claw 4 lengths
Anterior primary branch	29	13.3	–	27.9	45.9	–	59.5	22.2	52.7	4.9	3.3	24.7	51.9
Anterior base + secondary branch	29	8.8	–	19.9	32.8	–	40.2	15.2	35.7	3.7	2.0	17.6	37.0
Anterior spur	16	2.6	–	8.5	7.4	–	18.1	5.1	12.0	1.7	2.7	?	?
Anterior branches length ratio	28	61%	–	76%	–	68%	–	4%	–	71%	–
Posterior primary branch	27	12.1	–	28.9	44.1	–	58.4	20.9	49.8	5.0	3.2	?	?
Posterior base + secondary branch	26	8.5	–	20.1	29.7	–	40.3	14.3	34.4	3.6	2.6	17.4	36.6
Posterior spur	25	3.0	–	9.8	11.6	–	19.2	6.7	15.9	2.0	1.8	7.0	14.7
Posterior branches length ratio	23	61%	–	81%	–	68%	–	4%	–	?	–

Material examined: Type series consisting of 96 specimens (population DE.001) and additional 93 specimens (15 from JP .010 population, 9 from population CH .002, and 69 from BG.058 population). See Table 1 and “ Type repositories” below for details . Integrative description. Females: Body yellowish. Eyes present in live specimens, dissolved after fixation in Hoyer’s medium in 50% of specimens (remained visible in 1/30 = 3% specimens of the German type series, 15/15 = 100% of the Japanese series, 9/ 9 specimens = 100% of the Swiss series, and in 14/ 23 specimens = 61% of the Bulgarian series). Cuticle with very small pseudopores (0.46 ± 0.06µm , detectable only under a high quality PCM) in the German and the Swiss population and with slightly larger (but still small, 0.62 ± 0.06 µm ) pseudopores in the Bulgarian and the Japanese population (detectable under a standard PCM). In the German and the Swiss population, the cuticle on the entire body appears smooth under PCM ( Fig. 2B ), but under SEM a weak outline of a single dorsal pseudoplate is visible in some specimens in the caudal part of the body ( Fig. 2 D–E). In the Bulgarian and Japanese populations, no pseudoplates were detected either under PCM or in SEM. Six peribuccal papillae present, with the ventral being the smallest. Six triangular peribuccal lamellae of unequal size, with the two lateral being noticeable smaller than the two dorsal and the two ventral, i.e. with the 4+2 configuration (identifiable only in SEM; Fig. 2G ). Two lateral papillae present. Buccal tube funnel-shaped ( Fig. 2F ). Primary branches with typically developed and clearly visible accessory points. All secondary branches with three points, i.e. with the [3-3]-[3-3] CC ( Fig. 2H and I ). Spurs on secondary branches of moderate length. Cuticular bars under claws I–III present in the majority of examined specimens (23/ 29 specimens = 79% in the German type population, 15/ 15 specimens = 100% in the Japanese population, 7/ 9 specimens = 78% in the Swiss population, and in 11/ 16 specimens = 69% in the Bulgarian population; Fig. 2H ). Males: No males were found in German, Swiss, or Bulgarian populations and culturing of isolated virgin females confirmed that the type population is parthenogenetic. However, males were found to appear spontaneously in an otherwise parthenogenetic culture of the Japanese strain ( Suzuki 2008 ). This suggests that the species is facultatively parthenogenetic with males appearing only occasionally. Juveniles: Morphologically identical to adults, except for the lack of the cuticular pseudopores. Hatchlings: Morphologically identical to adults, except for the lack of cuticular bars under claws I–III in the majority of examined hatchlings (14/ 15 specimens = 93%), and the absence of cuticular pseudopores. Ontogenetic variability: No developmental variability in the CC. Pseudopores visible only in adults. Cuticular bars under claws I–III mostly absent in hatchlings but usually present in juveniles and adults. Eggs: Oval, yellow, smooth and laid in the exuviae, up to 18 