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 4. Pseudechiniscus lacyformis sp. nov. Roszkowska, Grobys, Bartylak & Kaczmarek ( Tables 5–6 , Figs 1 , 10–12 ) Pseudechiniscus sp. 4 ( Grobys et al. 2020 ) Material examined: 15 animals ( holotype (female) and 14 paratypes ( 11 females , 3 males ) mounted on microscope slides in Hoyer’s medium and 4 prepared for barcoding. Description Animals (measurements and statistics in Table 5 and 6 ) Females. Body ( Fig. 10A ) 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 spherical cephalic papillae [secondary clava]), only lateral cirrus A present (with finger-like clavae near the base [primary clava]) ( Fig. 10 A–C). Cephalic papillae smaller than clava. FIGURE 10. Pseudechiniscus lacyformis sp. nov. , female — A—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); asterisk indicates visible lateral, small plate-like structure separated from the scapular plate (scp) (holotype, PCM); B—characteristic pattern on the ventral side of the body; asterisk indicates female gonopore (holotype, PCM); C—focus on scapular plate (scp); empty arrow indicates a median longitudinal fold dividing scapular plate (scp) into two parts, filled arrow indicates stripes (folds) on plates (holotype, PCM); D—focus on pseudosegmental plate (psp); filled arrows indicate stripes (folds) on plates (holotype, PCM); E—focus on characteristic pattern on the ventral side of the body (holotype, PCM). Scale bars in micrometres [μm]. FIGURE 11. Pseudechiniscus lacyformis sp. nov. , female — A—granulation and papillae (arrow) on leg IV (holotype, PCM); B—claws and granulation of leg I (holotype, PCM). Scale bars in micrometres [μm]. FIGURE 12. Pseudechiniscus lacyformis sp. nov. , male — A—dorsal image of the entire animal; (paratype, PCM); B—focus on head and scapular plate (scp) (paratype, PCM); C—characteristic pattern on the ventral side of the body (paratype, PCM); D—focus on characteristic pattern on the ventral side of the body (patch of granulation IV) (paratype, PCM); E—claws and granulation on leg III (paratype, PCM). Scale bars in micrometres [μm]. TABLE 5. Measurements [in µm] and sp values of selected morphological structures of females of Pseudechiniscus lacyformis 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 (scp) plate 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	12	152	–	201	680	–	775	170	734	15	29	195	762
Scapular plate length	12	20.4	–	26.0	–	23.1	–	1.8	–	25.6	–
Head appendages lengths
Cirrus internus	11	10.6	–	14.0	48.4	–	53.9	12.0	51.5	1.2	1.7	13.4	52.3
Cephalic papilla	12	3.7	–	5.5	16.2	–	21.2	4.2	18.0	0.5	1.3	4.7	18.4
Cirrus externus	11	14.1	–	19.4	66.5	–	77.8	16.6	71.4	1.9	4.2	19.0	74.2
Clava	10	5.1	–	6.4	23.4	–	26.8	5.8	24.8	0.4	1.1	6.0	23.4
Cirrus A	11	26.5	–	35.0	114.1	–	142.5	29.8	128.6	2.9	10.5	29.2	114.1
Cirrus A /Body length ratio	11	15%	–	20%	–	18%	–	2%	–	15%	–
Cirrus int / ext length ratio	10	69%	–	78%	–	72%	–	3%	–	71%	–
Papillae on leg IV length	12	3.2	–	4.2	14.7	–	17.2	3.7	16.0	0.3	0.8	4.2	16.4
Claw 1 heights
Branch	11	7.8	–	9.4	34.4	–	41.1	8.7	37.6	0.6	2.0	9.3	36.3
Spur	10	1.3	–	1.6	5.1	–	7.0	1.4	6.2	0.1	0.6	1.6	6.3
Spur/branch height ratio	10	15%	–	18%	–	17%	–	1%	–	17%	–
Claw 2 heights
Branch	11	7.0	–	9.0	30.0	–	39.3	8.1	35.0	0.6	2.7	8.2	32.0
Spur	11	1.3	–	1.7	5.4	–	7.2	1.5	6.4	0.1	0.5	1.6	6.3
Spur/branch height ratio	11	16%	–	20%	–	18%	–	1%	–	20% –	–
Claw 3 heights
Branch	11	7.3	–	8.9	31.2	–	39.7	8.2	35.1	0.6	2.6	8.0	31.3
Spur	10	1.3	–	1.6	5.1	–	6.8	1.5	6.2	0.1	0.5	1.5	5.9
