Echiniscus quadrispinosus quadrispinosus Richters, 1902

Kaczmarek, Łukasz, Kayastha, Pushpalata, Gawlak, Magdalena, Mioduchowska, Monika & Roszkowska, Milena, 2022, An integrative redescription of Echiniscus quadrispinosus quadrispinosus Richters, 1902 (Heterotardigrada, Echiniscidae) from the terra typica in Taunus Mountain Range (Europe; Germany), European Journal of Taxonomy 823 (1), pp. 102-124 : 105-118

Echiniscus quadrispinosus quadrispinosus Richters, 1902

Figs 1–7 , Tables 1–4 , Supp. file 2

Material examined

Neotype GERMANY • Taunus Mountains Range, near Königstein im Taunus; 50°11′49″ N, 08°27′15″ E; 485 m a.s.l.; 2 Sep. 2019; moss from stone, mixed forest; Johenn Sholl leg.; slide GR1/5; Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań.

Paraneotypes GERMANY • 33 ♀♀, 22 ♂♂, 35 juvs, 15 undefined, 3 larvae; same collection data as for neotype; slides GR 1/1, 1/2, 1/4, 1/5, 1/7; Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań • 9 ♀♀, 6 ♂♂, 4 juvs, 2 undefined, 1 larvae; same collection data as for neotype; slides GR 1/3, 1/6; Institute of Systematics and Evolution of Animals, Polish Academy of Sciences .

Remarks

Animals were mounted on microscope slides in Hoyer’s medium, 45 animals prepared for SEM, and 15 prepared for molecular analyses. However, DNA sequences were obtained from two females and one male only (exoskeleton, No GR5/S, GR8/S and GR10/S) which was later mounted on microscope slide in Hoyer’s medium.

Description of the neotypic population

Females (measurements and statistics in Table 1 , Supp. file 2 and Figs 1–4 )

Body orange in live specimens and transparent/light yellow after preparation. Eyes red. Apart from the head appendages which include internal and external cirri and cylindrical cephalic papillae (secondary clava), only appendage A with clava (primary clava) near its base is present ( Fig. 1D ). Dorsal and lateral appendages in the shape of short, and long filaments and/or spines are present at positions A-B-C-Cd- D-Dd- E ( Fig. 1A–C ). However, appendage B may be absent, at least on one side of the body, for more details see Morphological variability below.

Dorsal plates well developed. Head and scapular plates not faceted. Under PCM, lateral portions of the scapular plate appear to be detached from the dorsal plate, forming small additional plates (one on each side of the body) ( Fig. 1E , arrowheads) divided from the lateral margin of the scapular plate by a thin bright stripe. This division is formed by the narrow stripe without pores, as it is clearly visible in SEM along with a small additional plate ( Fig. 1C , arrowhead). Paired plates I and II are divided into two parts – narrow anterior part and a wider posterior part – by smooth stripe without sculpture ( Fig. 2A , arrows). Anterior parts most often divided longitudinally into two parts ( Fig. 2A–B , indented arrowheads). Median plate 1 and 2 divided into anterior part ( Fig. 2A , asterisks) and posterior part ( Fig. 2A , filled arrowhead; which is especially visible in lateral position), m3 undivided ( Fig. 2A , empty arrow). The terminal plate with two notches ( Fig. 2A , empty arrowhead).

All dorsal plates, covered with double sculpture under PCM ( Fig. 2A–E ), i.e., regular polygonal or roundish black ‘granules’, i.e., endocuticular pillars (0.6–1.0 µm in diameter on scapular plate and similar in size also on other plates) and white roundish or oval pores which especially in larger specimens may be merged (seen as white spots, 1.0–4.2 µm in diameter on scapular plate and similar in size also on other plates); but see below for more details. Pores absent on anterior parts of m1 and m2 ( Fig. 2A , asterisks), with typical double sculpture present on the posterior parts ( Fig. 2A , filled arrowheads). Central part of terminal and scapular plates with cross-like pattern, and on terminal plate a transverse line of the cross is an extension of notches ( Fig. 2C–E ). Sometimes, on the scapular plate, the transverse line is absent and then only the longitudinal line visible in the middle of this plate or an additional transverse line is present and the plate is poorly divided into six rectangles ( Fig. 2C–D ). Under SEM the plates with regularly distributed pores ( Fig. 2F ), which means that where the white pores visible under PCM are absent (i.e., neck plate, lateral portions of the scapular plate and anterior parts of paired plates) the plates appear to be smooth or with poorly visible granulation ( Fig. 2F ).

