Fractonotus verrucosus ( Richters, 1900 ) Gąsiorek & Morek & Stec & Blagden & Michalczyk, 2019

Gąsiorek, Piotr, Morek, Witold, Stec, Daniel, Blagden, Brian & Michalczyk, Łukasz, 2019, Revisiting Calohypsibiidae and Microhypsibiidae: Fractonotus Pilato, 1998 and its phylogenetic position within Isohypsibiidae (Eutardigrada: Parachela), Zoosystema 41 (6), pp. 71-89 : 77-82

Fractonotus verrucosus ( Richters, 1900) n. comb.

Macrobiotus ornatus var. verrucosus Richters, 1900: 41 (terra typica: Taunus, Germany).

Macrobiotus scabrosus Murray, 1911: 10 (locus typicus: Clare Island, Ireland).

Hypsibius verrucosus – Thulin 1911: 29 (Kiruna, Lapland, Sweden). — Marcus 1928: 180 (Vannsee, Berlin, Germany). — Cuénot 1932: 77 (the Vosges, France and Switzerland). — da Cunha 1947, 1948: 6, 2 (Serra d’Arga, Serra do Buçaco, Serra da Estrela, Serra da Lousã, Portugal).

Calohypsibius verrucosus – Thulin 1928: 239 (Sweden).

Calohypsibius scabrosus Thulin, 1928: 239 (Sweden) .

Hypsibius scabrosus Cuénot, 1932: 77 (the Vosges, France and Switzerland). — da Cunha 1947, 1948: 6, 2 (Serra d’Arga, Serra do Buçaco, Serra da Estrela, Serra da Lousã, Portugal).

Hypsibius (Calohypsibius) verrucosus – Marcus 1936: 285 (Schwarzwald, Germany). — Franceschi 1951 -1952: 12 (Val Camonica, Italy). — Mihelčič 1953: 247 (Tirol, Austria). — Fontoura 1981: 18 (Viseu, Arga, Amarante, Portugal).

Hypsibius placophorus da Cunha, 1943: 1 (locus typicus: Cabril do Ceira, Portugal); 1947, 1948: 2, 2 (Serra d’Arga, Serra do Buçaco, Serra da Estrela, Serra da Lousã, Portugal) n. syn.

LOCALITIES. — Scotland. Creag Meagaidh (56°57’10’’ N, 4°30’35’’ W; 291 m a.s.l.; collection date: 1.X.2014), lichens from moorland rocks ; Scotland, Hebrides, Isle of Lewis, Loch nan Muilne (58°21’08’’ N, 6°35’14’’ W; 27 m a.s.l.; collection date: 29.VII.2015), moss and lichen mix from stones on the lakeshores ; Invermoriston, Loch Ness (57°12’39’’ N, 4°35’59’’ W; 20 m a.s.l.; 25.X.2015; Brian Blagden leg.), moss and lichen mix from stones on the lakeshores .

MATERIAL EXAMINED. — 23 individuals, UJ (19 specimens, including one simplex, on slides GB.005.03-12, GB.008.01-3, GB.028.01- 2 and 4 specimens on two SEM stubs) ; 2 individuals, MNHN (slides GB.005.01-2) ; 3 individuals, NHMD (slides GB.008.04- 5) ; 2 individuals, UAM (slides GB.028.03-4) ; 1 individual, CU (slide GB.028.02) .

ETYMOLOGY (NOT PROVIDED IN THE ORIGINAL DESCRIPTION). — The name most likely refers to the rugged cuticular surface of the species (from Latin verruca = wart).

DIFFERENTIAL DIAGNOSIS. — Fractonotus verrucosus n. comb. can be distinguished from F. caelatus and F. gilvus n. comb. ( Fig. 2A, B ) by the presence of plaques (absent in the latter species). It also differs from F. gilvus n. comb. by shorter, stouter claws (anterior and posterior primary branches 4.1-6.4 μm [N=10] and 4.3-7.4 μm long [N=18], respectively, in Fractonotus verrucosus n. comb. vs 7.0- 13.0 μm [N=21] and 10.5-16.5 μm long [N=21], respectively, in F. gilvus n. comb.; compare Fig. 11 A-D).

