Integrative description of five Pseudechiniscus species (Heterotardigrada Echiniscidae: the suillus-facettalis complex) Author Roszkowska, Milena Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61 - 614 Poznań, Poland. Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61 - 614 Poznań, Poland mil.roszkowska@gmail.com Author Grobys, Daria Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61 - 614 Poznań, Poland Author Bartylak, Tomasz Author Gawlak, Magdalena The Institute of Plant Protectio-National Research Institute, Wegorka 20, 60 - 318 Poznan, Poland Author Kmita, Han- Na Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61 - 614 Poznań, Poland Author Kepel, Andrzej Polish Society for Nature Conservation " Salamandra ", Stolarska 7 / 3, 60 - 788 Poznan, Poland Author Kepel, Marta Polish Society for Nature Conservation " Salamandra ", Stolarska 7 / 3, 60 - 788 Poznan, Poland Author Parnikoza, Ivan National Antartic Scientific Center of Ukraine, Boulevard Tarasa Shevchenka 16, 01601 Kyiv, Ukraine. Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine, Kyiv, Ukraine Author Kaczmarek, Łukasz Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61 - 614 Poznań, Poland text Zootaxa 2020 2020-04-15 4763 4 451 484 journal article 22886 10.11646/zootaxa.4763.4.1 4ef09580-992d-4f9f-b9d1-ee4c4eef41ad 1175-5334 3762014 urn:lsid:zoobank.org:pub:0DE45665-F3A9-474B-B438-1022FABB6BD1 5. Pseudechiniscus indistinctus sp. nov. Roszkowska, Grobys, Bartylak & Kaczmarek ( Table 7 , Figs 1 , 13–14 ) Pseudechiniscus sp. 5 ( Grobys et al. 2020 ) Material examined: 77 animals ( holotype and 76 paratypes (all females)) mounted on microscope slides in Hoyer’s medium, 97 animals prepared for SEM and 7 prepared for barcoding. Description Animals (measurements and statistics in Table 7 ) Females. Body ( Fig. 13 A–B) orange-yellow in living specimens (transparent after mounting on slides), black eyes present after mounting on slides. Apart from the head appendages (cirri interni and externi and elongated cephalic papillae [secondary clava]), only lateral cirrus A present (with finger-like clavae near the base [primary clava]) ( Fig. 13 A–B, D). FIGURE 13. Pseudechiniscus indistinctus sp. nov. , female — A–B—dorsal image of the entire animal; empty arrow indicates a median longitudinal fold dividing scapular plate (scp) into two parts, filled indented arrowheads indicate transverse folds dividing plates m1 and m2 in two portions, filled arrows indicate stripes (folds) on plates s1 and s2, empty indented arrowhead indicates straight pseudosegmental plate (psp), filled arrowhead indicates Y-shaped bifurcated ridges on caudal plate (cap) (holotype, PCM and paratype, SEM respectively); C–D—characteristic pattern on the ventral side of the body; asterisks indicate female gonopore, indented arrowhead indicates lateral, small plate-like structure separated from the scapular plate (scp) (paratypes, PCM and SEM, respectively). Scale bars in micrometres [μm]. FIGURE 14. Pseudechiniscus indistinctus sp. nov. , female — A—focus on upper ends of cuticular pillars connected by striae in dorsal plates (cap) (paratype, PCM); B—granulation and papillae (arrow) on leg IV (paratype, PCM); C—claws and granulation of leg II (paratype, PCM). Scale bars in micrometres [μm]. Dorsal plates with small hemispherical granules/upper ends of cuticular pillars (dots by PCM) 0.4–1.0 μm in diameter (except of the cap), densely (spaces between granules 0.4–1.3 μm) and uniformly distributed and joined by very thin striae forming indistinct hexagonal pattern (which are in fact a thin stripes positioned under epicuticle) ( Fig. 14A ). Granules/upper ends of cuticular pillars slightly larger in the centre and anterior parts of the plates and much larger (1.1–1.3 μm in diameter) on the caudal plate (cap), especially in the central area between Y-shaped bifurcated ridges ( Fig. 13A ). Dorsal plates typical for the genus Pseudechiniscus (cephalic plate (cp), neck plate (np), scapular plate (scp), median plates (m1, m2, m3), paired segmental plates (s1, s2), pseudosegmental plate (psp) and caudal plate (cap), see also Dorsal and ventral plates and sculpture in Grobys et al. 2020 ) well developed. The cp with W-shaped pattern divided into five parts ( Fig. 13A ). The scp divided by transversal fold, which forms a long narrow stripe in posterior part of the plate. Narrow stripe sometimes divided by three longitudinal folds which form four plate parts/ subplates ( Fig. 13A ). The entire scp divided by median longitudinal fold into two parts ( Fig. 13 A–B, empty arrow). Additionally, lateral portions of the scp appear detached from the dorsal plate, forming small plate-like structures separated from the scp by a thin bright stripe ( Fig. 13C , indented arrowhead). Plates m1 and m2 divided in two portions by transverse fold, plate m3 undivided ( Fig. 13 A–B, filled indented arrowheads). Laterally to the median plates, lateral intersegmental plates (lip) present. On the plates s1 and s2 darker stripes also visible ( Fig. 13 A–B, filled arrows). The psp divided by a longitudinal fold. Posterior margin of the psp straight, i.e. without projections, teeth or spines ( Fig. 13 A–B, empty indented arrowhead). The cap concave with two Y-shaped bifurcated ridges ( Fig. 13 A–B, filled arrowhead). Ventral cuticle with tiny granulation (formed by dense granules/upper ends of cuticular pillars, 0.1–0.3 μm) forming unique pattern ( Figs 1 , 13 C–D). Ventral PGs present (granulation 0.3–0.6 μm in diameter, spaces between granules 0.4–0.7 μm) with configuration PG:I-II-III-IV-V-VI-VIII a ( Figs 1 , 13 C–D). The PG VI (in line of leg III) is not uniformly granulated, i.e. line of granules is present in the center of the PG and two smooth areas clearly visible below and above of this line ( Fig. 1 ). The female gonopore with the typical six-petal rosette. ( Fig. 13 C–D, asterisks). The outer cuticle on legs I–III with round patches of granulation (with larger granules but more sparse in the centre and smaller and denser in peripheral parts), on leg IV with uniform wide stripes of granulation (slightly larger in the centre of these stripes) ( Fig. 14 B–C). Triangular spine on leg I and dentate collar on leg IV absent. A fingerlike papillae on leg IV present ( Fig. 14B , arrow). External claws of all legs smooth, internal with spurs directed downwards, identical in legs I–IV ( Fig. 14C ). Males. Unknown. Juveniles. Unknown. Larvae. Unknown. DNA sequences We obtained good quality sequences for the applied molecular markers: – COI sequence (GenBank: MN528471 ), 622 bp long; – ITS-2 sequence (GenBank: MN537867 ), 434 bp long. Etymology. The specific epithet ‘ indistinctus ’ means, in Latin, ‘indistinct’ and refers to the delicate and poorly marked ventral sculpture. Type locality. 58°33’33’’N , 06°26’24’’E , 273 m asl: Norway , Rogaland Province , Lund Region, Førland Sletthei Landscape Protection Area, S Førlandsvatnet, Betula pubescens forest, lichen ( Cladonia squamosa (Scop.) Hoffm., 1796 ), 12.08.2018 , coll. Terje Meier, Elisabeth Stur, Karstein Hårsaker, Tommy Prestø, Lasse Topstad. Type depositories. Holotype : slide NR255/17 and 10 paratypes (slides: NR255/*, where the asterisk can be substituted by any of the following numbers: 17, 17/S, 18/S, 19/S) are deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, 61- 614 Poznań, Poland ; 66 paratypes (slides: NR255/*, where the asterisk can be substituted by any of the following numbers: 3, 4, 8, 10, 11, 14, 15, 16, 19, 21) are deposited at the collection NTNU University Museum, NO-7491 Trondheim, Norway . TABLE 7. Measurements [in µm] and sp values of selected morphological structures of females of Pseudechiniscus indistinctus sp. nov. Roszkowska, Grobys, Bartylak & Kaczmarek mounted in Hoyer’s medium (N—number of specimens/ structures measured, RANGE refers to the smallest and the largest structure among all measured specimens, SD—stand-ard deviation,?—trait oriented unsuitably for measurement, sp— ratio of the length of a given structure to the length of the scapular plate (scp) expressed as a percentage ( sp = length of structure × 100⁄ length scapular plate (scp)).

