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 3. Pseudechiniscus angelusalas sp. nov. Roszkowska, Grobys, Bartylak & Kaczmarek ( Tables 2 and 4 , Figs 1 , 7–9 ) Pseudechiniscus sp. 3 ( Grobys et al. 2020 ) Material examined: 10 animals ( holotype (female) and 9 paratypes ( 8 females , 1 male )) mounted on microscope slides in Hoyer’s medium and 5 prepared for barcoding. Description Animals (measurements and statistics in Tables 2 and 4 ) Females. Body ( Fig. 7 A–B) 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]). Cephalic papillae smaller than clava. FIGURE 7. Pseudechiniscus angelusalas 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 m1 and m2, empty indented arrowhead indicates straight pseudosegmental plate (psp), filled arrowhead indicates Y-shaped bifurcated ridges on caudal plate (cap) (paratype, PCM); B—characteristic pattern on the ventral side of the body (holotype, PCM); C—focus on upper ends of cuticular pillars connected by striae in dorsal plates (paratype, PCM); D—characteristic pattern on the ventral side of the body (holotype, PCM). Scale bars in micrometres [μm]. FIGURE 8. Pseudechiniscus angelusalas sp. nov. , female — A—granulation on leg III (paratype, PCM); B—granulation and papillae (arrow) on leg IV (paratype, PCM); C—claws of leg III (PCM, paratype). Scale bars in micrometres [μm]. FIGURE 9. Pseudechiniscus angelusalas sp. nov. , male — A—lateral view of the entire animal; (paratype, PCM); B—focus on dorsal plates with visible upper ends of cuticular pillars (paratype, PCM); C—focus on ventral patches of granulation IV and VI (paratype, PCM); D—granulation on leg III (paratype, PCM). Scale bars in micrometres [μm]. Dorsal plates with small hemispherical granules/upper ends of cuticular pillars (dots by PCM) 0.3–0.7 μm in diameter, densely (spaces between granules 0.4–0.6 μm) and uniformly distributed and joined by very thin and delicate (poorly visible) striae forming indistinct hexagonal pattern (which are in fact a thin stripes positioned under epicuticle) ( Fig. 7C ). Granules/upper ends of cuticular pillars slightly larger in the centre of the plates. Dorsal plates typical for the genus Pseudechiniscus (cephalic plate (cp), neck plate (np), 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. The scp divided by transversal fold which form a long narrow stripe in posterior part of the plate. This narrow stripe is sometimes divided by three longitudinal folds which form four plate parts/subplates ( Fig. 7A ). Besides, the entire scp divided into two parts by median longitudinal fold ( Fig. 7A , empty arrow). Additionally, lateral portions of the scp appear detached from the dorsal plate, forming small platelike structures separated from the scp by a thin bright stripe. Plates m1 and m2 divided in two portions by transverse fold, plate m3 undivided ( Fig. 7A , filled indented arrowheads). Laterally to the median plates, lateral intersegmental plates (lip) present. On the plates s1 and s2 darker stripes also visible ( Fig. 7A , filled arrows). The psp divided by a longitudinal fold. Posterior margin of the psp straight, i.e. without projections, teeth or spines ( Fig. 7A , empty indented arrowhead). The cap concave with two Y-shaped bifurcated ridges ( Fig. 7A , filled arrowhead). Ventral cuticle with tiny granulation (formed by dense granules/upper ends of cuticular pillars, 0.1–0.2 μm) forming unique pattern ( Figs 1 , 7B, D ). Ventral PGs present (granulation 0.2–0.4 μm in diameter, spaces between granules 0.2–0.3 μm) with configuration PG:I-II-III-IV-VI-VIII a ( Figs 1 , 7B ). The female gonopore with the typical six-petal rosette. 