Spur/branch height ratio	10	16%	–	19%	–	18%	–	1%	–	19%	–
Claw 4 heights
Branch	11	7.9	–	10.6	35.9	–	43.6	9.0	39.0	0.8	2.6	9.2	35.9
Spur	6	1.3	–	1.7	6.3	–	7.3	1.6	6.6	0.1	0.4	1.6	6.3
Spur/branch height ratio	6	16%	–	19%	–	17%	–	1%	–	17%	–

Dorsal plates with small hemispherical granules/upper ends of cuticular pillars (dots by PCM) 0.4–0.9 μm in diameter, densely (spaces between granules 0.5–0.9 μm) and uniformly distributed and not joined by striae ( Fig. 10 C–D). Granules/upper ends of cuticular pillars slightly larger in the centre of the plates. On all dorsal plates, sometimes poorly visible darker stripes also present ( Fig. 10A , C–D, filled arrows). Dorsal plates typical for the genus Pseudechiniscus (cephalic plate (cp), scapular plate (scp), median plates (m1, m2, m3), paired segmental plates I and II (s1, s2), pseudosegmental plate (psp) and the 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. 10A ). The scp divided by transversal fold which forms a long narrow stripe in posterior part of the plate. Narrow stripe divided by three longitudinal folds forming four plate parts/subplates ( Fig. 10A, C ). Besides, the entire scp divided by median longitudinal fold into two parts ( Fig. 10A, C , 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. 10A , asterisk). Plates m1 and m2 divided in two portions by transverse fold, plate m3 undivided ( Fig. 10A , filled indented arrowheads). Laterally to median plates, lateral intersegmental plates (lip) present. On the plates s1 and s2 darker stripes also visible ( Fig. 10A , filled arrows). The psp divided by a longitudinal fold. Posterior margin of the psp plate straight, i.e. without projections, teeth or spines ( Fig. 10A , empty indented arrowhead). The cap concave with two Y-shaped bifurcated ridges ( Fig. 10A , filled arrowhead). Ventral cuticle with tiny granulation (formed by dense granules/upper ends of cuticular pillars, 0.2–0.4 μm) forming unique pattern ( Figs 1 , 10B, E ). Ventral PGs present (granulation 0.4–0.8 μm in diameter, spaces between granules 0.4–0.8 μm) with configuration PG:I-II-III-IV-VI-VIII a ( Figs 1 , 10B ). The female gonopore with the typical sixpetal rosette. ( Fig. 10B ). TABLE 6. Measurements [in µm] and sp values of selected morphological structures of males of Pseudechiniscus lacyformis 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
µm	sp	µm	sp	µm		sp
Body length	3	158	–	170	735	–	810	163	769	6	38
Scapular plate length	3	20.8	–	22.0	–	21.3	–	0.6	–
Head appendages lengths
Cirrus internus	3	12.9	–	14.1	61.4	–	64.1	13.3	62.5	0.7	1.4
Cephalic papilla	3	3.4	–	4.6	16.2	–	20.9	3.9	18.5	0.6	2.4
Cirrus externus	3	17.5	–	19.5	84.1	–	88.6	18.2	85.7	1.1	2.6
Clava	3	5.5	–	6.2	26.2	–	29.8	5.9	27.9	0.4	1.8
Cirrus A	3	26.9	–	29.3	129.3	–	139.5	28.5	134.0	1.4	5.1
Cirrus A /Body length ratio	3	17%	–	18%	–	17%	–	1%	–
Cirrus int / ext length ratio	3	72%	–	74%	–	73%	–	1%	–
Body appendages lengths
Papillae on leg IV length	3	3.6	–	3.9	17.1	–	17.8	3.7	17.6	0.2	0.4
Claw 1 heights
Branch	3	8.2	–	8.8	37.3	–	42.3	8.4	39.5	0.3	2.6
Spur	3	1.4	–	1.8	6.7	–	8.7	1.6	7.5	0.2	1.0
Spur/branch height ratio	3	17%	–	20%	–	19%	–	2%	–
Claw 2 heights
Branch	3	8.0	–	8.6	36.8	–	41.3	8.2	38.8	0.3	2.3
Spur	3	1.4	–	1.5	6.4	–	7.2	1.4	6.7	0.1	0.4
Spur/branch height ratio	3	17%	–	18%	–	17%	–	0%	–
Claw 3 heights
Branch	3	7.8	–	8.6	35.5	–	41.3	8.2	38.5	0.4	2.9
Spur	3	1.4	–	1.6	6.7	–	7.7	1.5	7.1	0.1	0.6
Spur/branch height ratio	3	17%	–	19%	–	18%	–	1%	–
Claw 4 heights
Branch	3	8.7	–	9.1	41.4	–	43.8	9.0	42.2	0.2	1.4
Spur	0	?	?	?	?	?	?
