Milnesium pseudotardigradum, Surmacz & Morek & Michalczyk, 2019

Surmacz, Bartłomiej, Morek, Witold & Michalczyk, Łukasz, 2019, Hylomus cervarius, Zoological Studies 58 (32), pp. 1-15 : 6-12

Order Apochela Schuster et al., 1980 Family Milnesiidae Ramazzotti, 1962 Genus Milnesium Doyère, 1840

Milnesium pseudotardigradum sp. nov. ( Figs. 2-6 , Table 3) urn:lsid:zoobank.org:act:0BE101DC-62F5-44FE-BEF1-06FDA9B4B157

Integrative description

Females (morphometrics and holotype measurements in Table 3): Milnesium of moderate length, up to 862 µm ( Fig. 3A, B ), yellowish. Eyes present in alive specimens, but dissolved in 80% of specimens (86/108 of the type series) mounted in Hoyer’s medium. Cuticle smooth without pseudopores or pseudoplates both in PCM ( Fig. 3E ) and SEM ( Fig. 3F ). The six rather short peribuccal papillae present around the mouth opening, with the ventral one being the smallest. Mouth opening with six triangular peribuccal lamellae of unequal size, i.e., the two lateral lamellae are significantly smaller than the pair of dorsal and lateral lamellae, the 4+2 configuration ( Fig. 3D ), which is detectable only under SEM. Two short lateral papillae present. Buccal tube funnel shape and of moderate width ( Fig. 3C ). Claws typical for this genus, primary branches with tiny, but well-visible accessory points ( Fig. 4C, D, H ). Typically internal and anterior secondary branches equipped with a basal spur, i.e., with a [2-3]-[3-2] CC ( Fig. 4C, D, G ). In the majority of specimens from the 4th life stages onwards (86% of the type series) additional spurs on external and/or posterior secondary branches may be present ( Fig. 4E, F ). These additional spurs are always smaller than the regular spurs ( Fig. 4H ), but they are characterised by a regular, normal shape, indicating they are not aberrations. The number of extra spurs in a single animal ranges from one to eight resulting in such cases in specimens with a [3-3]-[3-3] CC. This means that the number of spurs can be uneven and different on left and right pairs of claws. Cuticular bars under claws I–III are always present.

Males: No males were found in the sample, and the culture confirmed that the type population is (at least facultatively) parthenogenetic.

Juveniles: Morphologically identical to adults, with the exception that juveniles never exhibit the additional spurs on external and posterior secondary branches of claws, i.e., the CC is always [2-3]-[3-2] ( Fig. 4C, D ). After fixation in Hoyer’s medium eyes dissolved in 22/25 (88%) of the juveniles.

Hatchlings: Morphologically similar to adults and juveniles, apart from the claws, which are characterised by the presence of spurs on each secondary branch i.e., the CC is always [3-3]-[3-3] ( Fig. 4A, B ). After fixation in Hoyer’s medium eyes dissolved in 9/11 (82%) of the hatchlings.

Ontogenetic variability ( Fig. 5 ): The species exhibits ontogenetic variability in CC. Hatchlings have a [3-3]-[3-3] CC, whereas older life stages, from juveniles onwards exhibit a [2-3]-[3-2] CC, thus the species is characterised by an early negative CC change. Moreover, the new species also exhibits a unique feature, never observed in any Milnesium species before: the irregular appearance of additional spurs on the internal and posterior claws in adults from the 4th life stage (second adult instar) onwards (the summary of changes is depicted in figure 4). The additional spurs are more often present on claws II–IV than on claws I (exact frequencies are presented in figure 5. No variability in cuticle sculpture, presence of cuticular bars or eyes was detected.

Eggs: Smooth, oval, slightly yellowish and laid within exuviae, up to 10 in a single clutch found in the culture.

DNA markers: All four markers were characterised by single haplotypes, the sequences were of the following lengths: 18S rRNA: 1032 bp (GenBank: MK484088), 28S rRNA: 786 bp (MK483997), ITS-2: 545 bp (MK484022), and COI: 580 bp (MK492297).

Type locality: 64°08'23"N, 21°52'09"W; 14 m asl: Iceland, Reykjavík Botanic Garden, lichen from stone.

Etymology: The name of the species highlights the similarity between the new species and M. tardigradum .

Type repositories: The series consists of the holotype (IS.008.74) and 107 paratypes (IS.008.03–30; 53 – 56; 70–73; 75–141). All the slides are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland.

Adult and juvenile phenotypic differential diagnosis

Milnesium pseudotardigradum sp. nov. is one of eight described species of Milnesium characterised by a [2-3]-[3-2] adult CC. Since in the culture we found only a single specimens with a [3-3]-[3-3] CC (<1% of all analysed specimens), the differential diagnosis for species with adult [3-3]-[3-3] CC is not provided. The new species differs from all species with a [2- 3]-[3-2] CC by the irregular appearance of external and posterior spurs from the 4th instars onwards (see above). In addition, M. pseudotardigradum sp. nov. differs specifically from:

M. barbadosense Meyer and Hinton, 2012 , known from type locality in Barbados, by relatively longer internal spurs on claws I–III, the difference is most visible in claws III (15.0– 23.5 in the new species vs 3.6–13.2 in M. barbadosense ).