CHARACTER	N	RANGE		MEAN		SD		Holotype
µm	sp	µm	sp	µm	sp	µm	sp
Body length	15	140	–	166	648	–	697	152	673	8	15	146	658
Scapular plate length	15	20.5	–	24.9	–	22.5	–	1.1	–	22.2	–
Head appendages lengths
Cirrus internus	14	7.0	–	9.2	34.1	–	38.5	8.3	36.7	0.5	1.2	8.1	36.5
Cephalic papilla	15	3.3	–	4.3	15.5	–	17.3	3.7	16.2	0.3	0.5	3.7	16.7
Cirrus externus	15	11.3	–	14.4	54.3	–	59.3	12.7	56.2	0.8	1.4	12.8	57.7
Clava	15	4.2	–	5.4	19.3	–	22.5	4.6	20.6	0.3	0.9	4.6	20.7
Cirrus A	13	27.6	–	34.2	123.0	–	144.9	30.2	133.7	2.1	7.2	30.4	136.9
Cirrus A /Body length ratio	13	18%	–	22%	–	20%	–	1%	–	21%	–
Cirrus int / ext length ratio	14	62%	–	70%	–	65%	–	2%	–	63%	–
Papillae on leg IV length	14	2.2	–	2.9	9.8	–	11.9	2.5	10.8	0.2	0.6	2.4	10.8
Claw 1 lengths
Branch	15	6.1	–	7.7	28.1	–	31.7	6.8	30.0	0.4	1.2	6.7	30.2
Spur	15	1.8	–	2.3	7.9	–	10.2	2.1	9.2	0.1	0.5	2.1	9.5
Spur/branch length ratio	15	28%	–	34%	–	31%	–	2%	–	31%	–
Claw 2 lengths
Branch	14	5.6	–	7.2	26.0	–	30.1	6.4	28.1	0.4	1.3	6.3	28.4
Spur	13	1.7	–	2.2	7.8	–	9.3	2.0	8.7	0.1	0.6	2.0	9.0
Spur/branch length ratio	13	28%	–	33%	–	31%	–	1%	–	32% –	–
Claw 3 lengths
Branch	14	5.9	–	7.1	26.8	–	29.6	6.4	28.3	0.4	1.0	?	?
Spur	14	1.8	–	2.2	7.9	–	9.8	2.0	8.9	0.1	0.6	?	?
Spur/branch length ratio	14	29%	–	34%	–	32%	–	2%	–	–	–
Claw 4 lengths
Branch	15	6.6	–	8.4	30.3	–	34.7	7.3	32.3	0.4	1.3	7.1	32.0
Spur	10	1.9	–	2.3	8.8	–	10.6	2.2	9.6	0.1	0.6	?	?
Spur/branch length ratio	10	29%	–	32%	–	30%	–	1%	–	–	–

Morphological differential diagnosis* *only measurements of adult females are used in differential diagnosis Pseudechiniscus indistinctus sp. nov. differs specifically from: 1. Pse. angelusalas sp. nov. , see Morphological differential diagnosis of Pse. angelusalas sp. nov. above. 2. Pse. beasleyi, by: the scp not divided in anterior part (the scp divided into four parts in Pse. beasleyi ), short- er cirri interni (7.0–9.2 μm in Pse. indistinctus sp. nov. vs 10.4–15.7 μm in Pse. beasleyi ), longer clavae (4.2–5.4 μm in Pse. indistinctus sp. nov. vs 3.1–3.9 μm in Pse. beasleyi ) and different claws length arrangement (shortest claws II and III, and longest claws IV in Pse. indistinctus sp. nov. vs claws I and II shortest and III and IV longest in Pse. beasleyi ) and shorter claws (see Table 7 in this paper and Table 2 in Li et al. 2007 ). 3. Pse. chengi, known only from China ( Xue et al. 2017 ), by: dorsal granules joined by striae, plates m1 and m2 divided in two portions by transverse fold (unndivided in Pse. chengi ), longer cirri A (27.6–34.2 μm [ sp=123.0– 144.9 ] in Pse. indistinctus sp. nov. vs 21.4–27.5 μm [ sp=96.0–111.1 ] in Pse. chengi ), higher cirrus A /body length ratio (18–22% in Pse. indistinctus sp. nov. vs 13–17% in Pse. chengi ) and shorter claws I–III and lower sp of claws I–III (see Table 7 herein and Table 2 in Xue et al. 2017 ). 4. Pse. clavatus , by: different shape of clavae (finger like in Pse. indistinctus sp. nov. vs club-shaped in Pse. clavatus ) and normally developed cephalic papillae (reduced in Pse. clavatus ). 5. Pse. dastychi sp. nov. , see Morphological differential diagnosis of Pse. dastychi sp. nov. above. 6. Pse. ehrenbergi sp. nov. see Morphological differential diagnosis of Pse. ehrenbergi sp. nov. above. 