Spur/branch height ratio	0	?	–	?	–	?	–

The outer cuticle on legs I–III with round patches of granulation (with larger granules but sparser 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. 11 ). Triangular spine on leg I and dentate collar on leg IV absent. A finger-like papillae on leg IV present ( Fig. 11A , arrow). External claws of all legs smooth, internal with spurs directed downwards, identical in legs I–IV ( Fig. 11B ). Males ( Fig. 12 ). Dorsal plates and body appendages arranged identically like in females. Dorsal granules/upper ends of cuticular pillars similar in size, shape and pattern to those in females ( Fig. 12 A–B). Ventral granulation pattern similar in shape and size to females, but less visible ( Fig. 12 C–D). Ventral PGs present (granulation 0.3–0.6 μm in diameter, spaces between granules 0.2–0.5 μm) with configuration PG:I-II-IV-VI-VIII a ( Fig. 12 C–D). The gonopore round and without the six-petal rosette and with the PG in anterior and lateral parts. Leg sculpture similar to females ( Fig. 12E ). Juveniles. Unknown. Larvae. Unknown. DNA sequences We obtained good quality sequences for the applied molecular markers: – COI sequence (GenBank: MN528472 ), 689 bp long; – ITS-2 sequence (GenBank: MN537868 ), 281 bp long. Etymology. The specific epithet ‘ lacy ’ means, in Latin, ‘lacy’ and refers to the complicated and lace-like structure of the ventral sculpture. Type locality. 59°46’12’’N , 10°28’17’’E , 72 m asl: Norway , Buskerud Province , Røyken Region, Bøsnipa Nature Reserve, at Slemmestadveien, calcareous Tilia cordata forest, lichen, 10.08.2018 , coll. Torbjørn Ekrem, Ingemar Jönsson, Terje Meier, Tommy Prestø, Lasse Topstad. Type depositories. Holotype : slide NR135/6/S and 8 paratypes (slides: NR135/*, where the asterisk can be substituted by any of the following numbers: 2, 6/S, 8/S, 9/S, 10/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 ; 6 paratypes (slides: NR255/*, where the asterisk can be substituted by any of the following num- bers: 1, 3) are deposited at the collection NTNU University Museum, NO-7491 Trondheim, Norway . Morphological differential diagnosis* *only measurements of adult females are used in differential diagnosis Pseudechiniscus lacyformis 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 ), longer clavae (5.1–6.4 μm in Pse. lacyformis 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. lacyformis sp. nov. vs shorter claws I and II, and longest claws III and IV in Pse. beasleyi ). 3. Pse. clavatus , by: different shape of clavae (finger-like in Pse. lacyformis sp. nov. vs club-shaped in Pse. clavatus ) and normally developed cephalic papillae (reduced in Pse. clavatus ). 4. Pse. dastychi sp. nov. , see Morphological differential diagnosis of Pse. dastychi sp. nov. above. 5. Pse. ehrenbergi sp. nov. see Morphological differential diagnosis of Pse. ehrenbergi sp. nov. above. 6. 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. lacyformis sp. nov. . See also Morphological differential diagnosis of Pse. suillus and Discussion in the paper Grobys et al. (2020) . 