Two poorly marked ventral rectangular plates, arranged transversally, are present below the head ( Fig. 3A, C , arrowheads). Another two rounded square plates are present on lateral sides of the gonopore ( Fig. 3B–C , arrows). Ventral cuticle possesses tiny and regular granulation (due to the presence of dense endocuticular pillars). Granulation is a little larger on the plates around the gonophore (0.3–0.4 µm diameter) than in other parts of the ventral cuticle, 0.1–0.2 µm diameter) ( Fig. 3D ).

Outer cuticle of legs I–III with clearly visible stripes of tiny and regular granulation (0.1–0.4 µm in diameter): a thin frontal stripe on the upper part of the leg ( Fig. 4A , empty arrow), a wide stripe in the central part of the leg covering frontal and lateral side of the leg ( Fig. 4A , empty arrowhead) and the most distal, thin stripe above claws on the ventral side of the leg ( Fig. 4A , filled indented arrowhead); white pores absent. On legs IV only frontal stripe on the upper part of the leg (just above the plate with dentate collar) ( Fig. 4C, E , empty arrows) and thin stripe above claws on the ventral side of the leg are present. Triangular spine on leg I ( Fig. 4B–C , filled indented arrowhead) and finger-like papilla on leg IV, present ( Fig. 4D–E , filled arrow). Legs IV with dentate collar with seven to ten sharp, triangular teeth and the plate with the same sculpture as dorsal plates ( Fig. 4D–E ). External claws of all legs I–IV smooth, internal with spurs directed downwards ( Fig. 4C–F )). The gonopore with the typical six-petal rosette ( Fig. 3C , asterisk).

Males (measurements and statistics in Table 2, Supp. file 2 and Fig. 5 )

Males are, in general, similar to females in the morphology of plates and chaetotaxy ( Fig. 5A, C–D ). However, there are differences in the lengths of some morphological structures (especially slightly shorter head appendages, i.e., cirri internal and external and body appendages) (compare values in Tables 1–2 ). On the dorsal side, regular polygonal or roundish black ‘granules’ 0.3–0.9 µm in diameter (on scapular plate and similar in size also on other plates) and white roundish or oval pores 1.0–3.1 µm in diameter (on scapular plate and similar in size also on other plates). Gonopore round and without the six-petal rosette ( Fig. 5B ).

Juveniles – four-clawed (measurements and statistics in Table 3 , Supp. file 2 and Fig. 6 )

In general, juveniles were similar to adults of both sexes in the morphology of plates ( Fig. 6A ). However, there are differences in chaetotaxy ( Fig. 6A–D ), in general, lack of filaments B. Moreover, appendages are shorter in juveniles than in adult females (compare values in Tables 1–3 ) ( Fig. 6A–D ). On the dorsal side, regular polygonal or roundish black ‘granules’ 0.4–0.9 µm in diameter (on scapular plate and similar in size also on other plates) and white roundish or oval pores 1.0–2.7 µm in diameter (on scapular plate and similar in size also on other plates). Gonopore absent.