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 legs IV with uniform wide stripes of granulation (slightly larger in the centre of these stripes) ( Fig. 8 ). Triangular spine on leg I and dentate collar on leg IV absent. A finger-like papillae on leg IV present ( Fig. 8B ). External claws of all legs smooth, internal with spurs directed downwards, identical in legs I–IV ( Fig. 8C ). Male ( Fig. 9 ). Dorsal plates and body appendages arranged identically like in females. Cirri interni and externi and cephalic papillae larger than in females (compare Tables 2 and 4 ). Cephalic papillae larger than clava. Dorsal granules/upper ends of cuticular pillars similar in size, shape and pattern to those in females but without striae ( Fig. 9 A–B). Only one male was studied its body being in lateral position with the ventral side not well visible. Ventral granulation uniform (granules/upper ends of cuticular pillars, 0.2–0.4 μm) without visible pattern or the pattern poorly visible. Ventral PGs present (granulation 0.4–0.6 μm in diameter, spaces between granules 0.3–0.8 μm) with configuration PG:I-II-IV-VI-VIII g ( Fig. 9C ). Patches of granulation more pronounced than in females. The gonopore round and without the six-petal rosette and with indistinct PG in anterior and lateral parts. Leg sculpture similar to females but with larger granulation ( Fig. 9D ). Juveniles. Unknown. Larvae. Unknown. DNA sequences We obtained good quality sequences for the applied molecular markers: – COI sequence (GenBank: MN528468 ), 691 bp long; – ITS-2 sequence (GenBank: MN537864 ), 474 bp long. Etymology. The specific epithet ‘ angelus alas ’ means, in Latin, ‘angel wings’ and refers to the similarity of some ventral structures to the angel wings. Type locality. 22°37’04.5”S , 46°43’14.1”E , 1,201 m asl: Madagascar , Fianarantsoa Province , Ivohibory forest, mosses from dead wood, 04.06.2017 , coll. Marta Kepel and Andrzej Kepel. Type depositories. Holotype : slide MAD87/10 and 9 paratypes (slides: MAD87/*, where the asterisk can be substituted by any of the following numbers: 1, 5, 10, 2/S 4/S, 5/S, 6/S, 7/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 . Morphological differential diagnosis* *only measurements of adult females are used in differential diagnosis Pseudechiniscus angelusalas sp. nov. differs specifically from: 1. Pse. beasleyi, by: the scp not divided in anterior part (the scp divided into four parts in Pse. beasleyi ), shorter cirri interni (6.0–7.3 μm in Pse. angelusalas sp. nov. vs 10.4–15.7 μm in Pse. beasleyi ), shorter cirri externi (9.1–10.4 μm in Pse. angelusalas sp. nov. vs 13.1–18.3 μm in Pse. beasleyi ), different claws length arrangement (shortest claws II and III, and longest claws IV in Pse. angelusalas sp. nov. vs claws I and II shortest and III and IV longest in Pse. beasleyi ) and shorter claws (see Table 4 in this paper and Table 2 in Li et al. 2007 ). 2. 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 ), higher sp of cephalic papillae ([ 18.3–20.8 ] in Pse. angelusalas sp. nov. vs [ 11.8–16.6 ] in Pse. chegi ), shorter cirri externi (9.1–10.4 μm in Pse. angelusalas sp. nov. vs 10.5–14.2 μm in Pse. chegi ), higher sp of cirri A ([ 129.2–152.2 ] in Pse. angelusalas sp. nov. vs [ 96.0 –111.1] in Pse. chengi ), higher cirrus A /body length ratio (19–22% in Pse. angelusalas sp. nov. vs 13–17% in Pse. chengi ) and shorter claws I–IV and lower sp of claws II–III (see Table 4 herein and Table 2 in Xue et al. 2017 ). 3. Pse. clavatus , by: different shape of clavae (finger-like in Pse. angelusalas 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. angelusalas 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 , 7B for Pse. angelusalas 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. angelusalas 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 cap in comparison with other dorsal plates in Pse. indistinctus sp. nov. ), higher sp of cephalic papillae ( 18.3–20.8 in Pse. angelusalas sp. nov. vs 15.5–17.3 in Pse. indistinctus sp. nov. ), shorter cirri externi (9.1–10.4 μm in Pse. angelusalas sp. nov. vs 11.3–14.4 μm in Pse. indistinctus sp. nov. ) and shorter spurs on all legs (1.3–1.6 μm in Pse. angelusalas sp. nov. vs 1.7–2.3 μm 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. angelusalas sp. nov. . See also Morphological differential diagnosis of Pse. suillus and Discussion in the paper Grobys et al. (2020) . 