Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011

Tumanov, Denis V., 2020, Integrative redescription of Hypsibius pallidoides Pilato et al., 2011 (Eutardigrada: Hypsibioidea) with the erection of a new genus and discussion on the phylogeny of Hypsibiidae, European Journal of Taxonomy 681, pp. 1-37 : 6-22

Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011

Figs 1–7

Material examined

Holotype

UKRAINE • Kherson Oblast, Ivano-Rybalchansky district of Chernomorsky biosphere reserve ; 46º27′25″ N, 32º8′56″ E; Jun. 2008; D.A. Korolesova leg.; moss on wood; UNICT 5430 .

Paratypes

UKRAINE • 1 spec. + 1 exuvium with eggs; same collection data as for holotype; UNICT 5430 • 4 specs + 2 exuviae with eggs; same collection data as for holotype; KNU Чер- 9 II .

Other material

AUSTRIA • 78 specs + 35 exuvia with eggs; Carinthia; 46.817818º N, 13.859837º E; 20 Aug. 2017; A. Smirnov leg.; moss on soil; GenBank: MK973069 , MN912103 , MK967961 to MK967964 , MN927181 , MN927182 , MN919385 , MN915220 , MN915221 , MK967241 , MN918533 ; SPbU 251(1– 13) , 251(28) .

CROATIA • 1 spec.; Park Šuma Golubinjak [Golubinjak Forest Park], Primorje-Gorski Kotar County; 45.35216º N, 14.76557º E; 10 Sep. 2005; O. Orlova leg.; moss on stone; SPbU 228(30) .

RUSSIA – St Petersburg • 1 spec. + 1 exuvium; Puskin City ; 59.72537º N, 30.39147º E; 15 May 2016; D. Tumanov leg.; moss on tree trunk; SPbU 234(10) . – Karelia • 3 specs + 1 exuvium; vicinity of Akkaharju village; 61.49584º N, 29.84775º E; 11 May 1994; D. Tumanov leg.; mosses and leaf litter from the overgrown lake; SPbU 113(2) .

Morphological redescription

MEASUREMENTS. Body elongated, of uniform width on the entire body length ( Fig. 1 ), with a blunt snout (morphometrics Tables 2–3).

COLOUR. Body uncoloured or whitish with green gut content. Most specimens with eyespots, usually well-discernible after slide mounting ( Fig. 1A ) but absent in some specimens.

CUTICULAR SCULPTURE. Dorsal cuticle sculpture consists of a system of transverse folds with smaller irregular folds between ( Figs 1B , 2 A–D). Cuticle sculpture better visible in the caudal region of the body

( Figs 2 C–D, 3A–B), well developed even in juveniles ( Fig. 4E ). Ventral surface with poorly developed foldings, visible in SEM only ( Fig. 1C ).

CEPHALIC SENSORY STRUCTURES. Cephalic body portion with a pair of elliptical sensory organs developed in the form of flat porous areas, separated from the body surface with a oval cuticular groove. These structures are scarcely visible in LM, but are well-discernible in SEM ( Fig. 3 C–D, black arrowheads). Two indistinctly demarcated porous areas are also developed in the fronto-lateral region of the head, on the each side of the mouth opening (visible in SEM only; Fig. 4 A–B, white arrowheads). Central concavity on the dorsal surface of the head ( Fig. 3C , white arrowhead) seems to be similar to the structures present in some Isohypsibioidea (see Gąsiorek et al. (2019c: 91, fig. 4b, d) and in Cryobiotus roswithae ( Dastych 2019).

MOUTH. Opening antero-ventral, surrounded by six peribuccal lobes (visible in SEM only; Fig. 4C ). In large specimens a line of elliptical structures is visible in LM around the mouth opening ( Fig. 4 G–H, black arrowheads), similar to those described for Acutuncus antarcticus (Richters, 1904) and Hypsibius murrayi (Richters, 1907) ( Dastych 1991, 2018). These structures are possibly the compressed peribuccal lobes.

