Minibiotus dispositus sp. nov.

urn:lsid:zoobank.org:act: 780E50E1-48E8-474C-8935-070FCD6B09AE

Figs 1–8; Tables 2–6; Supp. files 1–3

Diagnosis

Minibiotus with smooth cuticle but with cuticular pores variously sized (0.9–3.1 µm) and shaped; under SEM, most pores are polygonal or multilobate (3–5 angles/lobes/arms); under PCM, pentagonal pores often appear round, and 5-lobate are rarely observable, only caudal or on legs. Dorsal pores arranged in a group of very cephalic and a group of very caudal pores, with in between a series of transverse bands; young specimens with 8 bands of 1–2 rows; senior specimens with 7 bands of about 2–5 less regular rows (band 8 joined to the very caudal pores). Ventral pores arranged in 7 transverse bands, starting posterior to legs I, of a single row each, but partially duplicated medially in senior specimens. Bucco-pharyngeal apparatus typical for the genus; oral cavity armature with three bands of teeth, better visible under SEM, with band I reduced; three macroplacoids (length sequence 3≤ 2<1) and an evident microplacoid in the pharynx. Robust double claws with short, robust accessory points and small, smooth lunules. Faint leg ‘cuticular bars’, divided on legs I–III, undivided on legs IV; no leg granulation, pulvini present on legs I–III. Moderate allometry regarding buccal tube width, macroplacoid and claw length.

Etymology

From the Latin word ‘ dispositus ’ = ‘ordered’, in the meaning of ‘with a pattern’, referred to the cuticular pores forming a pattern.

Material examined

In total, 51 animals (undetermined sex; 31 senior and 20 young specimens) and 6 eggs mounted on microscope slides in Polivinil lactophenol medium; 10 additional specimens mounted on SEM stubs; one specimen (voucher) used for DNA analysis.

Holotype ARGENTINA • senior spec.; Salta Province, Salta City; 24°47′18″ S, 65°24′38″ W; 1150 m a.s.l.; 2 May 2014; Rocha and Doma leg.; moss and lichen growing on Handroanthus Mattos; UNLPam 1088(3).

Paratypes ARGENTINA • 2 senior specs; same data as for holotype; 2 May 2014; MCNS Tar. 000026(1), Tar. 000026(4) • 1 egg; same data as for holotype; 2 May 2014; MCNS Tar. 000027(1) • 2 senior specs; same data as for holotype; 5 Jun. 2022; UNICT 6010, 6011 • 1 young spec.; same data as for holotype; 2 May 2014; UNICT 6012 • 1 egg; same data as for holotype; 2 May 2014; UNICT 6013 • 26 senior specs; same data as for holotype; 2 May 2014; UNLPam 643(3), 654(1), 655(1), 655(2), 656(1), 659(1), 659(2), 1037(4), 1038(2), 1042(2), 1049(1) to 1049(3), 1050(2), 1050(3), 1056(2) to 1056(4), 1085(1), 1085(3), 1085(4), 1087(3), 1088(4), 1089(3), 1090(1), 1090(2) • 19 young specs; same data as for holotype; 2 May 2014; UNLPam 1033(1), 1034(4), 1035(1), 1035(2), 1036(2), 1038(4), 1040(3), 1041(1), 1041(3), 1044(3), 1046(1) to 1046(4), 1047(1) to 1047(3), 1062(2), 1063(4) • 4 eggs; same data as for holotype; 2 May 2014; UNLPam 348(1), 348(2), 644(1), 1222(3) .

General morphological description of the animals

Body length 97–342 µm (Fig. 1; Tables 3, 5), yellowish before mounting, transparent after mounting. Eyespots, small and very caudal, present (Fig. 1B).

Smooth cuticle with pores of different shapes. Under PCM, apparently, the smaller pores (around 1 µm) are usually roundish (though often with irregular margins), or few elliptical, while the larger typically triangular, quadrangular, trilobate or quadrilobate (Fig. 2); the pore size is 0.9–3.1 µm on the dorsum (with the biggest on the head, on the mid-dorsal line along the body, and on the caudal extremity), 1.3–3.4 µm on the legs, 1.0–1.8 µm on the ventral cuticle. Coherently, dorsal pores, on average bigger, appear more often non-roundish, while the ventral ones on average smaller, appear more often roundish. Exceptionally, some caudal or leg pore may appear pentagonal- or, extremely rarely, star-shaped (with five lobes/arms; Fig. 2) under PCM, but this occurs only in few specimens and in only 1–2 pores in each of these few specimens.

