Hypsibius repentinus, Yuen & Chen & Yuen & Liu & Leung & Ko & Chan & Somani & Herrmann & Jung & Tokas & Croghan & Traxer & Chiu & Chew & Teoh & Gauhar & Li & Ng, 2021

Hypsibius repentinus View in CoL sp. nov.

( Figs 1–4)

Holotype. Female ; Sweden, Härjedalen, approx. 62°8′52″N 13°57′23″E, ca. 400 m a.s.l., Lake Norderåssjön, bottom near shore, plant debris, 30 Aug. 2017, Raquel Pereira leg. (SPbU 254(32)). GoogleMaps

Paratypes. 42 females, 2 exuviae with eggs, 10 cysts, same data as for holotype (SPbU 254(1) to 254(31) and 254(33) to 254(35)) GoogleMaps ; 5 adults, 15 cysts, same data as for holotype, SEM stub (SPbU_Tar20). All the type specimens are kept at St Petersburg State University GoogleMaps .

Morphological description. Body elongate, slightly widened in the region of legs III ( Fig. 1), with a blunt snout (morphometrics: Table 2 and Electronic supplementary material 2).

Colour. Body transparent or whitish. Live specimens with black eyespots, becoming invisible after mounting in Hoyer’s medium ( Fig. 1A).

Cuticular sculpture. Cuticle looking smooth under LM; rugose sculpture better developed dorsally, visible over body surface under SEM ( Figs 1B–D, 2A, B). No external cephalic sensory structures ( Fig. 2B).

Mouth. Opening anteroventral, on developed mouth cone; mouth opening surrounded by simple cuticular fold, without distinct peribuccal lobes or papulae ( Fig. 2C). In some specimens, a line of elliptical structures visible around mouth opening under LM ( Fig. 2F: black arrowhead), similar to those described in Notahypsibius pallidoides , Acutuncus antarcticus ( Richters, 1904) and Hypsibius murrayi ( Richters, 1907) ( Dastych, 1991, 2018; Tumanov, 2020).

Bucco-pharyngeal apparatus of Hypsibiinae model ( Fig. 2D). Oral cavity armature with a ring of small teeth located in anterior part (visible under SEM only; Fig. 2C, E). Dorsal and ventral apophyses for insertion of stylet muscles similar in shape and size ( Fig. 2G, H). Buccal tube rigid, often slightly bent ventrally in caudal part. Stylet furcae typically-shaped. Pharyngeal bulb spherical, with well-developed apophyses, two elongate macroplacoids and large septulum ( Fig. 2D, I, K). No microplacoids or pseudoseptulum.

First macroplacoid slightly longer than second; both macroplacoids with distinct (well discernible under LM) constrictions: in the middle of first macroplacoid and slightly closer to caudal part of second macroplacoid ( Fig. 2D, I–M: black arrows).

Legs and claws. All legs with well-developed claws, increasing in size from legs I to IV ( Figs 1A, 3A–F). Claws of Hypsibius - type with external and internal claws of each leg evidently dissimilar. All claws with developed accessory points, free apices of accessory points of external claws I–III usually positioned lateral to main branch and poorly discernible under LM ( Fig. 3A, E). Bases of all claws smooth, slightly widened. Claws of legs I–III with poorly developed smooth lunules (= pseudolunules, according to Gąsiorek et al., 2017) ( Fig. 3B: black arrows). Claws of legs IV with more developed lunules (usually larger and better visible at bases of anterior claws; Fig. 3D: black arrow) and a cuticular bar between the bases of the anterior and the posterior claws ( Fig. 3C, D, black arrowheads). Legs I–III without cuticular bars near claw bases but with an elongated bulge located near base of internal claw (visible under SEM only; Fig. 3E: white arrowhead).

Cysts. Numerous cysts were observed in the culture on the late stage of its development. Cysts oval (mean length 113.2 ± 10.3 µm, mean width 69.9 ± 3.3 µm; n = 10), with thickened cuticle forming more or less developed circular transverse folds ( Fig. 4A–C). Legs contracted, not raised above body surface ( Fig. 4A, C), with strongly modified claws being shorter and much more massive than claws of active forms ( Fig. 4D, E). No additional layers of cuticle visible below cyst wall.

Eggs. One to four white subspherical eggs with smooth shell laid in exuviae, 58.0–63.0 µm in diameter (mean 60.6 ± 2.3 µm; n = 5).

Reproduction mode. No males were observed, which suggest a parthenogenetic type of reproduction.