in a single clutch were found in laboratory culture. DNA markers: All sequences were of a very good quality. The 18S rRNA and 28S rRNA, sequenced only in the German type population, were 1070 bp ( MH000383 ) and 817 bp ( MH000385 ) long, respectively. In ITS-2, two haplotypes were found: H1 was shared by the German, the Japanese and the Swiss population (528 bp long, MH000386 ), whereas H2 was found in the Bulgarian population 528 bp, MH000387 ). The p-distance between the two ITS-2 haplotypes was 0.8%. The COI marker exhibited three haplotypes: H1 shared by the German and the Swiss population (658 bp, KU513422 ), H 2 in the Japanese population (580 bp, MK628723 ), and H 3 in the Bulgarian population (647 bp, MH000381 ). The p-distances between the COI haplotypes were as follows: 0.5% (H1 vs H2 and H1 vs H3), and 0.3% (H2 vs H3). Sequences with marked differences are provided in Appendix 1. Morphology and genetic markers: The sample size of four populations does not allow us to formulate strong conclusions on the relationship between genetic markers and animal morphology. Nevertheless, it should be noted that populations with COI H1 (DE.001 and CH .002) exhibited statistically smaller pseudopores than populations with COI H2 (JP.010) and H3 (BG.058): 0.46 ± 0.06 µm (DE.001) vs 0.62 ± 0.06 µm (BG.058), t 28 =7.450, p <0.001. No associations were observed between ITS-2 haplotypes and phenotypic taxonomic traits. Type locality: 48°33'42''N , 09°03'48''E ; 377 m asl : Germany , Tübingen , Bebenhausen ; forest; moss on soil . Etymology: The name of the new species originates from the Latin “ inceptor ”, meaning “an initiator”, or “a pioneer”, as this species was among the very first tardigrade laboratory models. Milnesium inceptum sp. nov. has been used in a number of studies, including first studies on molecular mechanisms underlying cryptobiosis. Type repositories: The type series consist of the holotype (slide DE.001.34) and 96 paratypes representing hatchlings, juveniles and adult females (slides DE.001.01–33). The holotype (DE.001.34) with 14 paratypes (DE.001.04–07; 32–33) are preserved at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland ; 18 paratypes (DE.001.08–13) are deposited in Department of Animal Taxonomy and Ecology , Adam Mickiewicz University , Poznań , Umultowska 89, 61-614 Poznań , Poland ; 18 paratypes are deposited in the Department of Zoology , Institute of Biomaterials and Biomolecular Systems , Stuttgart University , Germany (DE.001.14–19) , 18 paratypes (DE.001.20–25) are stored in Marine Biology & Ecology Research Centre , Plymouth University , Drakes Circus , Plymouth , PL4 8AA, United Kingdom ,, one paratype (DE.001.34) is deposited in Natural History Museum , Cromwell Road , London SW 7 5BD, United Kingdom , 18 paratypes (DE.001.26–31) are deposited in Department of Ecology and Environmental Conservation , Faculty of Biology , University of Plovdiv , Tzar Assen 24, BG-4000 Plovdiv , Bulgaria and the remaining 9 (DE.001.01–03) are deposited in the collection of Binda & Pilato , Museum of the Department of Biological , Geological and Environmental Sciences , Section of Animal Biology “Marcello La Greca”, University of Catania , Italy . TABLE 5. Measurements (in µm) and the pt values of selected morphological structures of 75 specimens of Milnesium inceptum sp. nov. from the type locality in Tübingen (Germany) and the additional localities from Hiyoshi (Japan), Zürich (Switzerland), and Kazanlak Valley (Bulgaria) mounted in Hoyer’s medium. Individuals were chosen to represent the entire body length range, with as equal representation of all available life stages as possible.

