Bryodelphax nigripunctatus Degma, Gasiorek , Voncina & Michalczyk, 2020

Gasiorek, Piotr, Voncina, Katarzyna, Degma, Peter & Michalczyk, Lukasz, 2020, Small is beautiful: the first phylogenetic analysis of Bryodelphax Thulin, 1928 (Heterotardigrada, Echiniscidae), Zoosystematics and Evolution 96 (1), pp. 217-236 : 217

Bryodelphax nigripunctatus Degma, Gasiorek, Voncina & Michalczyk sp. nov. Figures 7 , 8 , 9 , 10 , 11 , 12 , Tables 5, 6

Locus typicus and type material.

39°57'00"N, 3°10'50"E, 160 m a.s.l.; near the road above Cala Figuera beach, Cap de Formentor, NE Mallorca, the Balearic Islands, Spain. Holotype (adult female; together with one male paratype in slide 716/45), allotype (adult male; slide 716/9) and 30 paratypes (9 females, 14 males, 3 specimens of unknown sex, 2 juveniles and 2 larvae; slides 716/1-5, 10, 13, 17 -20, 25, 27-29, 32-34, 41, 45, 47-48, 50-51) deposited in the Department of Zoology, Comenius University in Bratislava; 13 paratypes (6 females, 6 males and one specimen of unknown sex; slides 716/26, 38, 40, 42-44, 46, 49) deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University; 9 paratypes (4 females and 5 males; slides 716/6-8, 11-12, 22-24, 35) deposited in the Natural History Museum of Denmark, University of Copenhagen; 9 paratypes (4 females and 5 males; slides 716/14-16, 21, 31, 36-37) deposited in the Department of Animal Biology, University of Catania. Single paratype (male) mounted on a SEM stub (14.19) deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University. Eight specimens used for DNA extraction. Bryodelphax nigripunctatus sp. nov. was not accompanied by other species in the sample.

Etymology.

From Latin niger = black + punctum = dot, spot. The name underlines evident epicuticular granules appearing dark in PCM and contrasting with other elements of dorsal sculpture. Adjective in the nominative singular.

Adults.

Body translucent without distinct colour and usually stout in females and more slender in males (Figs 7 , 8 ), bs = 43.0-49.2% (x̅ = 45.6%, N = 8) in females and 37.5-44.7% (x̅ = 39.8%, N = 10) in males. Eyes absent or not visible after mounting in Hoyer’s medium. Cephalic papillae and clavae elliptical with rounded apex. Cephalic papilla is relatively broader in males than in females. Both cephalic papillae and primary clavae relatively longer in males than in females (Figs 7A, B , 10C, D ; compare the sp of the cephalic papilla and clava in Tables 5 , 6 ). Cirri interni always shorter than cirri externi and both with poorly-developed cirrophores. Cirri A reach around 1/5-1/4 of the total body length (Tables 5 , 6 ). Unappendaged. Cuticular sculpture consists of large epicuticular granules, true pores and intra-cuticular pillars (Fig. 10 ). In PCM, these structures appear, respectively, as conspicuous large dark spots, smaller bright spots and fine dark and dense punctuation (pseudogranulation). Epicuticular granules of irregular shape and size (up to ca. 1.6 μm) are merged together (as visible in SEM, Figs 8 - 10 ) and arranged in rows along the margins of all plates, although they are least visible or absent in posterior median plate 1 and posterior median plate 2. Moreover, in the cervical (neck) plate, the row or a double row of granules is also present along its transverse axis. Granules in rows appear as dark spots in PCM (Fig. 7A-C ). Similar rows of granules cover also folds which create the faceting of the scapular and caudal plate: median and two lateral longitudinal folds (at the level of cirrophores A) together with 3-4 posterior transverse folds on the scapular plate and two longitudinal folds dividing the caudal plate into three parallel portions (Figs 7A, B , 8 , 9A, C ). Finally, short longitudinal rows of granules divide both paired segmental plates and posterior portions of anterior m1-2 plates into left and right portions (Figs 7A, B , 8 , 9B ). Scattered granules also irregularly cover the surface of the cephalic, paired, caudal, anterior m1-2 and anterior parts of scapular plates (Figs 7A, B , 8 , 9 , 10A ). Round, focusable pores (0.3-0.4 μm in diameter) are unequally distributed on dorsal plates in spaces between the scattered granules, on pedal plates IV and between transverse rows of granules in the scapular plate, but they are absent on the rows of granules (Figs 7C , 8 , 9A-C , 10B ). The density of pores varies between the sexes and elements of armour, with the largest pores present on the segmental plate II, anterior m2 and the scapular plate (21-40 pores/100 μm 2, x̅ = 29, 14-28 pores/100 μm 2, x̅ = 22 and 18-31pores/100 μm 2, x̅ = 26, respectively, N = 15 in females and 7-35 pores/100 μm 2, x̅ = 29, 0-34 pores/100 μm 2, x̅ = 25 and 11-33 pores/100 μm 2, x̅ = 25, respectively; N = 15 in males) and lowest density on caudal plate (16-27 pores/100 μm 2, x̅ = 22 in females and 7-28 pores/100 μm 2, x̅ = 20 in males; N = 15). On the scapular plate (in the area delimited with lateral longitudinal rows of granules), lines of pores tend to alternate with transverse rows of granules (Figs 7A-C , 8A , 9A , 10A, B ). Regularly distributed round intra-cuticular pillars (0.1-0.2 μm in diameter) reinforce the entire cuticle (also under the granules), but they are well-visible only in the cephalic, scapular, both paired segmental, caudal, anterior median 1 and anterior median 2 plates (Fig. 10B ), as well as on pedal plates IV. On the remaining cuticle, they are weakly (venter) or scarcely (legs) detectable.

