Milnesium guanyinensis sp. nov. (Eutardigrada: Apochela) from Yunnan, China Author Yuan, Zhimin College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. Author Liu, Qiuju 0000-0002-3309-0685 College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. & liuqj 1126 @ 126. com; https: // orcid. org / 0000 - 0002 - 3309 - 0685 Author Wang, Yan 0000-0002-3990-4985 College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. & 1957511977 @ qq. com; https: // orcid. org / 0000 - 0002 - 3990 - 4985 1957511977@qq.com Author Liu, Lijie 0000-0002-6223-5994 College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. & liulijie 0803 @ 163. com; https: // orcid. org / 0000 - 0002 - 6223 - 5994 Author Chen, Yan 0000-0001-9112-1278 College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. & 827949195 @ qq. com; https: // orcid. org / 0000 - 0001 - 9112 - 1278 827949195@qq.com Author Li, Xiaochen 0000-0001-7859-4564 College of Life Sciences, Shaanxi Normal University, Xi’an, 710100, P. R. China. & xiaochen @ snnu. edu. cn; https: // orcid. org / 0000 - 0001 - 7859 - 4564 xiaochen@snnu.edu.cn text Zootaxa 2023 2023-03-02 5249 3 378 392 http://dx.doi.org/10.11646/zootaxa.5249.3.4 journal article 10.11646/zootaxa.5249.3.4 26efc1b6-1832-4671-b5a5-c1d10f2b3da1 1175-5326 7691105 A20B0FA1-0BA8-4BB9-B166-7FE56991ECB9 Milnesium guanyinensis sp. nov. Fig. 2–8 , Table 1–2 Description of the new species Animals (measurements and statistics in Table 1 and Table 2 ) Adults and juveniles (from the second instar onwards; morphometrics and holotype measurements in Table 1 .) Body brownish and slender ( Fig. 2B , 3B ). Eyes present in living specimens, quickly dissolving after fixation in Hoyer’s medium. In juveniles, cuticle with a robust reticular design with thick reticulum and pit-shaped meshes (the krzysztofi type ) on the whole dorsum except for the head ( Fig. 6B, E ). In adults, the reticulum is weakly developed, thus it may not be visible under PCM ( Fig. 6C ), although it is always identifiable in SEM. Reticulum mesh reaches 0.50–0.70 μm in diameter. The species is characterised by numerous pseudoplates ( Fig. 3B–C ) arranged in nine transverse rows, which are visible both in PCM and UVM. Some of the pseudoplates are difficult to observe (marked with dotted line in Fig. 3C ); the exact arrangement is as follow: (I) situated anteriorly to legs I; central rectangular pseudoplate (divided longitudinally) + two smaller roundish lateral pseudoplates; (II) situated in line with legs I, large rectangular pseudoplate (divided into four uneven portions, with the anterior ones being smaller) + two smaller lateral rectangular pseudoplates; (III) situated between legs I and II, large central elliptical pseudoplate (divided longitudinally) + two lateral about elliptical pseudoplates; (IV) situated in line with legs II, central, undivided rectangular pseudoplate with poorly visible anterior portion + a pair of lateral roundish pseudoplates; (V) situated between legs II and III, central, rectangular pseudoplate (divided longitudinally) + a pair of lateral roundish pseudoplates; (VI) situated in line with legs III, large central trapezoid pseudoplate (divided into six roughly equal potions) + a pair of lateral roundish pseudoplates; (VII) situated just posterior to legs III, central rectangular pseudoplate (divided longitudinally) with poorly visible posterior portion + a pair of lateral roundish pseudoplates, each with poorly visible pair of roundish anterior pseudoplates; (VIII) the largest, most complex, trapezoid pseudoplate (divided into eight parts: a central triangle and seven quadrangles) + two roundish lateral pseudoplates; (IX) four pseudoplates arranged transversally (internal trapezoid and the lateral roundish). FIGURE 2. General morphology of Milnesium guanyinensis sp. nov. seen in PCM. A—hatchling habitus (paratype). B— juvenile habitus (holotype). Scale bars in μm. TABLE 2. Measurements and pt values of selected morphological structures of 20 hatchlings of Milnesium guanyinensis sp. nov. All available specimens were measured.