CHARACTER	N	RANGE		MEAN		SD
µm	pt	µm	pt	µm	pt
Body length	75	326–998	1136–1841	628.4	1493	181	164
Peribuccal papillae length	48	4.4–13.1	14.9–24.0	8.2	19.0	2.1	1.7
Lateral papillae length	69	3.4–11.2	11.8–21.6	7.1	16.5	2.1	2.1
Buccal tube
Length	75	25.8–56.2	–	41.6	9.1
Stylet support insertion point	73	17.1–36.4	59.0–71.6	26.9	65.5	5.4	2.4
Anterior width	75	8.4–23.8	28.5–45.2	15.5	36.8	4.4	4.0
Standard width	73	7.1–21.2	23.1–41.7	13.5	32.2	4.2	4.5
Posterior width	75	7.4–22.1	25.2–42.7	13.8	32.6	4.1	4.4
Standard width/length ratio	73	23%–42%	–	32%	5%
Posterior/anterior width ratio	75	74%–101%	–	87%	7%
Claw 1 lengths
External primary branch	65	11.0–26.1	34.2–51.4	18.5	43.2	4.2	3.7
External base + secondary branch	61	8.3–19.7	26.8–38.0	12.8	32.0	3.1	2.8
External spur	31	2.1–6.8	7.6–14.8	4.4	11.3	1.3	1.8
External branches length ratio	53	67%–81%	–	73%	4%
Internal primary branch	70	10.0–24.8	32.5–50.2	17.5	42.0	4.2	3.5
Internal base + secondary branch	57	8.2–21.2	24.4–38.8	12.7	31.5	3.5	3.1
Internal spur	43	3.0–8.5	10.8–16.8	5.7	13.8	1.7	1.5
Internal branches length ratio	54	68%–88%	–	73%	5%
Claw 2 lengths
External primary branch	72	10.8–28.4	37.5–56.8	19.6	46.7	4.8	4.5
External base + secondary branch	59	8.8–22.8	27.4–41.8	13.7	33.5	3.4	2.9
External spur	36	2.9–8.2	9.2–16.6	5.2	12.9	1.6	2.0
External branches length ratio	57	64%–83%	–	70%	4%
Internal primary branch	70	10.6–27.6	38.8–55.0	18.8	45.5	4.8	4.3
Internal base + secondary branch	52	8.3–21.0	27.3–40.7	13.3	32.9	3.7	2.9
Internal spur	42	3.2–20.1	11.8–36.8	6.8	16.4	2.8	4.0
Internal branches length ratio	49	59%–81%	–	70%	5%

......continued on the next page TABLE 5. (Continued)

CHARACTER	N	RANGE		MEAN		SD
µm	pt	µm	pt	µm	pt
Claw 3 lengths
External primary branch	69	11.7–27.7	38.8–54.5	19.6	46.9	4.7	4.3
External base + secondary branch	64	8.5–21.9	27.6–42.4	13.9	33.9	3.3	3.0
External spur	37	3.3–8.7	8.9–16.8	5.1	12.4	1.6	1.8
External branches length ratio	59	64%–90%	–	71%	6%
Internal primary branch	70	10.6–27.2	37.3–55.7	18.7	45.5	4.6	4.4
Internal base + secondary branch	42	8.3–20.5	25.9–39.5	13.0	33.2	3.8	2.9
Internal spur	44	3.3–11.0	11.7–21.4	6.6	16.2	2.1	2.4
Internal branches length ratio	40	62%–86%	–	71%	6%
Claw 4 lengths
Anterior primary branch	67	13.3–34.1	44.2–65.7	23.1	55.5	5.4	4.7
Anterior base + secondary branch	63	8.8–24.2	31.7–46.2	15.5	37.5	4.0	3.2
Anterior spur	45	2.6–11.1	7.4–22.8	6.0	14.1	2.3	3.9
Anterior branches length ratio	59	61%–76%	–	67%	4%
Posterior primary branch	64	12.1–32.7	44.1–65.7	22.5	54.3	5.5	5.8
Posterior base + secondary branch	59	8.5–22.8	29.7–43.8	15.2	36.7	4.0	3.6
Posterior spur	48	3.0–10.7	11.6–20.6	6.9	16.4	1.9	2.4
Posterior branches length ratio	55	60%–81%	–	66%	4%

Phenotypic differential diagnosis. Milnesium inceptum sp. nov. has the [3-3]-[3-3] CC and “smooth” cuticle ( i.e. cuticle smooth in SEM and with minute pseudopores, but with no sculpturing, such as reticulation, on cuticle surface), which places it in the largest group of Milnesium species that share these characteristics (19 species). Nevertheless, M. inceptum sp. nov. differs from: • M. alpigenum Ehrenberg, 1853 only reported from the type locality in Italy—please see the section “Delineation of M. alpigenum and M. inceptum sp. nov. ” below for a detailed differential diagnosis between these two pseudocryptic species. • M. antarcticum Tumanov, 2006 , only reported from the Antarctic ( Smykla et al. 2012 ), by the maximal length of the buccal tube (<57.0 µm in the new species vs >67.0 µm in M. antarcticum ), by a lower buccal tube standard width ( 7.1–21.2 µm in the new species vs 25.9–31.8 µm in M. antarcticum ), and by a statistically lower pt of the stylet support insertion point ( 59.0–71.6 , on average 65.5 in the new species vs 70.0–73.7 , on average 71.5 in M. antarcticum ; t 80 = 20.590, p<0.001). • M. argentinum Roszkowska, Ostrowska & Kaczmarek, 2015 , recorded from Argentina , by the appearance of cuticle (faint pseudopores visible only with a high quality PCM on the caudal part of the dorsal cuticle in the new species vs well-visible pseudopores in M. argentinum on the entire dorsum with a standard PCM), the maximal length of the buccal