Cephalic plate with an anterior chalice-like incision. Each segmental and median plate consists of the anterior and the posterior portion separated each from other with a transverse bright poreless stripe in PCM. Therefore, paired segmental plates are subdivided into the narrower anterior (ca. 1/3-2/5 of the plate length) and the wider posterior portions, trapezoidal anterior median plate 1 is subdivided just behind its anterior margin, pentagonal anterior m2 (the largest amongst the median plates) is divided at approximately equally-long portions, triangular anterior portion of median plate 3 is ca. two times as long (along median body axis) as the posterior one with rounded posterior margin (dividing transverse line of anterior median plates 2-3 correspond with posterior corners of paired segmental plates). Pentagonal posterior median plates 1-2 subdivided at portions of about same lengths. On each body side, the first two pairs of supplementary lateral platelets are connected with anterior and posterior median plates 1-2 and the third pair is connected with the posterior portion of m3 and with the anterior edge of caudal plate. Anterior platelets of each pair have very distinctly-thickened lateral (lower) margins (Figs 7A, B , 8 , 9 ).

Venter with transverse rows of weakly-developed plates unevenly sculptured with epicuticular granules similar to those on the dorsal plates, but a bit smaller. There are three rows of ventral plates in females (the plate formula III:2-2-1) and two rows in males (II:2-2) (Fig. 7D ). The outer surface of legs with a narrow well-visible proximal pulvinus and a wide weak distal pedal plate placed in the central part of the leg. A single row of small epicuticular granules (similar to those on ventral plates) on the distal edge of pulvini in legs I-III (rarely, the second row also on their proximal edge). Pedal plates I-III sculptured usually with three (sometimes with more) transverse rows of epicuticular granules, which can be either shortened or connected at their ends (Figs 7A-C , 8B ). The pedal plate IV sculptured with distinct intra-cuticular pillars and scattered pores and distally hemmed with dentate collar. The collar with sharp teeth, always longer than the width of their bases and with the distance between teeth similar to their basal widths, although some pairs of teeth can be merged (Fig. 11 ). Papilla or spine on legs I-III absent, papilla on legs IV well developed. Claws slender, claws IV always slightly longer than claws I-III. External claws smooth, internal ones with a small spur pointing downwards and placed very close to the claw bases (Figs 7A-C , 7D , insert).

Juveniles.

In appearance as adults, but smaller (111-112 μm) and with ventral plates just marked with rows of granules. Selected measurements of a shorter specimen: cephalic papilla 3.1 μm, scapular plate 14.9 μm long, claws I-III 5.1-5.6 and claws IV 6.5 μm long.

Larvae.

83-85 μm long. Dorsal plates (especially median ones) mostly with poorly-delineated margins, supplementary lateral platelets absent. Epicuticular granules less numerous than in adults, concentrated mainly on posterior margins of the cephalic, scapular, both paired and caudal plates. Cuticular pores less numerous than in adults, but intracuticular pillars, stripes of granules on the outer surface of legs, papilla on legs IV and dentate collar IV well developed. Ventral plates not visible in laterally orientated larvae. Claws with spurs formed as in adults. Some measurements of shorter specimen: cephalic papilla 2.5 μm, claws II-III 4.6- 5.3 and claws IV 6.4 μm long.