INTEGRATIVE DESCRIPTION

Animals (see Table 3 for measurements)

Body stubby, typically slightly rose in live animals, transparent in mounted specimens. Dorsum strongly sculptured from the first instar, although with substantial ontogenetic quantitative and qualitative variability in this trait ( Fig. 1 A- F). Juveniles with ten transverse bands of numerous tu- bercles that increase in size towards the caudal end of the body, but fully formed plaques never present, legs covered with fine tubercles ( Fig. 1A ). All ten bands not always easily identifiable under PCM in juveniles. In young adults, plaques present in bands 6-10, with the most prominent plaques in bands 8-10 ( Fig. 1B ). In older adults, smooth spaces between the transverse bands becoming narrow and sometimes merge into larger areas ( Fig. 1 C-F). Plaques larger and more numerous than in young adults, and typically developping in bands 4-10, but the most evident plaques present in the caudal part of the body ( Fig.1 C-F). Tubercles more or less round or oval ( Figs 3A, B ; 5A, B ), gradually increasing in size from juveniles to adults, and becoming scabrous with age (compare Figs 1 A-F; 5A, B). Plaques, on the other hand, typically smooth and only sometimes slightly rough ( Fig. 5C, D , arrowheads); under stereomicroscope strongly opalescent. Plaques arranged symmetrically in respect to the longitudinal body axis, although deviations

A

from symmetry are not rare ( Fig. 1C, D ). In adults, seven pairs of central plaques and four lateral plaque pairs. Central plaques triangular in shape, with their apices directed laterally and outwards. In rows where only central plaques are present, plaques slimmer and longer than in rows with lateral plaques. Central plaques present in bands aligned with legs I-III as well as in bands between those legs. First three pairs of lateral plaques in line with legs I-III and the last pair of double lateral plaques situated between legs III and IV ( Fig. 1E ). Plaque configuration VII:4-2-4-2-4-2-6. Cephalic elliptical organs present but not easy to identify, given the rich cuticular sculpturing ( Fig. 7A ). Eyes absent in live animals.

Buccal apparatus of the Fractonotus - type ( Fig. 7B, C, E ), i.e. with a long ventral AISM, and the dorsal AISM subdivided into the proximal, weakly developed thickening, and the distal, small blunt hook ( Fig. 9A ). Mouth opening surrounded by six large and soft peribuccal lobes (visible only under SEM, Fig. 6A ). Oral cavity armature, visible only under SEM, consisting of a single row of minute conical teeth located on the ring fold ( Fig. 8A ). Two distinct porous areas on the lateral sides of the buccal crown are visible in SEM only ( Fig. 8B ). Stylet furcae of the modified Hypsibius shape, i.e. with very broad and trapezoid bases, thick arms and rounded apices ( Figs 7B , 8D , 10A ). Buccal tube with slight lateral thickenings posterior to the stylets supports ( Figs 7B, C, E ; 8C ). Round bulbus with large pharyngeal apophyses (almost as large as the placoids), and two granular macroplacoids ( Figs 7B, C, E ; 8E, F ). In PCM, macroplacoids without constrictions, however slight central constrictions in both macroplacoids detectable under SEM ( Fig. 8E, F ).

Claws of the modified Isohypsibius - type ( Figs 11 A-C; 12A, B). Specifically, claw bases triangular, especially pronounced in claws IV ( Figs 11C , 12B ). Claw branches V-shaped, elongated and strongly curved. Apparent accessory points on the primary branches ( Figs 11 A-C; 12A, B). Weakly developed pseudolunulae present, particularly visible under the internal and anterior claws ( Fig. 11A, C ). Claw septa and cuticular bars on legs absent.

Eggs

Roundish and smooth, deposited in exuviae (up to two eggs per exuvia recorded).