Larvae – two-clawed (measurements and statistics in Table 4, Supp. file 2 and Fig. 7G–H )

All head and body appendages much shorter than in adults and juveniles (compare values in Tables 1–4 ) ( Fig. 7G–H ). Moreover, morphology and sculpture of plates are also different. Anterior parts of the paired plates not divided. Although, sculpture on dorsal plates composed of regular polygonal or roundish black ‘granules’ and white roundish or oval pores as in adults and juveniles. However, on head plate, anterior parts of paired plates and on entire median plate m3, pores completely absent and black ‘granules’ poorly marked. On scapular plate pores are distributed mainly on the margins of the plate and almost absent in the centre. On the dorsal side, regular polygonal or roundish black ‘granules’ 0.4–0.9 µm in diameter (on scapular plate) and white roundish or oval pores 0.7–1.4 µm in diameter (on scapular plate). Also, chaetotaxy different than adults and juveniles, i.e., A-Cd- Dd- E ( Fig. 7G ). Gonopore absent ( Fig. 7H ).

Eggs

Smooth, light orange and deposited in the exuviae up to 6 in one exuvium.

DNA sequences

We obtained good quality sequences for the applied molecular markers:

18S rRNA: GenBank: MZ798389 - MZ798396 , 771 bp long;

28S rRNA: GenBank: MZ816972 - MZ816979 , 715-747 bp long;

ITS-2: GenBank: MZ816980 - MZ816987 , 464 bp long;

COI: GenBank: MZ798397 - MZ798404 , 688 bp long.

Morphological variability

A strict chaetotaxy was analysed in 36 females, 23 males, 35 juveniles, 3 larvae. In all adult specimens, both females and males typical chaetotaxy, i.e., A-B-C-Cd- D-Dd- E was observed ( Fig. 1A–B ), whereas, in juveniles appendages B are most often absent (chaetotaxy: A-C-Cd- D-Dd- E). The other dorsal and lateral appendages were in general shorter in juveniles than in adults. In all studied larvae, chaetotaxy was always A-Cd- Dd- E and all appendages were much shorter than in juveniles and adults (compare values in Tables 1–4 ). Moreover, some modifications in chaetotaxy were observed in juveniles and adults.

In two juveniles appendages B were present on both sides of the body and in five juveniles appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) ( Fig. 6A–B ). Moreover, in one juvenile appendages Cd and Dd were present only on one side of the body, and in another one Dd was present on both sides and Cd only on one side (chaetotaxy: A-C-Cd- D-Dd- E) ( Fig. 6C–D ).

In two females appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) ( Fig. 7A ). Two females had additional small spines near the base of normally developed appendages B (chaetotaxy: A-B-C-Cd- D-Dd- E) ( Fig. 7B–D ). In other two females only appendage Cd on one side of the body was present and appendages Dd were completely absent (chaetotaxy: A-B-C-Cd- D-E) ( Fig. 7E–F ). One female had very short appendage B on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E). In another one a very short appendage B on one side of the body was present and appendages Cd and Dd were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E). Finally, in one female appendages Cd and Dd were present on only one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E).

In four males appendages B were present only on one side of the body (chaetotaxy: A-B-C-Cd- D-Dd- E) ( Fig. 5C–D ) and in two males appendages B were absent on both sides (chaetotaxy: A-C-Cd- D-Dd- E) ( Fig. 5A ).

Based on these assumptions chaetotaxy formula for adults and juveniles of this species is in general A-(B)- C-Cd- D-Dd- E and for larvae A-Cd- Dd- E. The other observed aberrations in chaetotaxy are only accidental.

Genetic variability

The obtained eight COI sequences (GenBank accession numbers: MZ798397 - MZ798404 ) of E. q. quadrispinosus consisted of four COI haplotypes. Haplotype 1 was found in the Norwegian (169/7 sequence, population code: 169) and German (GR8 and GR10 sequences, population code: GR) populations whereas haplotypes 2, 3 and 4 were identified in different Norwegian populations (haplotype2 – 169/8sequence, population code: 169; haplotype 3– 184/3 and 184/8 sequences, population code: 184; haplotype 4 –187/4 and 187/7 sequences, population code: 187). The value of uncorrected genetic p-distances between obtained COI haplotypes ranged from 0.2% to 0.8%. In turn, the analysis of the p-distances between E. q. quadrispinosus and compared 20 taxa of the genus Echiniscus ranged from the most similar 1.2% for E. quadrispinosus (GenBank accession number: JX683821 , Vincente et al. 2013) to the least similar 21.8% for E. tantulus Gąsiorek, Bochnak, Vončina & Kristensen, 2020 (GenBank accession number: MT107427 , Bochnak et al. 2020), with an average p-distance of 14.4%.