Eggs.

Not found.

DNA sequences.

Two 18S rRNA haplotypes (MT333472-3), single 28S rRNA haplotype (MT333465).

Phenotypic differential diagnosis.

Having ventral plates, Bryodelphax nigripunctatus sp. nov. belongs to the weglarskae group. Within the group, only B. amphoterus and B. maculatus have a reduced number of ventral plate rows to two or three, as in the new species. Consecutively, B. nigripunctatus sp. nov. differs from:

B. amphoterus, known from Croatia (Istria) and Greece (Crete) (McInnes 1994), by: the mode of reproduction (dioecy in the new species vs. parthenogenesis in B. amphoterus), the presence of lateral supplementary platelets (absent in B. amphoterus), the presence of epicuticular granules on dorsal and ventral plates (absent in B. amphoterus), a different number of ventral plate rows in females (ventral plate formula III:2-2-1 in females of the new species vs. II:2-2 in B. amphoterus) and by the lack of spurs on external claws (extremely small spur very difficult to observe just near the base in B. amphoterus);

B. maculatus, known from Tunisia and Greece, by: the mode of reproduction (dioecy in the new species vs. parthenogenesis in B. maculatus), large contrasting granules on dorsal plates (granules not contrasting and clearly visible only in SEM in B. maculatus), the absence of patches or transverse stripes of epicuticular granules on ventral cuticle between legs (present in B. maculatus), a smaller maximal pore density (21-40 pores per 100 μm 2 in segmental II plate in the new species females vs. 48-61 pores per 100 μm 2 in the same plate in B. maculatus), a relatively longer internal peribuccal cirrus (sp is 33-43% in females of the new species vs. 21-30% in B. maculatus) and by relatively longer claws II-IV (sp for claws II is 36-44%, for claws III 36-46%, for claws IV 38-48% in females of the new species vs. 29-36%, 30-34% and 32-38%, respectively in B. maculatus).

Genotypic differential diagnosis: p -distances between the new species and the remaining Bryodelphax spp., for which DNA sequences are available, were as follows:

18S rRNA: from 0.4% (B. maculatus, KY609137 and MT333471) to 2.9% (B. australasiaticus sp. nov., MT333468);

28S rRNA from 4.2% (B. instabilis, MH414965) to 8.1% (B. decoratus sp. nov., MT333462, MT333463).

Figures

Figure 7. Habitus of adults of Bryodelphax nigripunctatus sp. nov. (PCM): A - female (holotype, dorsolateral view); B - male (allotype, dorsal view); C - female (paratype, lateral view; Roman numerals signify epicuticular belts of granules on legs); D - male (allotype, ventral view, Roman numerals point out reduced ventral armature; insert with the claws II, arrowhead indicates spur on internal claw). Scale bars: 50 µm.

Figure 8. Habitus of adult male of Bryodelphax nigripunctatus sp. nov. (paratype, SEM): A - dorsal view; B - lateral view. Scale bars: 50 µm.

Figure 9. Detailed morphology of Bryodelphax nigripunctatus sp. nov. (paratype, SEM): A - frontal part of the body; B - central part of the body; C - caudal part of the body. Scale bars: 10 µm.

Figure 10. Details of cuticular sculpturing of Bryodelphax nigripunctatus sp. nov.: A - microscope focused on the epicuticular granules (allotype, NCM); B - microscope focused on the intracuticular pillars (allotype, NCM); C - frontal part of the body (allotype, PCM); D - frontal part of the body (holotype, PCM). Scale bars in µm.

Figure 11. Variability of the dentate collar IV in Bryodelphax nigripunctatus sp. nov. (paratypes, PCM). Scale bars: 10 µm.

Figure 12. Schematic arrangement of the ventral plates in all members of the weglarskae group (species are arranged in order of the increasing reduction of the ventral armature). Known cases of ontogenetic variability and sexual dimorphism are depicted. Following species taken from Kaczmarek et al. (2012): B. aaseae, B. iohannis, B. parvuspolaris, B. sinensis and B. weglarskae; Gasiorek et al. (2017 a): B. maculatus; Gasiorek & Degma (2018): B. instabilis.