Milnesium inceptum
sp. nov.
Figure 2
View FIGURE 2
, Tables 4–5
View TABLE 4
View TABLE 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).
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
View 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
View FIGURE 2
), 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
View FIGURE 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
View FIGURE 2
). Two lateral papillae present. Buccal tube funnel-shaped ( Fig. 2F
View FIGURE 2
). 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
View FIGURE 2
). 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
View FIGURE 2
).
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
View Materials
) and 817 bp (
MH000385
View Materials
) 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
View Materials
), whereas H2 was found in the Bulgarian population 528 bp,
MH000387
View Materials
). 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
View Materials
), H 2 in the Japanese population (580 bp,
MK628723
View Materials
), and H 3 in the Bulgarian population (647 bp,
MH000381
View Materials
). 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
GoogleMaps
.
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
.
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
View FIGURE 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
View Materials
–2) and two COI (
EU244603
View Materials
–4) (all by Schill, unpublished). The GQ403683 and
EU244604
View Materials
sequences originated from Germany and represent the same laboratory strain that was utilised herein to describe the new species. The sequences
GQ403682
View Materials
and
EU244603
View Materials
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
View Materials
, present study) and the least similar being an undetermined species from the USA (
GQ925696
View Materials
, Chen et al. unpublished) as well as an undetermined species from South Georgia in the sub-Antarctic (
EU266922
View Materials
, 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
View Materials
, Mallatt et al. unpublished) and another undetermined species also from the USA (
JX888540
View Materials
–1, Adams et al. unpublished) and the least similar being an undetermined species from Spain (
FJ435779
View Materials
–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
View Materials
, Morek et al. 2019) and the least similar being
M. tardigradum
s.s. from France (
MG923555
View Materials
, Morek et al. 2019).
• COI: 17.8%–25.8% (19.7% on average), with the most similar being
M. dornensis
from Romania (
MG923566
View Materials
, Morek et al. 2019) and an undetermined species from the USA (
KX306950
View Materials
, Fox et al., unpublished) whereas the least similar being two undetermined species from the Antarctic (
KP013601
View Materials
and
KP013598
View Materials
, Velasco-Castrillón et al. 2015).