SEM reveals that pores are actually never perfectly rounded; instead, few small pores are truly elliptical, a minority (of any size) is irregular, while the rest are all (both dorsal and ventral, smaller and larger) polygonal or multilobate, from three to five angles/lobes/arms (Fig. 2); pores in an unsuitable position, and/or too small, may give the false impression to be irregularly roundish under SEM also, and, obviously, the lower magnification of PCM gives the impression of seeing a more common shape, especially where pores are smaller.

There is no clear distinction between polygonal and multilobate pores, since there are many intermediate shapes (e.g., between triangular and three-lobated/armed, or between quadrangular and four-lobated/ armed), and, very probably, each pore may partially appear more polygonal or multilobate depending on the cuticle distention or contraction. Pentagonal pores are less common but more easily detectable under SEM with respect to PCM (several of them can be seen on each specimen), while properly star-shaped (with five arms/lobes) are quite rare: some of the specimens mounted for SEM apparently lack them, while the others may show one or few of them (Fig. 2).

Pores are arranged in transverse bands made of one or more transverse rows (more regular in young specimens). The cuticle along the body, as visible in most eutardigrades, forms transverse folds marking the division into the five body segments (head plus four segments of the trunk), and, additionally, each segment is subdivided into 2 ‘subsegments’, one more anterior, and one more posterior, by an additional transverse cuticular fold. In this way, there are ten cuticular subsegments of the body clearly followed by the dorsal (Tables 2, 4), and partially ventral, pore arrangement.

Smaller (97–156 µm, called young) and larger (180–342 µm, called senior) specimens show differences in the pore number and arrangement on each subsegment, but we concluded they must belong to the same species (additionally to having been found in the same sample, first sampling) for the following reasons: 1) only one egg type was found; 2) most morphological characters were the same; 3) regarding the more detailed characters for which they differed, the two groups had clear body size distinction (97–156 µm vs 180–342 µm) with no exceptions; 4) the main difference regarded the pore number and pattern, but this was consistent with ontogenetic changes documented in other species (e.g., Minibiotus pentannulatus Londoño, Daza, Lisi & Quiroga, 2017), and the pattern of the bigger specimens was still perfectly comparable with that of the smaller, just more complicated due to the appearance of more pores; 5) the other differences were metric, but they all appeared consistent with allometric growing (buccal tube becoming wider, and placoids and claws becoming longer) already known in eutardigrades, especially macrobiotids. Besides, specimens from resampling (only large individuals) matched perfectly the morphology and the morphometry of the large animals obtained from the first sampling.

As already mentioned, noticeable differences in the pore number and arrangement on each subsegment can be seen between young and senior specimens; for this reason, their pattern is indicated separately in the subsequent paragraphs with the pertinent tables and illustrations. It must be stressed that the determination of bands, rows and pore number and shape, was obtained through the observation of all specimens of each group (i.e., young and senior specimens); the bands of pores and the average pattern were always recognisable, but the precise number, disposition and shape of the pores composing the rows, or other patterns, had some degree of variability (especially in senior specimens); for this reason, descriptions and illustrations (Figs 6–7) are partially schematic trying to take into account the average situation of most specimens and have to be intended more as a tendency than an exact, constant reality.

Mouth antero-ventral; peribuccal papulae present (Fig. 3C–E, SEM), very probably corresponding to reduced lamellae (see Stec et al. 2020a). Oral cavity armature (Fig. 3) with three bands of teeth. The first band, very reduced and visible only under SEM, is located at the basal zone of peribuccal papulae and composed of a single row of small cone-shaped teeth fused to form a continuous ring ridge around the oral cavity (Fig. 3D). The second band of teeth comprises one row of rather large, globular-shaped, separate teeth (Fig. 3D), partially visible under light microscopy (Fig. 3A–B PCM and DIC), depending on the animal size and quality of the preparation: it may be also invisible or appearing as an irregular line since teeth are not distinguishable from one another; this second band of teeth is instead very obvious under SEM (Fig. 3D–E). Third band of teeth (buccal crests) difficult to see under light microscopy; under SEM, in our specimens it was possible to see only the dorsal portion, made of three little-protruding ridges, with two lateral, and one medial bearing two sharpened teeth (Fig. 3D–E); ventral portion not visible under SEM in our specimens, but supposedly more developed than the dorsal one, since slightly better visible under DIC, showing two lateral crests and a median tooth (Fig. 3B).