MOLECULAR MARKERS

The sequences for all DNA markers were of a good quality. The sequenced fragments were of the following lengths: 1.727 bp (18S rRNA; MG800855 ), 819 bp (28S rRNA; MG800856 ), and 499 bp (ITS-2; MG800857 ). All markers, including the specimen without cuticular plaques, were represented by single haplotypes. The p-distances between 18S haplotypes of all available isohypsibioid species and Fractonotus verrucosus n. comb. ranged from 2.0% ( I. prosostomus Thulin, 1928 , EF620404 from Denmark) to 7.1% ( Hexapodibius micronyx Pilato, 1969 , HQ604915 from Italy), with an average distance of 5.2%. As our 28S rRNA primers obtain a different gene fragment to the one sequenced by previous authors, comparisons of this gene were not possible. Matrices with p-distances are provided in the Supplementary Material 2.

REMARKS

The vast part of the Richters Collection has been lost, thus the type material (if ever existed) is not available for examination. Moreover, no specimens from Germany were examined in this study, therefore the neotype series is not established. Hence, until the redescription from the terra typica in Germany is available, we propose to consider the description of the Scottish specimens only as the current perception of the species.

PHYLOGENETIC POSITION OF FRACTONOTUS AMONG OTHER ISOHYPSIBIIDAE

Both Bayesian Inference and Maximum Likelihood methods unreservedly located Fractonotus within Isohypsibioidea ( Fig. 13 ), thus corroborating the phenotypic analysis (see above). The genus Isohypsibius s.s. (i.e. I. prosostomus and its closest relatives) appears paraphyletic with respect to Fractonotus . However, in general, all isohypsibioid lineages clearly remain in polytomy, with only the occasional sound Bayesian posterior probabilities characterising clades with morphologically similar taxa. Therefore, the exact relationships between different isohypsibioid clades remain unsolved.

Figures

FIG. 2. — Fractonotus gilvus (Biserov, 1986) n. comb., adults showing intraspecific variability in cuticular sculpturing (A, dorsal and B, dorsolateral view, paratypes). Scale bars: 20 μm.

FIG. 11. — Comparison of Fractonotus-type (A-D, F), Calohypsibius-type (E), Microhypsibius-type (G), and Isohypsibius-type (H, I) claws (all but Fig. 11F PCM): Fractonotus verrucosus (Richters, 1900) n. comb.: A, claws I, arrowhead indicates weakly developed pseudolunula; B, claws II, interrupted line designates isohypsibioid claw curvature; C, claws IV, arrowhead indicates weakly developed pseudolunula; Fractonotus gilvus (Biserov, 1986) n. comb.; D, claws III, arrowhead indicates weakly developed pseudolunula; Calohypsibius ornatus (Richters, 1900); E, claws IV; Fractonotus caelatus (Marcus, 1928); F, claws IV; Microhypsibius truncatus Thulin, 1928; G, claws III (DIC); Isohypsibius prosostomus (Thulin, 1928); H, claws II; Isohypsibius coulsoni Kaczmarek, Zawierucha, Smykla & Michalczyk, 2012; I, claws II. Scale bars: 10 μm.

FIG. 1. — Fractonotus verrucosus (Richters, 1900) n. comb., ontogenetic series (Fig. 1F: differential interference contrast [DIC], the remaining photos are phase contrast microscope [PCM]): A, juvenile habitus (lateral view); B, young adult habitus (dorsal view); C-F, old adult habitus, variability in the development of large tubercles (dorsal views); E, adult habitus with the symmetrical arrangement of plaques (Calohypsibius placophorus sensu da Cunha 1943, dorsolateral view); F, adult habitus (a specimen from Borgholm, Öland from the Thulin collection, lateral view, DIC). Scale bars: 20 μm.

FIG. 3. — Fractonotus verrucosus (Richters, 1900) n. comb., habitus (SEM): A, lateral view; B, dorsal view; Calohypsibius ornatus (Richters, 1900), habitus (SEM): C, lateral view; D, dorsal view. Scale bars: A, 30 μm; B-D, 20 μm.

FIG. 5. — A-D, Fractonotus verrucosus (Richters, 1900) n. comb.; E, F, Calohypsibius ornatus (Richters, 1900), cuticle morphology (SEM): Fractonotus-type: A, smooth, obtuse tubercles of the smallest specimens (Calohypsibius scabrosus sensu Murray 1911, Calohypsibius verrucosus sensu da Cunha 1947); B, scabrous tubercles occurring in the caudal part of the smallest specimens and on the whole body of adults; C, D, dorsal plaques of adults (Calohypsibius placophorus sensu da Cunha 1943, Calohypsibius verrucosus sensu Thulin 1911,Marcus 1928 and Cuénot 1932), arrowheads indicate coarse portions of the plaques; Calohypsibius-type: E, irregular, multangular tubercles of the cephalic part; F, rows of tubercles of the trunk in close-up. Scale bars: A, B, F, 2 μm; C-E, 20 μm.