tube (up to 57 µm in the new species vs up to 74 µm in M. argentinum ), and by a lower pt of the primary branches IV ( 44.1–65.7 in the new species vs 28.4–36.4 in M. argentinum ). • M. asiaticum Tumanov, 2006 , recorded from Kirghizstan ( type locality), China ( Beasley & Miller 2007 ), Estonia ( Zawierucha et al. 2014 ), and the Svalbard archipelago ( Kaczmarek et al. 2012 ), by a statistically lower pt of primary branches IV ( 44.1–65.7 , on average 54.8 in the new species vs 63.9–76.0 , on average 69.7 in M. asiaticum ; t 54 = 26.040, p<0.001). • M. barbadosense Meyer & Hinton, 2012 , only reported from the type locality in Barbados , by a lower pt of the stylet support insertion point ( 59.0– 71.6 in the new species vs 71.6–82.1 in M. barbadosense ), and by a higher pt of the primary branches IV ( 44.1–65.7 in the new species vs 28.4–42.2 in M. barbadosense ). • M. beatae Roszkowska, Ostrowska & Kaczmarek, 2015 , only reported from the type locality in Argentina , by the appearance of cuticle (faint pseudopores visible only with a high quality PCM on the caudal part of the dorsal cuticle in the new species vs well-visible pseudopores in M. argentinum on the entire dorsum with a standard PCM), and by more elongated buccal tube (standard width/length ratio 23 – 42% in the new species vs standard width/length ratio 58–66% in M. beatae ) • M. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014 , recorded from North Carolina and Tennessee , USA , by the more slender buccal tube (standard width/length ratio 23 – 42% in the new species vs standard width/length ratio 54–64% in M. bohleberi ). • M. brachyungue Binda & Pilato, 1990 , reported from the type locality in Chile and south Argentina ( Roszkowska et al. 2016 ), by a higher pt of primary branches of claws I–III ( 32.5–56.8 in the new species vs 22.9–27.1 in M. brachyungue ) and by the pt of primary branches IV ( 44.1–65.7 in the new species vs 33.1 in M. brachyungue ). • M. burgessi Schlabach, Donaldson, Hobelman, Miller & Lowman, 2018 , recorded from Kansas , USA , by a higher pt of the buccal tube standard width ( 23.1–41.7 in the new species vs 52.9–68.5 in M. burgessi ) and by the lower pt of primary branches IV ( 44.1–65.7 in the new species vs 66.6–96.2. in M. burgessi ). • M. dornensis Ciobanu, Roszkowska & Kaczmarek, 2015 , recorded from Romania ( type locality), Poland ( Kaczmarek et al. 2018 ) and Tunisia ( Gąsiorek et al. 2017b ), by the appearance of cuticle (faint pseudopores visible only with a high quality PCM on the caudal part of the dorsal cuticle in the new species vs well-visible pseudopores in M. dornensis on the entire dorsum with a standard PCM), and by a statistically lower pt of buccal tube standard width ( 23.1–41.7 , on average 32.2 in the new species vs 37.8–51.6, on average 44.1 in M. dornensis ; t 22 = 10.686, p<0.001). • M. eurystomum Maucci, 1991 , recorded from Greenland ( type locality), Argentina and Chile ( Maucci 1996 ), and Mongolia ( Kaczmarek & Michalczyk 2006 ), by a more slender buccal tube (standard width/length ratio 23 – 42% in the new species vs standard width/length ratio 62–65% in M. eurystomum ). • M. longiungue Tumanov, 2006 , reported from the Himalayas ( India , type locality) and China ( Beasley & Miller 2007 ), by the presence of accessory points on primary branches, a lower pt of primary branches III ( 37.3–55.7 in the new species vs 57.1–73.5 in M. longiungue ), and by the lower pt of primary branches IV ( 44.1–65.7 in the new species vs 81.8–92.4 in M. longiungue ). • M. minutum Pilato & Lisi, 2016 , only reported from the type locality in Sicily , by a statistically lower pt of the buccal tube standard width ( 23.1–41. 7, on average 32.2 in the new species vs 38.6–42.4 , on average 41.1 in M. minutum ; t 3 = 7.990, p=0.002). • M. sandrae Pilato & Lisi, 2016 , only reported from the type locality in Hawaii , by a higher pt of the stylet support insertion point ( 59.0–71.6 , on average 65.5 in the new species vs 58.0–60.5 , on average 58.9 in M. sandrae ; t 22 = 8.506, p<0.001) and by a lower pt of the buccal tube standard width ( 23.1–41. 