BUCCO- PHARYNGEAL APPARATUS. Hypsibiinae model sensu Pilato & Binda 2010 ( Fig. 4F ). Oral cavity armature with a ring of small teeth located it its anterior part followed by the second band of larger,

irregular teeth (visible in SEM only; Fig. 4D ). Dorsal and ventral apophyses for the insertion of the stylet muscles (AISM) are evidently dissimilar. Dorsal AISM are shorter and higher than ventral, with thickened anterior margin ( Fig. 5 A–C). A short thickening of the buccal tube wall is present posteriorly to both these apophyses (the ventral poorly visible; Fig. 5A , black arrowheads). Buccal tube rigid, bent ventrally in its caudal part ( Fig. 5A ). Stylet furcae typically shaped (sensu Pilato & Binda 2010) ( Fig. 4F ). Pharyngeal bulb spherical ( Fig. 5D , black arrowhead), with well-developed apophyses, two elongated macroplacoids, and a small dot-like structure interpreted here as a septulum (following Pilato et al. 2011) ( Fig. 5A , white arrow), connected to the second macroplacoid with a thin cuticular line (often scarcely visible) ( Fig. 5D , black arrow). No microplacoids. Posteriorly to the septulum, an indistinct thickening of the cuticular lining similar to “pseudoseptulum” described in Diphascon mirabilis Dastych, 1984 and Hypsibius iskandarovi Tumanov, 1997 is present ( Dastych 1984; Tumanov 1997) ( Fig. 5D , white arrowhead). First macroplacoid longer than second with a slight constriction in the middle ( Fig. 5 E–F, black arrowhead). Second macroplacoid can also have a poorly developed subterminal constriction (visible in SEM only) ( Fig. 5F , black arrow).

LEGS AND CLAWS. All legs with well-developed claws, increasing in size from legs I to legs IV ( Fig. 6 A–I). Legs IV evidently swollen dorsally, above the claws ( Fig. 3A , white arrowheads). Claws similar to the Ramazzottius- type claws (sensu Pilato & Binda 2010 and Guidetti et al. 2019b) with external and internal claws of each leg strongly dissimilar. External claws with massive base+ secondary branch complex, where the base is at least as long as the secondary branch and only slightly curved, while the secondary branch is thinner than the base and connected with it at a nearly right angle without forming a smooth ark ( Fig. 6 A–B, I). Thin and long primary branch connected to the base+secondary branch complex far from the claw’s basal point (length of the claw base is equal or slightly exceeds the length of the secondary branch). The connection point is shifted laterally and located near the evident crest developed on the lateral surface of the claw base ( Fig. 6G, I , white arrowheads). Basal part of the primary branch flexible, with thinned walls ( Fig. 6 A–B, black arrowheads). External and posterior claws of H. pallidoides differ from typical Ramazzottius- type claws only in having primary branches wider with less pronounced differentiation between rigid distal and soft basal parts. Primary branches are connected with the base by a filamentous structure (not always visible in LM, Fig. 6D , black arrow), but no distinct light-refracting unit is present. Internal claws much shorter than external ones, without flexible parts, with developed internal structure, consisting of the system of cavities and septae ( Fig. 6A , D–E). All claws with developed accessory points and widened smooth bases ( Fig. 6 A–I). Claws of legs I–III with very poorly developed smooth lunules (or pseudolunules, according Gąsiorek et al. 2017) ( Fig. 6A , white arrow), usually not discernible on the external claws. Claws of legs IV with welldeveloped wide lunules ( Fig. 6E, I ). Posterior claws with thickened region on the lunule margin, visible in LM as a dark line, which can create the impression of the presence of a cuticular bar between the bases of the anterior and posterior claws ( Fig. 6 E–F, black arrows). Legs I–III without cuticular bars near the claw bases, but with an elongated bulge located near the base of the internal claw. In SEM, the pulvinus is similar in appearance to the typical cuticular bar of Hypsibioidea , but in LM no zone of thickened cuticle is visible ( Fig. 6C, H , white arrows). Also, poorly developed pulvini are visible on the inner side of the legs ( Fig. 6C, H , white asterisks)

EGGS. One to six white subspherical eggs are laid in the exuvium ( Fig. 7A ), 59.4–71.9 µm in diameter (65.96 ± 3.71; N = 20). Egg shell in LM appears sculptured with minuscule granules, visible only with PhC or DIC in high magnification ( Fig. 7 B–C, E–F). In fact, these granules are inner pillar-like structures in the egg shell ( Fig. 7D ).