9. Pse. lacyformis sp. nov. , by: different ventral pattern Figs 1 , 7B for Pse. angelusalas sp. nov. vs Figs 1 , 10B for Pse. lacyformis sp. nov. ), dorsal granules joined by striae, smaller body size (113.0–143.0 μm in Pse. angelusalas sp. nov. vs 152.0–201.0 μm in Pse. lacyformis sp. nov. ), shorter cirri interni (6.0–7.3 μm, [ sp=34.4–36.6 ] in Pse. angelusalas sp. nov. vs 10.6–14.0 μm, [ sp=48.4–53.9 ] in Pse. lacyformis sp. nov. ), shorter cirri externi (9.1– 10.4 μm, [ sp=49.0–54.3 ] in Pse. angelusalas sp. nov. vs 10.6–14.0 μm, [ sp=48.4–53.9 ] in Pse. lacyformis sp. nov. ), shorter papillae on leg IV (2.0–2.4 μm, [ sp=10.9–11.6 ] in Pse. angelusalas sp. nov. vs 3.2–4.2 μm, [ sp=14.7–17.2 ] in Pse. lacyformis sp. nov. ) and lower spur/branch length ratio of all claws (23–26% in Pse. angelusalas sp. nov. vs 15–20% in Pse. lacyformis sp. nov. ). 10. Pse. megacephalus , by: different shape of cephalic papillae (elongated in Pse. angelusalas 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 7B for Pse. angelusalas 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-VI-VIII a in Pse. angelusalas sp. nov. vs PG:I-II-III-IV-VI-VIII g in Pse. suillus ), dorsal granules joined by striae, shorter cirri interni (6.0–7.3 μm, [ sp=34.4–36.6 ] in Pse. angelusalas sp. nov. vs 8.7–11.1 μm, [ sp=44.0–49.6 ] in Pse. suillus ), shorter cirri externi (9.1–10.4 μm, [ sp=49.0–54.3 ] in Pse. angelusalas sp. nov. vs 12.0–16.8 μm, [ sp=62.1–75.0 ] in Pse. suillus ), shorter papillae on leg IV (2.0–2.4 μm, [ sp=10.9–11.6 ] in Pse. angelusalas sp. nov. vs 3.3–4.3 μm, [ sp=14.7–18.4 ] in Pse. suillus ) and shorter claws (see Table 4 herein and Table 4 in Grobys et al. 2020 ). TABLE 4. Measurements [in µm] and sp values of selected morphological structures of females of Pseudechiniscus angelusalas 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	9	113	–	143	625	–	698	129	668	11	25	113	646
Scapular plate length	9	17.4	–	20.9	–	19.3	–	1.4	–	17.5	–
Head appendages lengths
Cirrus internus	8	6.0	–	7.3	34.4	–	36.6	6.7	35.3	0.4	0.8	6.4	36.6
Cephalic papilla	8	3.2	–	4.2	18.3	–	20.8	3.7	19.3	0.3	0.8	3.2	18.3
Cirrus externus	8	9.1	–	10.4	49.0	–	54.3	9.8	51.7	0.4	2.0	9.5	54.3
Clava	8	3.9	–	4.7	22.4	–	25.7	4.5	23.5	0.3	1.3	4.5	25.7
Cirrus A	8	24.1	–	28.0	129.2	–	152.2	25.9	136.3	1.5	8.2	24.3	138.9
Cirrus A /Body length ratio	8	19%	–	22%	–	20%	–	1%	–	22%	–
Cirrus int / ext length ratio	8	66%	–	71%	–	68%	–	2%	–	67%	–
Papillae on leg IV length	7	2.0	–	2.4	10.9	–	11.6	2.2	11.3	0.2	0.3	?	?
Claw 1 heights
Branch	8	5.6	–	6.3	28.2	–	33.7	5.9	30.8	0.3	1.8	5.9	33.7
Spur	6	1.3	–	1.5	7.1	–	8.2	1.4	7.6	0.1	0.4	?	?
Spur/branch height ratio	6	23%	–	25%	–	25%	–	1%	–	–	–
Claw 2 heights
Branch	8	5.3	–	5.8	25.8	–	31.0	5.5	28.7	0.2	1.7	5.3	30.3
Spur	6	1.3	–	1.5	6.7	–	8.0	1.4	7.3	0.1	0.5	1.3	7.4
Spur/branch height ratio	6	25%	–	26%	–	26%	–	1%	–	25%	–
Claw 3 heights
Branch	8	5.3	–	5.7	25.4	–	31.0	5.5	28.8	0.1	1.9	5.4	30.9
Spur	7	1.3	–	1.6	7.1	–	8.6	1.5	7.7	0.1	0.5	1.5	8.6
Spur/branch height ratio	7	25%	–	29%	–	27%	–	2%	–	28%	–
Claw 4 heights
Branch	7	5.7	–	7.0	31.1	–	35.9	6.3	33.2	0.4	1.8	6.1	34.9
Spur	4	1.3	–	1.6	7.5	–	8.2	1.5	7.8	0.1	0.3	?	?
Spur/branch height ratio	4	23%	–	25%	–	23%	–	1%	–	–	–

12. Pse. xiai , known only from China ( Wang et al. 2018 ), by: different ventral pattern ( Figs 1 and 7B for Pse. angelusalas sp. nov. vs Figs 1B, F and 2E in Wang et al . (2018) for Pse. xiai ), dorsal granules joined by striae, smaller body size (113.0–143.0 μm in Pse. angelusalas sp. nov. vs 179.0–215.0 μm in Pse. xiai ), higher cirrus A / body length ratio (19–22% in Pse. angelusalas sp. nov. vs 13–16% in Pse. xiai ), shorter papillae on leg IV (2.0–2.4 μm in Pse. angelusalas sp. nov. vs 2.5–5.1 μm in Pse. xiai ) and shorter claws (see Table 4 herein and Table 1 in Wang et al. 2018 ). Genotypic differential diagnosis The ranges of genetic between Pse. angelusalas sp. nov. and species of the genus Pseudechiniscus , for which DNA sequences are available in GenBank, are as follows: COI : 24.5–28.5% (26.7% on average), with the most similar being Pse. dastychi sp. nov. ( MN528469 , present study) and the least similar being Pse. aff. suillus ( MK804900 , Cesari et al. 2020 ). ITS-2: 12.8–39.3% (29.1% on average), with the most similar being Pse. ehrenbergi sp. nov. ( MN537866 , present study) and the least similar being Pse. suillus ( MN537863 ).