In the conservative 18S rRNA gene fragment we observed no differences between our eight sequences from the German and Norwegian populations (GenBank accession numbers: MZ798389 - MZ798396 ) and sequences of E. quadrispinosus deposited in NCBI (GenBank accession number: MK529684 ). In turn, the uncorrected genetic p-distances between the other 21 taxa of the genus Echiniscus showed that the least similar was E. belloporus Gąsiorek & Kristensen, 2018 (GenBank accession number: MK529674 , Gąsiorek et al. 2019a) with a genetic distance value of 3.1% and an average p-distance was 1.4%.

The analysis of the p-distances between our eight sequences of 28S rRNA from the German and Norwegian populations (GenBank accession numbers: MZ816972 - MZ816979 ; two groups of sequences, i.e., the first consisted of GR8, GR10, 169/8, 169/9, 184/3, 184/8, 187/1 sequences and the second – one 187/8 sequence) indicated that the genetic distance was 1%. Comparison with other 19 taxa of the genus Echiniscus , for which GenBank sequences are available, are as follows: the most similar was E. quadrispinosus (GenBank accession number: MK529714 , Gąsiorek et al. 2019a) with 1% value of the p-distance and the least similar was E. belloporus Gąsiorek & Kristensen, 2018 (GenBank accession numbers: MK529702 , Gąsiorek et al. 2019a) – 5.4%, with an average p-distance of 2.5%.

No genetic differences were observed between our eight ITS2 sequences from the German and Norwegian populations (GenBank accession numbers: MZ816980 - MZ816987 ). The ranges of uncorrected genetic p-distances between our sequences and the other 10 species of the genus Echiniscus indicated that the most similar was E. virginicus Riggin, 1962 (GenBank accession number: MN545756 , Gąsiorek et al. 2019b) – 0.42% and the least similar was E. blumi Richters, 1903 (GenBank accession number: EF620383 , Jørgensen et al. 2007) – 34.5%, with an average p-distance of 21.8%. There were no available ITS2 sequences of E. quadrispinosus in the GenBank database.

Establishing of the neotype and paraneotypes of E. q. quadrispinosus

The search for the type material of E. q. quadrispinosus in various collections did not bring positive results. We can probably assume that the type material of E. q. quadrispinosus no longer exists. Taking into consideration that accurate diagnoses of the species were poorly provided in the past, it is necessary to establish a neotype series of this species. For this reason, we designated the neotype and 108 paraneotypes of E. q. quadrispinosus which agree with the original description and were collected in the terra typica in the Taunus Mountain Range ( Germany). The neotype series was deposited at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznań and Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Poland. All the above-mentioned statements are in accordance with the International Commission on Zoological Nomenclature (ICZN) acts dedicated to the establishment of neotype series.

Figures

Fig. 1. Echiniscus quadrispinosus Richters, 1902, ♀, habitus. A*–B. Dorsal view of the entire animal with typical chaetotaxy A-B-C-Cd-D-Dd-E (PCM and SEM, respectively). C. Lateral view; arrowhead indicates additional plate divided fromthe lateral margin of the scapularplate (SEM).D. Headand scapular plates and head appendages visible in SEM; empty arrow indicates external cirri, filled arrow indicates internal cirri, indented arrowhead indicates cephalic papillae, empty arrowhead indicates appendage A and filled arrowhead indicates clava. E. Lateral view of head and scapular plates; arrowhead indicates additional plate divided from the lateral margin of scapular plate (PCM). * = manually assembled deepfocus image. Scale bars in micrometres (μm).