7 in the new species vs 44.9–48.0 in M. sandrae ). • M. shilohae Meyer, 2015 , only reported from the type locality in Hawaii , by a lower pt of the stylet support insertion point ( 59.0– 71.6 in the new species vs 75.5–77.5 in M. shilohae ) and by a lower pt of the buccal tube standard width ( 23.1–41.7 in the new species vs 47.1–55.9 in M. shilohae ). • M. swansoni Young, Chappell, Miller & Lowman, 2016 , only reported from the type locality in the USA , by a higher number of peribuccal lamellae (six in the new species vs four in M. swansoni ; but note that the number of peribuccal lamellae in M. swansoni was determined only with a PCM) and by a lower pt of the buccal tube standard width ( 23.1–41. 7, on average 32.2 in the new species vs 39.2–42.2 , on average 40.3 in M. swansoni ; t 12 = 10.325, p<0.001). • M. tumanovi Pilato, Sabella & Lisi, 2016 , only reported from the type locality in Crimea, by a higher pt of the stylet support insertion point ( 59.0– 71.6 in the new species specimens being 326–998 µm long vs 52.3 in M. tumanovi in a specimen 774 µm long) and by a lower pt of the buccal tube standard width ( 23.1–41.7 in the new species in specimens 326–998 µm long vs 55.1 in M. tumanovi in a specimen 774 µm long). • M. validum Pilato, Sabella, D’Urso & Lisi, 2017 , only reported from the type locality in the Antarctic; according to measurements presented in the description of M. validum all pt ranges overlap, but a comparison between specimens of similar body length ( 393–513 µm in the new species and 424–482 µm in M. validum ) shows that M. inceptum sp. nov. has a shorter buccal tube (27.1–39.0 µm in the new species vs 44.1–55.6 in M. validum ), moreover the two species differ in the shape of the secondary branches (typical in the new species vs robust in M. validum , compare Fig. 2 H–I here and Fig. 6B–D in Pilato et al. 2017 ), and in the shape of spurs (moderate length and of normal width in the new species vs long and very thin in M. validum ). • M. zsalakoae Meyer & Hinton, 2010 , recorded from Arizona and New Mexico ( USA ), by the presence of accessory points on primary branches, by a lower pt of primary branches I–III ( 32.5–56.8 in the new species vs 64.4–88.6 in M. zsalakoae ) and by a lower pt of primary branches IV ( 44.1–65.7 in the new species vs 94.8– 102.9 in M. zsalakoae ). Genotypic differential diagnosis: Four sequences deposited in GenBank prior to this publication, labelled as “ M. tardigradum ”, in fact represent M. inceptum : two ITS-2 ( GQ403681 –2) and two COI ( EU244603 –4) (all by Schill, unpublished). The GQ403683 and EU244604 sequences originated from Germany and represent the same laboratory strain that was utilised herein to describe the new species. The sequences GQ403682 and EU244603 originated from Japan and represent the Japanese strain, also used in the present study. The ranges of uncorrected p-distances between the new species and sequences of other congeners are as follows: • 18S rRNA: 1.1%–3.9% (2.9% on average), with the most similar being M. alpigenum , ( MG996146 , present study) and the least similar being an undetermined species from the USA ( GQ925696 , Chen et al. unpublished) as well as an undetermined species from South Georgia in the sub-Antarctic ( EU266922 , Sands et al. 2008 ). • 28S rRNA: 0.4%–8.8% (6.0% on average), with the most similar being an undetermined species from the USA ( AY 210826 , Mallatt et al . unpublished) and another undetermined species also from the USA ( JX888540 –1, Adams et al. unpublished) and the least similar being an undetermined species from Spain ( FJ435779 –80, Guil & Giribet 2012 ). • ITS-2: 19.6%–22.8% (20.3% on average), with the most similar being M. tardigradum s.s. from Hungary and Poland ( MG923553 , Morek et al. 2019 ) and the least similar being M. tardigradum s.s. from France ( MG923555 , Morek et al. 2019 ). • COI: 17.8%–25.8% (19.7% on average), with the most similar being M. dornensis from Romania ( MG923566 , Morek et al. 2019 ) and an undetermined species from the USA ( KX306950 , Fox et al ., unpublished) whereas the least similar being two undetermined species from the Antarctic ( KP013601 and KP013598 , Velasco-Castrillón et al . 2015 ).