Bucco-pharyngeal apparatus of the Minibiotus type (Fig. 4A–B). Buccal tube rigid with anterior and posterior dorsal bends; well-developed ventral lamina. Buccal tube wall with cribrous areas, at least anteriorly (Fig. 3E). Pharyngeal bulb oval, with triangular apophyses, three granular macroplacoids and a small microplacoid. The macroplacoid length sequence is 3≤ 2 <1. The first macroplacoid tapering anteriorly (Fig. 4B). All macroplacoids without constrictions. Robust double claws with short and robust accessory points and small, smooth lunules (Figs 4C–E, 5). Faint leg cuticular bars (i.e., muscular attaches), divided on legs I–III, undivided on legs IV, but not visible on all specimens under PCM (Fig. 4C–E); clearly visible under SEM (Fig. 5). No granulation on the legs, pulvini present on legs I–III (Fig. 5A, C).

Young specimens (Fig. 6; Tables 2–3; Supp. file 1)

Arrangement of dorsal and dorso-lateral pores (Fig. 6; Table 2): the smaller specimens have a group of cephalic pores (in subsegment 1) forming a pattern but not exactly ‘rows’; from subsegment 2 to 9 pores are arranged in 8 bands (one per subsegment) made of transverse rows, and, lastly, subsegment 10 has pores not arranged in proper rows and continuing onto the dorsal part of legs IV.

Describing textually in detail the complete dorsal/dorso-lateral pore arrangement would be long and complicated, with continuous necessity to compare the text with Figures and Tables; for this reason, we refer to these latter (Fig. 6; Table 2) for the description of the dorsal/dorso-lateral pore arrangement.

Leg pores (Fig. 6, Table 2): on the external side of each leg (I–IV) there is a big, usually lobate (three or four lobes) pore, but sometimes just triangular/quadrangular (usually triangular or three-armed on legs I–II, while usually quadrangular or four-armed on legs III–IV); legs III show few additional, smaller and more caudal pores, while legs IV show 3–4 additional, dorsal pores.

Arrangement of ventral pores (Fig. 6): ventral cuticle may show 1–2 medial pores, aligned longitudinally, on the caudal portion of the head (subsegment 2), and, normally, seven rows (each representing also a band) each on subsegments 3–9 (subsegment 10 without ventral pores), organized as follows: two rows are present on each of the first three segments of the trunk (subsegments 3–8), while only one on the hind segment (subsegment 9); four medial pores are present in all seven rows, but the three rows just behind each of legs I–III (subsegments 3, 5 and 7) show some (usually three) additional, smaller, pores lying more laterally just at the base of the legs; instead, the four rows consisting of only the four medial, bigger pores, are the interlegs 4, 6, 8 and 9.

Morphometry is reported in Table 3.

Senior specimens (Figs 1, 7; Tables 4–5; Supp. file 2)

In the senior specimens, the dorsal and dorso-lateral pore pattern (Figs 1, 7), and, partially, the ventral one, is basically a complication of that of the young, with an increase of the pore number (and their rows) and also introducing more variability and less ‘order’ especially in those that should be the pore transverse rows.

Arrangement of dorsal and dorso-lateral pores: the bigger pores are kept rather similar in shape and size from the earlier life stage(s), with rather good correspondence especially on the head and on the legs, while additional pores appear, usually smaller, resulting in the above-mentioned increase in row number and decrease in row clearness especially on the rest of the dorsum.

As stated for the young, we refer to Figures and Tables (Figs 1, 7; Table 4) for the detailed description of the dorsal/dorso-lateral pore arrangement of the senior specimens, with all the more reason since the pattern is indeed more complicated. In general, there is a group of cephalic pores (subsegment 1), 7 bands of pores (subsegments 2–8) more or less organized in transverse rows, while the eighth band that was recognisable in the smaller specimens (subsegment 9), here is joined to the group of caudal pores (subsegment 10 plus dorsum of legs IV), so that the dorsal and dorso-lateral cuticle of the whole hind segment (plus legs IV) show a unique, large, caudal group made of many pores with no clear rows recognisable.

Arrangement of leg pores: on the external side of legs I–III there are some large, usually lobate (with 3–4 lobes) pores, but sometimes just triangular/quadrangular; legs IV show several triangular, quadrangular and lobate/star-shaped (with 3–5 lobes/arms) pores.