Fig. 2. Echiniscus quadrispinosus Richters, 1902, ♀. A. Close-up of the dorsal plates; arrows indicate smooth stripes dividing paired plates I and II into anterior and posterior part; indented arrowheads indicate longitudinal division of anterior part of paired plates; asterisks indicate anterior part of median plates I and II whereas filled arrowheads indicate posterior parts; empty arrow indicates median plate 3; empty arrowhead indicates notches on terminal plate (PCM). B. Close-up of the paired plate II; indented arrowhead indicates longitudinal division of anterior part of the plate (PCM). C–D. Close-up of the scapular plate sculpture (PCM). E. Close-up of the terminal plate sculpture (PCM). F. Close-up of the scapular plate (SEM). Scale bars in micrometres (μm).

Fig. 3. Echiniscus quadrispinosus Richters, 1902, ♀. A. Two ventral plates below the head, arrowheads (PCM). B. Two ventral rounded plates on the lateral sides of the gonopore, arrows (PCM). C. Lateral view of the entire animal; arrowheads indicate plates below the head; arrows indicate plates on the lateral sided of the gonopore; asterisk indicates gonopore (SEM). D. Ventral sculpture visible in PCM. Scale bars in micrometres (μm).

Fig. 4. Echiniscus quadrispinosus Richters, 1902, ♀. A*. Leg I outer cuticle with clearly visible stripes of tiny and regular granulation: a thin frontal stripe on the upper part of the leg (empty arrow), a wide stripe in the central part of the leg covering frontal and lateral side of the leg (empty arrowhead) and the most distal, thin stripe above claws on the ventral side of the leg (filled indented arrowhead) (PCM). B. Spine on leg I (arrowhead) (PCM). C. Spine on leg I (arrowhead) and thin frontal stripe on the upper part of the leg (empty arrow) (SEM). D*. Claws IV with dentate collar and finger-like papilla (filled arrow) (PCM). E. Vlaws IV with dentate collar and finger-like papilla (filled arrow); empty arrow indicates thin frontal stripe on the upper part of the leg (SEM). F. Claws of the II leg (PCM). * = manually assembled deep-focus image. Scale bars in micrometres (μm).

Fig. 5. Echiniscus quadrispinosus Richters, 1902, ♂. A*. Dorsal view of the entire animal with appendages B absent on both sides (chaetotaxy: A-C-Cd-D-Dd-E). B*. Two ventral rounded plates on the lateral sides of the gonopore (arrows); asterisk indicates gonopore. C–D. Lateral view of male with appendage B present only on one side of the body (chaetotaxy: A-B-C-Cd-D-Dd-E); arrowhead indicates presence and lack of appendage B. * = manually assembled deep-focus image. All PCM. Scale bars in micrometres (μm).

Fig. 6. Echiniscus quadrispinosus Richters, 1902, juvenile. A–B. Lateral view of the animal with appendage B present only on one side of the body (arrowhead) (chaetotaxy: A-B-C-Cd-D-Dd-E). C–D. Lateral view with appendage Cd present only on one side of the body (filled arrows) and two short spines present instead of appendage B (empty arrows). E–F. Close-up to the two short spines present instead of appendage B (empty arrows). All PCM. Scale bars in micrometres (μm).

Fig. 7. Echiniscus quadrispinosus Richters, 1902. A–F. Examples of different chaetotaxy found in examined population. A*. Female with appendage B present only on one side of the body (empty arrow) (chaetotaxy: A-B-C-Cd-D-Dd-E). B–D. Close-up to the spines near the base of normally developed appendages B (filled arrows). E–F. Presence of appendage Cd only on one side of the body (filled arrowheads) and lack of appendagesD d (chaetotaxy:A-B-C-C d-D-E). G. Larvae, dorsal view (chaetotaxy: A-C d-Dd-E). H. Larvae, ventral view. * = manually assembled deep-focus image. All PCM. Scale bars in micrometres (μm).