DNA sequences

Sequences of good quality for the 4 aforementioned molecular markers were obtained from five specimens: 2 paragenophores and 3 hologenophores (voucher specimens 251(09), 251(10) and 251(87)). Each gene was represented by single haplotype.

COI sequence (GenBank: MK967241 ), 688 bp long.

18S rRNA sequence (GenBank: MN912103 ), 1777 bp long:

28S rRNA sequence (GenBank: MK967961 ), 1618 bp long:

ITS-2 sequence (GenBank: MN927181 ), 486 bp long:

All obtained sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the following accession numbers: MK967241 , MN915220 , MN915221 , MN918533 , MN919385 (COI), MK973069 , MN912103 (18S rRNA); MK967961 , MK967962 , MK967963 , MK967964 (28S rRNA); MN927181 , MN927182 (ITS-2).

Phylogenetic analysis

In the 18S DNA phylogenetic analysis, the order Hypsibioidea was highly supported and divided into two well-supported clades: clade I, embracing the family Ramazzottiidae, Marley, McInnes & Sands, 2011 , and clade II, comprised of taxa currently attributed to the families Hypsibiidae Pilato, 1969 , Calohypsibiidae Pilato, 1969 and Microhypsibiidae Pilato, 1998 ( Fig. 8 ). Clade II was further divided into two subclades well-supported with Bayesian analysis, but weakly supported or unsupported with ML analysis. The first subclade included the families Microhypsibiidae s. str. (genus Microhypsibius Thulin, 1928 ) and Calohypsibiidae s.str. (genus Calohypsibius Thulin, 1928 ) (see Gąsiorek et al. 2019a for a discussion on the taxonomic composition of these two families), together with two genera of unclear taxonomic position, Acutuncus and Mixibius . The second subclade was divided into three subclades with unclearly resolved phylogenetic relationships. The first of these subclades included the species representing the subfamily Pilatobiinae Bertolani, Guidetti, Marchioro, Altiero, Rebecchi & Cesari, 2014 , Hypsbius pallidoides , and the species attributed to Hypsibius convergens (Urbanowicz, 1925) by Guil & Giribet (2012). The second one included the species of the subfamily Itaquasconinae Rudescu, 1964 and the third one was comprised of two well-supported lineages, the subfamilies Diphasconinae Dastych, 1992 and Hypsibiinae Pilato, 1969 .

Analyses of the concatenated18S+ 28S sequences resulted in a phylogeny with the same tree configuration, but with slightly weaker support of the clades (see Supplementary file SM.02). This weakened support is possibly a consequence of the small number of sequences available for such analysis.

Comparison with the original description

Morphometry of specimens from all analysed populations (including the type series) corresponds well with the data from the original description ( Pilato et al. 2011). Small differences in the values of the stylet supports insertion point pt index (54.2–55.2 in the original description vs 56.9–63.3 in the material investigated) and the length of the first macroplacoid (3.8–4.2 µm (pt 15.5–17.0) vs 2.2–3.8 µm (11.2– 15.9) respectively) should be considered as the result of some differences in the measuring process, taking into account that my own measurements of the type series specimens are concordant with those of the specimens from the other populations (see Table 2).

It was stated in the original description ( Pilato et al. 2011) that H. pallidoides had a smooth cuticle, but high quality LM and SEM observations revealed the presence of a cuticular sculpture ( Figs 2 A–D, 3A– B). It is poorly visible in the type series specimens because of the intensive staining of soft tissues with acetocarmine during the slide preparation.