M. beasleyi Kaczmarek, Jakubowska and Michalczyk, 2012 , known only from type locality in Turkey, by the appearance of cuticle (smooth dorsal cuticle in the new species vs well-visible pseudopores in M. beasleyi on the entire dorsum) and by relatively longer internal spurs on claws III (14.1–23.4 in the new species vs 7.7–14.3 in M. beasleyi ).

M. krzysztofi Kaczmarek and Michalczyk, 2007 , reported from Costa Rica and Peru ( Kaczmarek et al. 2014), by the appearance of cuticle (smooth dorsal cuticle in the new species vs reticulated in M. krzysztofi ) and by relatively longer internal spurs on claws III (15.0– 23.5 in in the new species vs 10.8 in M. krzysztofi ).

M. lagniappe Meyer, Hinton and Dupré, 2013 , known from the United States, by the appearance of cuticle (smooth dorsal cuticle in the new species vs pseudoporous in M. lagniappe ) and by a smaller relative buccal tube standard width (25.5–50.8 in in the new species vs 63.4–77.9 in M. lagniappe ).

M. reductum Tumanov, 2006 , known from Kyrgyzstan and China ( Yang 2007), by the presence of accessory points on claw primary branches.

M. reticulatum Pilato, Binda and Lisi, 2002 known from the type locality on Seychelles, by the absence of gibbosities and cuticular reticulation on the dorsum.

M. tardigradum Doyère, 1840 , reported from integratively identified European localities ( Michalczyk et al. 2012a; Morek et al. 2019a) and a single locality in Asia (Morek and Michalczyk, in press), by statistically higher pt values for claw spur heights: Claw I internal spur (10.3–22.0, on average 17.3 in the new species vs 8.70–22.05, on average 14.7 in M. tardigradum ; t 260 = 8.283, p <0.001); claw II internal spur (14.1–23.4, on average 19.5 in the new species vs 9.6–22.5, on average 16.6 in M. tardigradum ; t 279 = 9.292, p <0.001), claw III internal spur (15.0–23.5, on average 19.5 in the new species vs 9.6–24.5, on average 16.4 in M. tardigradum ; t 268 = 9.951, p <0.001), claw IV anterior spur (12.9–23.1, on average 18.9 in the new species vs 10.7–21.8, on average 16.0 in M. tardigradum ; t 257 = 9.323, p <0.001).

M. tetralamellatum Pilato and Binda, 1991 , known from Tanzania, by a higher number of peribuccal lamellae (six in the new species vs four in M. tetralamellatum ) and by a more posterior position of stylets support insertion points (61.8–75.9 in in the new species vs 61.9–64.37 in M. tetralamellatum ).

M. vorax Pilato, Sabella and Lisi, 2016 , reported from Sicily, by a lower pt of the buccal tube standard width (25.5–49.4 in in the new species vs 59.5–60.3 in M. vorax ).

Hatchling phenotypic differential diagnosis

Milnesium pseudotardigradum sp. nov. hatchlings differ from the described species with a [3-3]-[3-3] CC by:

The position of stylet support insertion point: 68.4–75.9 in hatchlings of the new species vs 63.6–66.9 in M. asiaticum Tumanov, 2006 , 64.3–68.1 in M. dornensis Ciobanu, Roszkowska and Kaczmarek, 2015 , 59.1–66.7 in M. longiungue Tumanov, 2006 , 63.0– 65.9 in M. minutum Pilato and Lisi, 2016 , 58.0– 60.5 in M. sandrae Pilato and Lisi, 2016 , 75.5–77.5 in M. shilohae Meyer, 2015 , 66.6–68.2 in M. swansoni Young, Chappell, Miller and Lowman, 2016 , 63.6–70.0 in M. tardigradum hatchlings, 52.3 in M. tumanovi Pilato, Sabella and Lisi, 2016 , 62.0– 65.1 in M. validum Pilato, Sabella, D’Urso and Lisi, 2017 , and 62.0– 64.8 in M. zsalakoae Meyer and Hinton, 2010 .

The pt of the buccal tube anterior width: 32.5– 46.1 in hatchlings of the new species vs 66.7–70.3 in M. beatae Roszkowska, Ostrowska and Kaczmarek, 2015 , 63.4–74.6 in M. bohleberi Bartels, Nelson, Kaczmarek and Michalczyk, 2014 , 56.2–75.5 in M. burgessi Schlabach, Donaldson, Hobelman, Miller and Lowman, 2018 , and 47.5–58.3 in M. shilohae .