FIG. 7. — A-E, Fractonotus spp., F-G, Calohypsibius spp. and H-I, Isohypsibius spp., elliptical organs and details of the buccal apparatus (PCM):Fractonotus verrucosus (Richters,1900) n. comb.: A, cephalic region,empty arrowheads indicate elliptical organs;B, dorsal view,incised arrowhead indicates buccal tube thickening, and incised empty arrowhead points to the furca;C, ventral view;Fractonotus gilvus (Biserov,1986) n. comb.; D, ventral view,incised empty arrowhead points to the furca;Fractonotus verrucosus (Richters,1900) n. comb.; E, lateral view, arrowhead indicates the posterior portion of dorsal apophysis in the shape of stumpy hook; F, Calohypsibius ornatus (Richters,1900), lateral view, arrowhead indicates the anterior portion of dorsal apophysis in the shape of stumpy hook; G, Calohypsibius schusteri Nelson & McGlothlin, 1996, arrowhead indicates the anterior portion of dorsal apophysis in the shape of stumpy hook, and arrow points to the visible peribuccal papulae; H, Isohypsibius prosostomus (Thulin, 1928); I, Isohypsibius coulsoni Kaczmarek, Zawierucha, Smykla, Michalczyk, 2012. Scale bars: 10 μm.

FIG. 9. — Schematic comparison of the AISM (lateral view) in: A, Fractonotus Pilato, 1998; B, Calohypsibius Thulin, 1928; C, Isohypsibius Thulin, 1928.

FIG. 6. — Fractonotus verrucosus (Richters,1900) n. comb.and Calohypsibius ornatus (Richters,1900),peribuccal structures (SEM):A, Fractonotus verrucosus n.comb., with six peribuccal lobes typical for Isohypsibiidae;B, Calohypsibius ornatus with six small and roundish peribuccal papulae typical for Calohypsibiidae.Scale bars:1 μm.

FIG. 8. — Buccal apparatus of Fractonotus verrucosus (Richters, 1900) n. comb. (SEM): A, oral cavity armature, the arrowhead indicates the row of conical teeth; B, the buccal crown and both dorsal and ventral apophyses for insertion of stylet muscles (AISM) in lateral view;C, buccal tube, empty arrowheads indicate lateral thickenings; D, stylet furca; E, pharynx in dorsal view; F, pharynx in lateral view. Scale bars: A, D, 0.5 μm; B, 1 μm; C, E, F, 2 μm.

FIG. 10. — Schematic comparison of stylet furcae in: A, Fractonotus Pilato, 1998; B, Calohypsibius Thulin, 1928; C, Isohypsibius Thulin, 1928.

FIG. 12. — A-B, Fractonotus verrucosus (Richters,1900) n. comb.;C-D, Calohypsibius ornatus (Richters,1900); E-F, Isohypsibius spp., claws (SEM):Fractonotus- type: A, claws III; B, claws IV; Calohypsibius-type; C, claws II; D, claws IV; Isohypsibius-type; E, Isohypsibius prosostomus (Thulin, 1928),claws I; F, Isohypsibius coulsoni Kaczmarek, Zawierucha, Smykla & Michalczyk, 2012, claws II. Scale bars: A, B, 3 μm; C, D, 1 μm; E, F, 4 μm.

FIG. 13. — The phylogenetic position of Fractonotus verrucosus (Richters, 1900) n. comb. on the Bayesian Inference and Maximum Likelihood cladogram constructed from currently available 18S rRNA sequences for Isohypsibioidea and Hypsibioidea, with a Milnesium spp. sequence as the outgroup (see also Table 2). Values above branches indicate posterior probability values (BI), whereas values under branches show bootstrap values (ML). Branches with support below 0.9 in BI (70% in ML) were collapsed.