Contrary to the absence of lunules in H. pallidoides stated by Pilato et al. (2011), my investigation determined that scarcely visible lunules on the claws of legs I–III and well-developed wide lunules on the claws of legs IV are present ( Fig. 6A, E ). In the original description of the species, Pilato et al. (2011) indicated the absence of a cuticular bar between the claw bases of legs IV, but considered this as unconfirmed. My observations revealed the presence of a thickened zone of the posterior claw lunule, located between the anterior and the posterior claw bases ( Fig. 6 E–F). This thickening can give the impression of a cuticular bar in the case when the main part of the lunule is not discernible.

While Pilato et al. (2011) described the eggs of H. pallidoides as being smooth, further scrutiny ascertained the presence of a granular pattern formed by the system of internal pillars in the egg shell of this species ( Fig. 7 B–G).

New phenotypic differential diagnosis

Hypsibius pallidoides is similar to the species of the genera Ramazzottius Binda & Pilato, 1986 and Cryoconicus Zawierucha, Stec, Lochowska-Cierlik, Takeuchi, Li & Michalczyk, 2018 in having claws of the Ramazzottius type; AISM asymmetrical with respect to the frontal plane; cephalic elliptical sensory organs and in laying ornamented eggs. It clearly differs from all species of those genera by having wider primary branches of the external and posterior claws, with less pronounced differentiation between rigid distal and soft basal parts; the dorsal AISM raised and thickened in its anterior margin, and eggs laid in the exuvium without external processes, but with pillars inside the egg shell only.

Hypsibius pallidoides is similar to the species of the genus Mixibius in having AISM asymmetrical with respect to the frontal plane, where the ventral apophysis is similar, but not identical, to the “semilunar hook” of Hypsibius ; dorsal apophysis more stumpy with a blunt and swollen caudal apex. Also a short median cuticular thickening caudal to both these apophyses is present (the ventral one slightly visible) ( Pilato & Binda 2010). It clearly differs from all species of this genus by having: cephalic elliptical sensory organs and Ramazzottius -like claws (external claws with elongated primary branches and less developed secondary branches).

The type of egg shell sculpture of Hypsibius pallidoides is similar to that of Acutuncus antarcticus , from the Antarctic region (see Dastych 1991 for a review of the old records) in that the sculpture,

.

formed by the pillars within the egg shell, presents as a dot-like pattern when observed in LM. Hypsibius pallidoides differs from A. antarcticus by having the Ramazzottius - type claws; AISM asymmetrical with respect to the frontal plane; a sculptured cuticle and a small dot-like septulum. The precise nature of the latter structure requires further investigation as its small size prevents it from being undoubtedly interpreted as microplacoid or septulum.

Hypsibius pallidoides is similar to the following species of the genus Hypsibius : Hypsibius allisoni Horning, Schuster & Grigarick, 1978 (known from New Zealand and South America ( Horning et al. 1978; Maucci 1988; Pilato et al. 2003)); H. murrayi (= H. heardensis Miller, McInnes & Bergstrom, 2005 ; known from Antarctica ( Dastych 2018)) and H. pachyunguis Maucci, 1996 (known from Greenland).

Hypsibius pallidoides clearly differs from the above mentioned species by having the Ramazzottius -like claws and by having cuticular sculpture. Additionally, H. pallidoides differs from:

Hypsibius allisoni by having a thinner buccal tube (external width up to 2.1 µm in H. pallidoides vs 4 µm in H. allisoni holotype) ( Horning et al. 1978).

Hypsibius murrayi by the absence of cuticular bars near the claw bases of legs I–III, by having a dot-like pattern of the egg shell, a smaller body length (up to 292 µm in H. pallidoides vs 338.0–603.0 µm in H. murrayi ) ( Dastych 2018).

Hypsibius pachyunguis by having less elongated macroplacoids (see Maucci 1996: 196, fig. 1; Tumanov 2018: 440, fig. 4a–b).

Two species of the genus Hypsibius are known as laying eggs with granulated chorion in exuvium, Hypsibius roanensis Nelson & McGlothlin, 1993 ( Guidetti et al. 1999) and H. cf. scabropygus ( Guidetti & Bertolani 2001) . Hypsibius pallidoides clearly differs from both of these species by having a septulum, the Ramazzottius -like claws, and a different cuticular sculpture.