7. Pse. facettalis , known from distant localities throughout the world ( McInnes 1994 ). Based on present study, an inaccurate description of this species makes it impossible to differentiate this taxon from Pse. indistinctus sp. nov. . See also Morphological differential diagnosis of Pse. suillus and Discussion in the paper Grobys et al. (2020) . 8. Pse. juanitae , known from Austria , Brazil ( type locality), Italy and Galapagos Islands ( McInnes 1994 ). Based on present study, an inaccurate description of this species makes it impossible to differentiate this taxon from Pse. indistinctus sp. nov. . See also Morphological differential diagnosis of Pse. suillus and Discussion in the paper Grobys et al. (2020) . 9. Pse. lacyformis sp. nov. , see Morphological differential diagnosis of Pse. lacyformis sp. nov. above. 10. Pse. megacephalus , by: different shape of cephalic papillae (elongated in Pse. indistinctus sp. nov. vs mushroom-like in Pse. megacephalus ), absence of papilliform projection between external buccal cirri and cirri A . 11. Pse. suillus , by: a small papilla-like structure on leg I absent, different ventral pattern ( Figs 1 and 13 C–D for Pse. indistinctus sp. nov. vs Figs 1 herein and 4C–D in Grobys et al . (2020) for Pse. suillus ), well-developed ventral patches of granulation, different ventral PG configuration (PG:I-II-III-IV-V-VI-VIII a in Pse. indistinctus sp. nov. vs PG:I-II-III-IV-VI-VIII g in Pse. suillus ), granules visibly larger on cap in comparison with other dorsal plates in Pse. indistinctus sp. nov. (granules on cap similar in size to other dorsal plates in Pse. suillus ), dorsal granules joined by striae, lower sp of cirri interni ( 34.1–38.5 in Pse. indistinctus sp. nov. vs 44.0– 49.6 in Pse. suillus ), lower sp of cephalic papillae ( 15.5–17.3 in Pse. indistinctus sp. nov. vs 19.1–24.3 in Pse. suillus ), lower sp of cirri externi ( 54.3–59.3 in Pse. indistinctus sp. nov. vs 62.1–75.0 in Pse. suillus ), shorter papillae on leg IV (2.2–2.9 μm, [ sp=9.8–11.9 ] in Pse. indistinctus sp. nov. vs 3.3–4.3 μm, [ sp=14.7–18.4 ] in Pse. suillus ) and higher spur/branch length ratio (28–34% in Pse. indistinctus sp. nov. vs 21–27% in Pse. suillus ). 12. Pse. xiai , known only from China ( Wang et al. 2018 ), by: different ventral pattern ( Figs 1 and 13 C–D for Pse. indistinctus sp. nov. vs Figs 1B, F and 2E in Wang et al. (2018) for Pse. xiai ), dorsal granules joined by striae, smaller body size (140.0–166.0 μm in Pse. indistinctus sp. nov. vs 179.0–215.0 μm in Pse. xiai ), higher cirrus A / body length ratio (18–22% in Pse. indistinctus sp. nov. vs 13–16% in Pse. xiai ) and shorter claws (see Table 7 herein and Table 1 in Wang et al. 2018 ). Genotypic differential diagnosis The ranges of genetic distances between Pse. indistinctus sp. nov. and species of the genus Pseudechiniscus , for which DNA sequences are available in GenBank, are as follows: COI : 25.9–30.9% (27.9% on average), with the most similar being Pseudechiniscus sp. ( KJ857005 , Velasco- Castrillón et. al. 2015 (described in GenBank as Echiniscus sp.); for more details see Discussion section in Grobys et al. 2020 ) and the least similar being Pse. aff. suillus ( MK804907 , Cesari et al. 2020 ). ITS-2: 6.8–38.0% (25.3% on average), with the most similar being Pse. suillus sp. nov. ( MN537863 ) and the least similar being Pse. angelusalas sp. nov. ( MN537864 , present study).