CHARACTER	N	RANGE µm			pt	MEAN µm pt		SD µm pt
Body length	20	256	–	370	1213	–	1741	322	1512	31	156
Peribuccal papillae length	19	3.3	–	6.2	15.8	–	28.2	4.2	19.7	0.8	3.9
Lateral papillae length	19	3.1	–	5.1	15.0	–	23.1	3.8	17.9	0.5	2.5
Buccal tube
Length	20	18.4	–	24.1	–	21.3	–	1.4	–
Stylet support insertion point	20	12.5	–	17.1	67.1	–	72.1	14.8	69.1	1.1	1.4
Anterior width	20	7.2	–	9.4	33.9	–	41.1	8.1	37.9	0.6	2.5
Standard width	20	6.0	–	8.4	28.4	–	39.6	7.3	34.3	0.7	3.6
Posterior width	20	6.0	–	8.2	27.9	–	37.3	7.1	33.5	0.6	2.7
Posterior/anterior width ratio	20	79%	–	94%	–	88%	–	4%	–
Claw 1 heights
External primary branch	19	9.3	–	13.0	41.8	–	55.6	10.7	50.3	1.1	4.1
External base + secondary branch	19	7.4	–	9.8	34.7	–	47.8	8.6	40.7	0.7	3.0
External branches length ratio	18	72%	–	98%	–	82%	–	8%	–
Internal primary branch	19	9.1	–	12.5	41.8	–	54.9	10.6	49.7	0.9	3.5
Internal base + secondary branch	20	7.5	–	10.1	34.2	–	46.8	8.7	40.8	0.7	3.6
Internal branches length ratio	19	66%	–	98%	–	83%	–	8%	–
Claw 2 heights
External primary branch	19	9.3	–	12.4	43.9	–	54.3	10.6	49.8	0.9	3.0
External base + secondary branch	19	8.2	–	10.5	37.9	–	47.8	9.0	42.4	0.7	2.5
External branches length ratio	16	73%	–	96%	–	86%	–	7%	–
Internal primary branch	20	9.0	–	12.4	41.2	–	56.1	10.4	48.8	1.0	4.4
Internal base + secondary branch	20	7.7	–	9.9	36.1	–	46.7	8.8	41.3	0.6	2.9
Internal branches length ratio	20	67%	–	98%	–	85%	–	8%	–
Claw 3 heights
External primary branch	20	9.4	–	12.3	42.9	–	55.7	10.9	51.1	0.8	3.5
External base + secondary branch	19	7.9	–	10.1	38.3	–	48.4	9.1	42.9	0.5	2.9
External branches length ratio	19	74%	–	93%	–	84%	–	6%	–
Internal primary branch	20	8.8	–	11.9	41.1	–	56.4	10.7	50.0	1.0	4.1
Internal base + secondary branch	20	7.9	–	9.8	36.8	–	48.4	9.0	42.1	0.5	3.2
Internal branches length ratio	20	74%	–	107%	–	85%	–	8%	–
Claw 4 heights
Anterior primary branch	20	9.1	–	13.0	45.2	–	57.3	10.8	50.7	0.9	2.9
Anterior base + secondary branch	20	8.2	–	10.7	39.8	–	49.5	9.4	44.1	0.6	2.5
Anterior branches length ratio	20	69%	–	97%	–	87%	–	7%	–
Posterior primary branch	20	9.3	–	12.7	46.5	–	55.3	11.0	51.5	0.8	2.6
Posterior base + secondary branch	20	8.4	–	11.2	41.6	–	50.2	9.6	45.2	0.7	2.6
Posterior branches length ratio	20	81%	–	98%	–	88%	–	6%	–

FIGURE 3. General morphology of Milnesium guanyinensis sp. nov. A—juvenile habitus with visible pseudoplates; UVM (paratype); B—adult habitus with visible pseudoplates; UVM (paratype); C—pseudoplate arrangement based on the observation of holotype and paratypes in PCM, UVM and SEM (drawn with MS PowerPoint 2016). Scale bars in µm. FIGURE 4. Buccal tube of Milnesium guanyinensis sp. nov. in PCM. A—hatchling habitus (paratype). B—juvenile habitus (paratype). C—adult habitus (paratype). Scale bars in μm. FIGURE 5. Buccal apparatus of Milnesium guanyinensis sp. nov. A—a lateral view of the buccal tube in juvenile (paratype, SEM); B—buccal apparatus with outspread stylet supports and stylets in adult (holotype, PCM); C—mouth opening, dorsal view with three peribuccal papillae visible in juvenile (paratype, PCM); D—mouth opening, ventral view with two peribuccal papilla and two cephalic papillae visible in juvenile (same as the paratype in Fig. 3C, PCM); Scale bars in μm. Buccal apparatus of the Milnesium type ( Fig. 4B – C , 5A–D ). Buccal tube in adults and juveniles wide and short (standard width on average 39% of its length) and cylindrical, wider anteriorly (posterior diameter on average 95% of the anterior diameter); Six peribuccal papillae and six peribuccal lamellae around the mouth opening present ( Fig. 5A – D ). Two cephalic papillae positioned laterally. The peribuccal papillae usually longer than the cephalic ones. Pharyngeal bulb elongated and pear-shaped, without placoids or septulum. Claws of the Milnesium type ( Fig. 7C – F , 8C – D ). Primary branches with thin accessory points; claw bases with rounded basal thickenings; the CC in adults and juveniles is [2-3]-[3-2] ( Fig. 5C–F, I–J ). A long cuticular bar present under the claws I–III ( Fig. 5A, C, E ). Hatchlings (morphometrics and measurements in Table 2 .) Morphologically similar to adults and juveniles, but with a better developed dorsal reticulation (denser and more visible; meshes 0.65–0.90 μm in diameter; Fig. 6A, D ) and a more vaguely outlined dorsal pseudoplates ( Fig. 2A , 3A ). Buccal tube in hatchlings is more slender (standard width on average 34% of its length) and cylindrical, slightly wider anteriorly (posterior diameter on average 88% of the anterior diameter). The [2-2]-[2-2] CC was observed in hatchlings ( Fig. 7A–B , 8A–B ). Other qualitative traits as in adults and juveniles. Ontogenetic variability . Based on the relationship between buccal tube and body length, the type series of Milnesium guanyinensis sp. nov. comprises hatchlings, juveniles and adults ( Fig. 9 ). The new species exhibits developmental changes in three crucial taxonomic traits: cuticular sculpturing, buccal tube shape and CC. The dorsal cuticle sculpturing becomes less clear in later life stages. Particularly, in PCM, the reticulum is most clearly visible in hatchlings, marginally less developed in juveniles, and it can be indistinct in adults or even not visible at all in large adults. In SEM, the dorsal cuticle sculpturing also fades with consecutive instars, but it is visible in all life stages ( Fig. 6 ). The buccal tube in hatchings is more slender than in adults and juveniles. The CC changes from [2-2]-[2-2] in hatchlings to [2-3]-[3-2] in juveniles and adults, i.e. the new species exhibits an early positive CC change. Remarks. Several irregular cuticular depressions present in the dorsal cuticle are present, with the most caudal one being always the largest ( Fig. 6F ). Eggs . Oval, yellow, smooth and laid in the exuviae; up to 7 in a single clutch were found in exuviae. Type locality. 23º2'52"N , 102º49'34"E ; 2 300 m asl: Guanyinshan , Ailao Mountains , Yuannan Province , China ; habitat: forest; substrate: mosses on a rock . Etymology. The new species is named after the Guanyinshan district of Yunnan , China , where it was discovered. FIGURE 6. Dorsal cuticle sculpturing of Milnesium guanyinensis sp. nov. A—hatchling (paratype, PCM). B—juvenile (paratype, PCM). C—adult (holotype, PCM). D—hatchling (paratype, SEM). E—juvenile (paratype, SEM). F—dorsal cuticle with irregular depressions of juvenile (paratype, SEM). Scale bars in μm. Type depositories. The series consists of the holotype (adult, GY050116) and 37 paratypes , 29 on microscope slides (GY050101–15, GY050117–20, GY050201–10) and 8 on an SEM stub (22.02 and 22.06). All slides and the SEM stub are deposited at Xiaochen Li’s tardigrade collection, Molecular Ecology, Department of Biology, College of Life Sciences of Shaanxi Normal University, China . FIGURE 7. Claws of Milnesium guanyinensis sp. nov. in PCM. A—claws III of a hatchling, with the [2-2] CC (paratype). B—claws IV of the hatchling, with the [2-2] CC (paratype). C—claws III of a juvenile with the [2-3] CC (paratype). D—claws IV of the juvenile, with the [2-3] CC (paratype). E—claws III of an adult, with the [2-3] CC (holotype). F—claws IV of an adult, with the [3-2] CC (holotype). Adults and juveniles differential diagnosis. Milnesium guanyinensis sp. nov. is characterised by six peribuccal lamellae, fine reticular design of dorsal cuticle and a [2-3]-[3-2] CC in adults. Thus, the new species is similar to five other species but differs specifically from: • M . cassandrae Moreno-Talamantes et al. , 2019 ( Mexico ), by cuticle with fine reticular design (adults cuticle sculptured with pseudopores, sparsely distributed and not forming a reticular design in M . cassandrae ); by a lower pt of the standard buccal tube width ( 37.5–41.5 in the new species vs 41.6–67.2 in M . cassandrae ). • M. decorum Morek et al. , 2022 ( Portugal ), by cuticular bars under claws I–III present (cuticular bars under claws I–III absent in the majority of specimens in M . decorum ); by a higher pt of stylet supports inserted on buccal tube ( 65.5–69.3 , mean 66.8 , in the new species vs 59.2–65.6 , mean 62.9 , in M . decorum , t 16 =-5.17, p <0.001). • M. krzysztofi Kaczmarek & Michalczyk, 2007 ( Costa Rica ), by eyes present and body brownish (eyes absent and body white in M . krzysztofi ); by the relatively shorter posterior primary branch on claws IV ( 51.7–57.1 , mean 53.7 in the new species vs 56.9–66.5 , mean 62.1 in M . krzysztofi , t 12 =-5.92, p<0.001); by the relatively longer posterior spur on claws IV ( 10.1–15.0 , mean 12.1 in the new species vs 8.0–10.1 , mean 9.1 in M . krzysztofi , t 12 =- 3.15, p<0.01). • M. pacificum Sugiura et al. , 2020 ( Japan ) by the brownish body and the reticulum mesh is sunken (body creamy white and the reticulum is tuberculate in M . pacificum ); by the relatively longer spurs on claws III ( 12.6– 15.5 in the new species vs 7.5–12.4 in M . pacificum ). • M. pelufforum Rocha et al. , 2022 ( Argentina ) by the brownish body (body reddish in M . pelufforum ); no spurs present on claws (small basal spurs present in M . pacificum ); by a lower pt of the standard buccal tube width ( 37.5–41.5 in the new species vs 55.2–64.0 in M. pelufforum ). FIGURE 8. Claws of Milnesium guanyinensis sp. nov. in SEM. A—claws III of a hatchling, with the [2-2] CC (paratype). B—claws IV of a hatchling, with the [2-2] CC (paratype). C—claws III of a juvenile, with the [2-3] CC (paratype). D—claws IV of a juvenile, with the [2-3] CC (paratype). Scale bars in μm. Hatchlings differential diagnosis . Milnesium guanyinensis sp. nov. first instar specimens are characterised by fine reticular design and a [2-2]-[2-2] CC, the new species is similar to four other congeners but differs specifically from: • M . cassandrae by eyes present and body brownish (eyes absent and white or transparent with light yellow brownish tones before fixation in M . cassandrae ); by a higher pt of stylet supports inserted on buccal tube ( 67.1–72.1 , mean 69.1 in the new species vs 56.5–70.5 , mean 66.3 in M . cassandrae , t 31 =- 3.36, p<0.01). • M . katarzynae Kaczmarek et al. , 2004 ( China ) by eyes present and body brownish (eyes absent and body white in M . katarzynae ), a higher pt of the standard buccal tube width ( 28.4–39.6 in the new species vs 21.7–26.6 in M . katarzynae ) and a lower pt of stylet supports inserted on buccal tube ( 67.1–72.1 in the new species vs 73.3–78.3 in M . katarzynae ). • M . pacificum by the brownish body and the reticulum mesh is shallow (body creamy white and the reticulum is tuberculate in M . pacificum ). • M . pelufforum by the brownish body (body reddish in M . pelufforum ); by a lower pt of the standard buccal tube width ( 28.4–39.6 in the new species vs 43.7–52.1 in M . pelufforum ).