Genotypic differential diagnosis

The ranges of uncorrected genetic p -distances between the studied population of Hypsibius pallidoides and species of the order Hypsibioidea for which sequences are available from GenBank (see Supplementary file SM.01) are as follows:

COI: 20.9%–26.7% (mean 23.0%), with the most similar being Pilatobius recamieri ( Richters, 1911) ( KX347530 , Gąsiorek et al. 2017), and the least similar being Diphascon puniceum (Jennings, 1976) ( KP013612 , Velasco-Castrillón et al. 2015).

18S rRNA: 2.0%–8.7% (mean 6.2%), with the most similar being Pilatobius recamieri ( KX347526 , Gąsiorek et al. 2017) and P. islandicus Buda, Olszanowski, Wierzgoń & Zawierucha, 2018 ( MH682258 , Buda et al. 2018), and the least similar being Diphascon puniceum ( EU266948 , Sands et al. 2008).

28S rRNA: 5.9%–18.7% (mean 11.2%), with the most similar being Mesocrista revelata Gąsiorek, Stec, Morek, Zawierucha, Kaczmarek, Lachowska-Cierlik & Michalczyk, 2016 ( KX347536 , Gąsiorek et al. 2016), and the least similar being Ramazzottius varieornatus Bertolani & Kinchin, 1993 ( MG432818 , Zawierucha et al. 2018).

ITS-2: 19.6%–45.1% (mean 38.2%), with the most similar being Pilatobius recamieri ( KX347528 , Gąsiorek et al. 2017), and the least similar being Ramazzottius subanomalus (Biserov, 1985) ( KU900019 , Stec et al. 2016b).

Full matrices with p -distances are provided in the Supplementary file SM.03.

Sequences of the 18S and 28S rRNA genes, attributed to the species “ Hypsibius convergens ” by Guil & Giribet (2012) are nearly identical to those of Hypsibius pallidoides (p -distances 0.0% and 1.1% respectively).

Phylogeny of Hypsibioidea and phylogenetic position of Hypsibius pallidoides

The results of phylogenetic analysis presented herein correspond well with the molecular phylogenies of Tardigrada reconstructed in the recent works of other researchers ( Guil & Giribet 2012; Bertolani et al. 2014; Guil et al. 2019), being most similar to the results of Bertolani et al. (2014). In comparison with the results of Guil et al. (2019), some differences in the tree topology may be attributable to a different approach taken in the selection of the compared sequences. In my opinion, the position of some sequences attributed to the species Acutuncus antarcticus within the cluster of species of Hypsibius ( Guil et al. 2019: fig. 2) and the unclear differentiation of the Itaquasconinae and Hypsibiinae lineages are artefacts, caused by the inclusion of sequences derived from pooled samples which could contain multiple species ( Sands et al. 2008), or by misidentifications of the sequenced specimens (see below).

My phylogenetic analysis confirmed the presence of the weakly supported but distinct basal clade within Hypsibiidae that includes the genera Acutuncus , Mixibius , Calohypsibius and Microhypsibius . With the addition of recently published data for two species of the genus Pilatobius Bertolani, Guidetti, Marchioro, Altiero, Rebecchi & Cesari, 2014 ( Gąsiorek et al. 2017; Buda et al. 2018), the Pilatobiinae clade, recognized in the analysis of Bertolani et al. (2014), became better supported in my analysis. Surprisingly, Hypsibius pallidoides (and a species attributed to H. convergens by Guil & Giribet (2012)) were distinctly placed within the Pilatobiinae clade, and even more interestingly within the genus Pilatobiotus itself, forming a cluster with the species P. patanei (Binda & Pilato, 1971) / P. islandicus / P recamieri , while the species P. ramazzottii (Robotti, 1970) and P. nodulosus (Ramazzotti, 1957) formed a separate paraphyletic group. Grouping of the species attributed to H. convergens with Pilatobius recamieri was obtained by Guil et al. (2019), but this result was not discussed by the authors. In an earlier publication ( Guil & Giribet 2012), the taxon misidentified with H. convergens was joined with Astatumen trinacriae (Arcidiacono, 1962) , but this result is likely an artefact because no species of Pilatobius were used in the analysis. In my opinion, extreme similarity of the 18S and 28S sequences of this species to the sequences of H. pallidoides (p -distances 0.0% and 1.1% respectively) should be considered as evidence of their identity on the genus level. Hypsibius pallidoides is morphologically similar to H. convergens and could be misidentified with this species, especially when temporary slides were used for the identification ( Guil & Giribet 2012), because of the poor visibility of the cuticular sculpture and septulum in living specimens.