Phenotypic comparison. Hypsibius repentinus sp.nov. belongs to the Hypsibius dujardini species– group, recognised here as a group of species with a smooth or slightly rugose cuticle, two macroplacoids and a septulum (or microplacoid) in the pharynx (see Discussion). This group currently contains eleven species: H. allisoni Horning, Schuster et Grigarick, 1978 , H. conwentzii Kaczmarek, Parnikoza, Gawlak, Esefeld, Peter, Kozeretska et Roszkowska, 2018 , H. dujardini , H. exemplaris Gąsiorek, Stec, Morek et Michalczyk, 2018 , H. iskandarovi Tumanov, 1997 , H. murrayi , H. pachyunguis Maucci, 1996 , H. septulatus Pilato, Binda, Napolitano et Moncada, 2004 , H. seychellensis Pilato, Binda et Lisi, 2006 , H. valentinae Pilato, Kiosya, Lisi et Sabella, 2012 , and H. vaskelae Tumanov, 2018 .

Hypsibius repentinus View in CoL sp. nov. clearly differs from H. conwentzii View in CoL , H. iskandarovi View in CoL , H. murrayi View in CoL , H. septulatus View in CoL and H. vaskelae View in CoL in the absence of cuticular bars on legs I–III, and from H. allisoni View in CoL and H. pachyunguis View in CoL , in having a large distinct septulum instead of small dot-like structures usually considered as microplacoids ( Horning et al., 1978; Pilato et al., 2012; Tumanov, 2018).

Hypsibius repentinus View in CoL sp. nov. differs from H. seychellensis View in CoL in having a wider buccal tube ( pt value for the external buccal tube width 7.9–10.9 in H. repentinus View in CoL sp. nov. vs. 6.3–6.4 in H. seychellensis View in CoL ) and in the presence of a constriction of the second macroplacoid visible under LM.

Hypsibius repentinus sp. nov. differs from H. valentinae in having a slightly wider buccal tube ( pt value for the external buccal tube width 7.9–10.9 in H. repentinus sp. nov. vs. 7.4–7.8 in H. valentinae ), the higher pt value for the stylet support inserting point ( 62.8–66.8 in H. repentinus sp. nov. vs. 61.3–62.5 in H. valentinae ), the shorter external claws of legs II ( pt value 41.6–56.3 in H. repentinus sp. nov. vs. 60.2–60.3 in H. valentinae ) and in the presence of a constriction of the second macroplacoid visible under LM.

Hypsibius repentinus sp. nov. is very similar to H. dujardini and H. exemplaris , differing from them only in the presence of a constriction of the second macroplacoid visible under LM.

It should be noted that a form with a very similar configuration of the buccal-pharyngeal apparatus, with an evident constriction on the second macroplacoid, was briefly described and illustrat- ed from Italy by Bertolani (1982) as belonging to H. dujardini . As with other numerous records of H. dujardini published prior to the redescription of this species ( Gąsiorek et al., 2018a), the real taxonomic status of this form is unclear, but it can be an evidence of the presence of H. repentinus in Italy.

DNA sequences . Sequences of good quality for the four molecular markers mentioned above were obtained from the two hologenophores: voucher specimens 254(17) and 254(18). Each gene was represented by a single haplotype. All obtained sequences were deposited in GenBank.

COI sequence (GenBank: MW549048 and MW549049 ), 645 and 648 bp long.

18S rRNA sequence (GenBank: MN927183 and MN927184 ), 852 and 830 bp long.

28S rRNA sequence (GenBank: MW549063 and MW549064 ), 800 and 802 bp long.

ITS-2 sequence (GenBank: MW549061 and MW549062 ), 457 and 469 bp long.

Molecular comparison. The ranges of uncorrected genetic p -distances between the studied population of Hypsibius repentinus sp. nov. and the species of the subfamily Hypsibiinae whose sequences are available from GenBank

D.V. Tumanov & G.S. Avdeeva. Integrative description of Hypsibius repentinus sp. nov.

(see Electronic supplementary material 1) are as follows:

COI: 17.4%–24.5% (mean 20.5%), with the most similar being Hypsibius cf. exemplaris 3 ( MW010376 View Materials : Zawierucha et al., 2020) and the least similar being Cryobiotus klebelsbergi (Mihelč ič, 1959) ( KT901831 View Materials : Dabert et al., 2015).

18S rRNA: 0.0%–4.1% (mean 2.0%), with the most similar being H. exemplaris ( HQ604943 View Materials : Gąsiorek et al., 2018a) and Hypsibius sp. ( EU266939 View Materials : Sands et al., 2008) and the least similar being H. scabropygus ( KC582831 View Materials : Dabert et al., 2014).

28S rRNA: 0.5%–3.2% (mean 2.2%), with the most similar being H. exemplaris ( MG800337 View Materials : Gąsiorek et al., 2018a) and the least similar being C. klebelsbergi ( KC582835 View Materials : Dabert et al., 2014).

ITS-2: 2.8%–11.6% (mean 7.2%), with the most similar being H. exemplaris ( MG800336 View Materials : Gąsiorek et al., 2018a) and the least similar being H. dujardini ( MG777531 View Materials : Gąsiorek et al., 2018a).

Full matrices with p -distances are provided in Electronic supplementary material 3.

Etymology. The species epithet repentinus is a Latin adjective, which means “unexpected”. It refers to an unexpected appearance of the species in the material prepared for culturing protists.