The pt of the of primary branches heights of all claws: 41.1–60.4 in hatchlings of the new species vs 22.9–33.1 in M. brachyungue Binda and Pilato, 1990 .

The pt of the claw I external base + secondary branch height: 26.2–34.26 in hatchlings of the new species vs 16.3–20.0 in M. antarcticum Tumanov, 2006 , 22.0– 25.2 in M. argentinum Roszkowska, Ostrowska and Kaczmarek, 2015 , 22.9 in M. brachyungue , 38.8– 55.2 in M. burgessi , and 35.0– 36.6 in M. swansoni Young, Chappell, Miller and Lowman, 2016 .

The posterior/anterior buccal tube width ratio: 68%–100% in hatchlings of the new species vs 62%– 65% in M. eurystomum Maucci, 1991 .

Buccal tube length: 21.6–24.2 µm in hatchlings of the new species vs 32.3–67.5 µm in M. alpigenum, Ehrenberg, 1853 , 24.4–46.1 µm in M. dornensis , 25.8–56.0 µm in M. inceptum Morek et al. 2019b , 25.7–26.4 µm in M. minutum , 28.6–36.3 µm in M. sandrae , 38.4–50.3 µm in M. shilohae , 44.1–71.0 µm in M. swansoni , and 44.1–71.0 µm in M. validum .

Molecular differential diagnosis

The ranges of uncorrected p -distances between the new species and available sequences for other congeners are as follows:

18S rRNA: 1.5%–3.8% (2.7% on average), with the most similar being M. tardigradum , (MK484076 from Russia Morek and Michalczyk, in press) and the least similar being M. cf. tardigradum (GQ925697, Chen et al., unpublished).

28S rRNA: 2.4%–9.4% (6.8% on average), with the most similar being M. variefidum (MK483988, found in the same lichen sample, Morek and Michalczyk, in press) and the least similar being an unidentified species from Madagascar (MK483999, Morek and Michalczyk, in press).

ITS-2: 7.7%–23.9% (16.6% on average with the most similar being M. tardigradum , (MK484011 from Russia, Morek and Michalczyk, in press) and the least similar being an unidentified species from Australia (MK484018, Morek and Michalczyk, in press).

COI: 11.4%–21.3% (16.6% on average), with the most similar being M. variefidum (MK492293, from the same sample, Morek and Michalczyk, in press), whereas the least similar being an unidentified species from Australia (MK492295, Morek and Michalczyk, in press).

The phylogenetic position of M. pseudotardigradum sp. nov., as well as the remaining two analysed species, is shown in figure 6.

Figures

Fig. 2. The graph showing the frequencies of number of additional spurs in 4th+ specimens of Milnesium pseudotardigradum sp. nov. on internal/ posterior claws of each pair of legs.

Fig. 3. Milnesium pseudotardigradum sp. nov. A, habitus, ventral view (holotype, PCM). B, habitus, dorsal view (paratype, SEM). C, Buccal apparatus (holotype, PCM). D, six peribuccal lamellae of unequal size, i.e., 4 + 2 configuration (paratype, SEM). E, smooth dorsal cuticle (holotype, PCM). F, smooth dorsal cuticle (paratype, SEM). All the scale bars in µm.

Fig. 4. Milnesium pseudotardigradum sp. nov. A, claws III of hatchling with [3-3]-[3-3] CC (paratype, PCM), the empty arrow indicate the external spur, which disappear after the first moult. B, claws IV of hatchling with [3-3]-[3-3] CC (paratype, PCM)), the empty arrow indicate the posterior spur, which disappear after the first moult. C, claws III of adult (paratype, PCM), the arrow indicates the accessory points. D, claws IV of adult (holotype, PCM), the arrow indicates the accessory points. E, claws III of adult (paratype, PCM) with additional spur indicated by empty arrow. F, claws IV of adult (paratype, PCM) with additional spur indicated by empty arrow. G, claws III of adult (paratype, SEM). H, claws IV of adult (paratype, SEM) with additional spur indicated by empty arrow. All the scale bars in µm.

Fig. 5. The scheme illustrating the developmental variability in Milnesium pseudotardigradum sp. nov. Note that this species undergoes the developmental change twice—between hatchling and juvenile stage—early negative CC change, and between adult stages, which is presented for the first time, and which we call the delayed CC change. The second change is incomplete, i.e., not all spurs on external and posterior secondary branches appear.

Fig. 6. Fragment of the Bayesian phylogenetic tree based on concatenated 18S rRNA + 28S rRNA + ITS-2 + COI nucleotide sequences obtained by Morek and Michalczyk (in press), showing the positions of the three Milnesium species analysed in the study (shaded in grey). Values at nodes represent Posterior Probability (PP) supports and scale bar shows the number of substitutions per site. For the entire tree as well as the details of the methods please see the reference.