As a result, in the case of H. pallidoides we have a distinct contradiction between the morphological and molecular taxonomical approaches. Analysis of the morphological traits of this species reveals similarities with Ramazzottiidae (i.e., presence of the cephalic elliptical organs, the Ramazzottius -like claws, asymmetry of the AISM), but, according to the analysis of the gene sequences, this species should be attributed to the subfamily Pilatobiinae . Its position in the obtained phylogenetic tree also supports the presumably paraphyletic nature of the genus Pilatobius , also inferred by Gąsiorek et al. (2018). To my knowledge, this is the first occurrence of such a distinct controversy between morphological and molecular taxonomy within Tardigrada. Previously, genetic analyses have supported

the erection of taxa recognized by traditional morphological analysis (e.g., genera Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar & Wolf, 2009 , Mesobiotus Vecchi, Cesari, Bertolani, Jönsson, Rebecchi & Guidetti, 2016 , Acantechiniscus Vecchi, Cesari, Bertolani, Jönsson, Rebecchi & Guidetti, 2016, family Ramazzottiidae , order Isohypsibioidea ) ( Guidetti et al. 2009; Vecchi et al. 2016; Sands et al. 2008) or provided an opportunity to resolve the phylogeny of a group when morphological data were insufficient (e.g., the clarification of the phylogenetic position of the genera Apodibius Dastych, 1983 and Haplomacrobiotus May, 1948 , revisions of Isohypsibioidea and Echiniscus C.A.S. Schultze, 1840 ) ( Dabert et al. 2014; Cesari et al. 2016; Gąsiorek et al. 2019b, 2019c). The presence of such controversy is a problem that has been acknowledged in current zoology since molecular methods began to be widely used ( Hillis 1987; Osawa et al. 2004; Smirnov et al. 2005; Cohen 2018). Various authors who have analysed this problem ( Hedges & Sibley 1994; Scotland et al. 2003; Osawa et al. 2004; Wiens 2004; Smith & Turner 2005) came to the conclusion that the best (if not the only) way to align the conflicting morphological and molecular phylogenies is to improve the morphological data by involving new characters in the analysis and by re-evaluating some characters already in use.

Taking into account the unique combination of the morphological features and the phylogenetic position of Hypsibius pallidoides distant from the remaining species of Hypsibius , as demonstrated by the analysis of the molecular data, the erection of the new genus Notahypsibius gen. nov. for the species H. pallidoides is proposed.

Taxonomic account

Figures

Fig. 1. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011, total view. A. Phase contrast (SPbU 251(82)). B. Dorsal view in SEM. C. Ventral view in SEM. Scale bars = 50 µm.

Fig. 2. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011, dorsal sculpture. A. Type series specimen (KNU Чер-9 II), PhC. B. Type series specimen (KNU Чер-9 II), DIC. C. Specimen from Austria (SPbU 251(82)), PhC. D. Specimen from Russia (SPbU 113(3)). Scale bars = 5 µm.

Fig. 3. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011. A. Dorsal view of the caudal body end, white arrowheads indicate the dorsal inflations of the hind legs, SEM. B. Dorsal sculpture of the caudal body region, SEM. C. Dorsal surface of the head region, black arrowheads indicate the elliptical sensory organs, white arrowhead indicates the central concavity on the dorsal surface of the head, SEM. D. Dorsal surface of the head region, black arrowheads indicate the elliptical sensory organs (SPbU 251(89)), PhC. Scale bars = 5 µm.