7. Pse. indistinctus sp. nov. , by: different ventral pattern ( Figs 1 , 10B for Pse. lacyformis sp. nov. vs Figs 1 , 13 C–D for Pse. indistinctus sp. nov. ), different ventral PG configuration (PG:I-II-III-IV-VI-VIII a in Pse. lacyformis sp. nov. vs PG:I-II-III-IV-V-VI-VIII a in Pse. indistinctus sp. nov. ), granules on the cap similar in size to other dorsal plates (granules visibly larger on the cap in comparison with other dorsal plates in Pse. indistinctus sp. nov. ), longer cirri interni (10.6–14.0 μm, [ sp=48.4–53.9 ] in Pse. lacyformis sp. nov. vs 7.0–9.2 μm, [ sp=34.1–38.5 ] in Pse. indistinctus sp. nov. ), higher sp of cirri externi ( 66.5–77.8 in Pse. lacyformis sp. nov. vs 54.3–54.9 in Pse. indistinctus sp. nov. ), higher sp of clavae ( 23.4–26.8 in Pse. lacyformis sp. nov. vs 19.3–22.5 in Pse. indistinctus sp. nov. ), longer papillae on leg IV (3.2–4.2 μm, [ sp=14.7–17.2 ] in Pse. lacyformis sp. nov. vs 2.2–2.9 μm, [ sp=9.8–11.9 ] in Pse. indistinctus sp. nov. ) and lower spur/branch length ratio of all claws (15–20% in Pse. lacyformis sp. nov. vs 28–34% in Pse. indistinctus sp. nov. ). 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. lacyformis sp. nov. . See also Morphological differential diagnosis of Pse. suillus and Discussion in the paper Grobys et al. (2020) . 9. Pse. megacephalus , by: different shape of cephalic papillae (spherical in Pse. lacyformis sp. nov. vs mushroom-like in Pse. megacephalus ), absence of papilliform projection between external buccal cirri and cirri A . 10. Pse. suillus , see by: a small papilla-like structure on leg I absent, different ventral pattern ( Figs 1 and 10B for Pse. lacyformis sp. nov. vs Fig. 1 herein and 4C–D in Grobys et al . (2020) for Pse. suillus ), well-developed ventral patches of granulation, different position of PG VIII (PG VIII placed above gonopore in Pse. lacyformis sp. nov. vs PG VIII placed around gonopore in Pse. suillus ) and lower spur/branch length ratio (15–20% in Pse. lacyformis sp. nov. vs 21–27% in Pse. suillus ). 11. Pse. xiai , known only from China ( Wang et al. 2018 ), by: different ventral pattern ( Figs 1 and 10B for Pse. lacyformis sp. nov. vs Figs 1B, F and 2E in Wang et al. (2018) for Pse. xiai ). 12. Pse. chengi, known only from China ( Xue et al. 2017 ), by: plates m1 and m2 divided in two portions by transverse fold (unndivided in Pse. chengi ), longer cirri interni (10.6–14.0 μm [ sp=48.4 –53.9] in Pse. lacyformis sp. nov. vs 3.2–8.5 μm [ sp=13.2 –38.1] in Pse. chegi ), higher sp of cirri externi ([ 66.5–77.8 ] in Pse. lacyformis sp. nov. vs [ 45.9–56.1 ] in Pse. chegi ), higher sp of cirri A ([ 114.1–142.5 ] in Pse. lacyformis sp. nov. vs [ 96.0 –111.1] in Pse. chengi ). Genotypic differential diagnosis The ranges of genetic distances between Pse. lacyformis sp. nov. and species of the genus Pseudechiniscus , for which DNA sequences are available in GenBank, are as follows: COI : 10.6–27.0% (21.1% on average), with the most similar being Pse. aff. xiai ( MK804894 –7, Cesari et al. 2020 ) and the least similar being Pse. indistinctus sp. nov. ( MN528471 , present study). ITS-2: 10.3–39.3% (26.7% on average), with the most similar being Pse. indistinctus sp. nov. ( MN537867 , present study) and the least similar being Pse. angelusalas sp. nov. ( MN537864 , present study).