Fig. 4. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011. A. Lateral view of the head region, white arrowhead indicates the anterior porous area, black arrowhead indicates the elliptical sensory organ, SEM. B. Enlarged view of the lateral surface of the head, white arrowhead indicates the anterior porous area, white arrow indicates the muscle attachment zone, SEM. C. Mouth opening with peribuccal lobes, SEM. D. Mouth opening with anterior ring of teeth, SEM. E. Dorsal sculpture of the juvenile, SEM. F. Bucco-pharyngeal apparatus (SPbU 251(82)), PhC. G. Dorsal view of the buccal cavity, black arrowhead indicates the circumoral elliptical structures (SPbU 251(82)), PhC. H. Ventral view of the buccal cavity, black arrowhead indicates the circumoral elliptical structures (SPbU 251(82)), PhC. Scale bars: A, F–H = 5 µm; B–C = 2 µm; D = 1 µm; E = 10 µm.

Fig. 5. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011, bucco-pharyngeal apparatus. A. Lateral view of the bucco-pharyngeal apparatus, black arrowheads indicate the thickenings of the buccal tube, white arrow indicates the septulum (SPbU 251(109)), PhC. B. Dorsal AISM (SPbU 251(82)), DIC. C. Ventral AISM (SPbU 251(82)), DIC. D. Pharyngeal bulb, black arrowhead indicates the pharynx outline, black arrow indicates the cuticular line connecting second macroplacoid with septulum, white arrow indicates the septulum, white arrowhead indicates the “pseudoseptulum” (SPbU 251(89)), PhC. E. Macroplacoids, black arrowhead indicates the constriction of the first macroplacoid (SPbU 251(89)), DIC. F. Cuticular structures of the pharynx, black arrowhead indicates the constriction of the first macroplacoid, black arrow indicates the constriction of the second macroplacoid, SEM. Scale bars: A–E = 5 µm; F = 2 µm.

Fig. 6 (opposite page). Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011, claws. Specimens SPbU 251(82) (A, C, E) and KNU Чер-9 II (B, D, F). A. Claws of leg I, black arrowhead indicates the flexible part of the external claw main branch, black arrows indicates the lunule of the internal claw, PhC. B. Claws of leg III, type series specimen, black arrowhead indicates the flexible part of the external claw main branch, PhC. C. Claws of leg II, white arrow indicates the elongated bulge near the base of the internal claw, white asterisk – pulvilla on the inner side of the leg, PhC. D. Claws of leg IV, type series specimen, black arrow indicates the filamentous structure connecting the external claw main branch with the claw base, PhC. E. Claws of leg IV, black arrow indicates the thickened region on the lunule margin, PhC. F. Claws of leg IV, type series specimen, black arrow indicates the thickened region on the lunule margin, PhC. G. Claws of leg II, white arrowhead indicates the lateral crest of the claw base, SEM. H. Claws of leg III, white arrow indicates the elongated bulge near the base of the internal claw, white asterisk – pulvilla on the inner side of the leg, SEM. I. Claws of leg IV, white arrowhead indicates the lateral crest of the claw base, SEM

Fig. 7. A–G. Hypsibius pallidoides Pilato, Kiosya, Lisi, Inshina & Biserov, 2011 eggs. A–D, specimen KNU Чер-9 II. A. Type series specimen mounted during the egg laying process, PhC. B. Type series egg shell, PhC. C. Type series egg shell, DIC. D. Type series egg shell structure with numerous internal pillars visible, DIC. E. Austrian population egg shell (SPbU 251(3)), PhC. F. Karelian population egg shell (SPbU 113(2)), PhC. G. Pushkin population egg shell (SPbU 235(28)), PhC. – H. Pilatobius recamieri (Richters, 1911), egg shell (SPbU 203(7)), PhC. Scale bars: A = 50 µm; B–H = 5 µm.

Fig. 8 (opposite page). The phylogeny of Hypsibioidea based on 18S rRNA seqences. Numbers at nodes indicate Bayesian posterior probability values (BI, first values) and bootstrap values (ML, second values). Black dots indicate the nodes supported by values of 1.0/100% with both methods. Scale bar and branch lengths refer to the Bayesian analysis.