Arrangement of ventral pores: ventrally, there is instead a good correspondence with the pattern of the young specimens; thus, to avoid repetition, we stress here only the difference, consisting of a tendency to complicate only the medial part of the transverse rows, which are partially multiplied with 4–12 pores forming in the centre a patch of pores sometimes similar to some geometric figure such as a rhombus, a circle, a square or a pentagon.

Morphometry is reported in Table 5. These senior specimens, in comparison to the young, have a slightly wider buccal tube (e.g., pt of external width [8.3–11.5] vs [6.4–8.7] in young) and longer macroplacoids (e.g., pt of macroplacoid row [32.7–39.4] vs [26.0–30.9] in young) and claws (e.g., pt of claw I external primary branches [28.7–35.9] and of claw IV posterior primary branches [38.0–46.0] vs [28.1–31.5] and [29.6–34.9] respectively in young).

Eggs (Fig. 8; Table 6; Supp. file 3)

Eggs are light orange in colour before mounting, spherical and laid freely. Processes in the shape of elongated cones, rarely bifurcate distally, usually ending at the tip in a filament (Fig. 8A–E). In some processes, single bubble-like structures can be seen inside the distal half portion of the processes (Fig. 8C). On the egg circumference 29–34 processes are present and about 135–177 in the hemisphere depending also on the egg size. Process bases without projections on the chorion (Fig. 8F), but this latter, between the process, has evident granulation (Fig. 8A–B, F). Quantitative data are reported in Table 6.

Body region s Body segmentsSubsegments and legsNumber of rowsNumber of poresPore shapeHeadHead2triangular144quadrilobated2triangular6various228 10variousSecond segment (segment I of the trunk), with legs I6–10Trunk6–103 (with legs I)4 plus lateral patch6–10various6–10lateral patch of about 7–12lateral patch of about 7–12Legs I1 bigThree – or four –armed428–10various8–106–105 (with legs II)6–104 plus lateral patch6–10various6–10Third segment (segment II of the trunk), with legs IIlateral patch of about 7–12Legs II1 bigThree – or four –armed pore628–10 8–10various4–10Fourth segment (segment III of the trunk), with legs III4–107 (with legs III)5 plus lateral patch4–10various4–104–10lateral patch of about 7–124–104–10lateral patch of about 7–12Body region s Body segmentsSubsegments and legsNumber of rowsNumber of poresPore shapeFourth segment (segment III of the trunk), with legs III4–107 (with legs III)5 plus lateral patch4–104–104–10variouslateral patch of about 7–12TrunkLegs III1 big more anterior some caudal smallerFour – armed various4–10834–104–10Fifth segment (segment VI of the trunk), with legs VI910 (with legs IV)continuous, big, caudal group made of many pores with no clear rows recognisable (also on the dorsum of legs IV)various One big, four – or five – armed, others of various shape4–10

Legs IV

CharacterNRangeMeanSDHolotypeµmptµm pt µm pt µmptBody length29 180 – 342 656 – 1047656 – 1047258 883 47 112 300 989Buccopharyngeal tubeBuccal tube length2924.6 – 33.629.0 – 2.2 – 30.3 –Stylet support insertion point2817.2 – 23.265.0 – 71.419.9 68.2 1.5 1.7 20.1 66.2Buccal tube external width282.2 – 3.68.3 – 11.52.8 9.7 0.4 0.8 3.5 11.5Buccal tube internal width281.1 – 2.34.2 – 7.41.6 5.5 0.4 1.0 2.2 7.4Ventral lamina length1613.0 – 16.045.1 – 53.714.6 50.6 1.0 2.3 16.0 52.8Placoid lengthsMacroplacoid 1292.6 – 4.09.8 – 13.43.3 11.3 0.4 0.9 3.6 11.7Macroplacoid 2292.1 – 3.58.1 – 11.62.7 9.2 0.3 0.7 2.9 9.6Macroplacoid 3291.9 – 3.37.5 – 11.12.6 8.9 0.3 0.7 2.9 9.6Microplacoid290.9 – 1.83.5 – 5.91.4 4.9 0.3 0.8 1.7 5.7Macroplacoid row288.5 – 12.532.7 – 39.410.5 36.1 1.2 2.1 11.9 39.2Placoid row289.7 – 14.939.4 – 47.812.5 43.0 1.4 2.8 14.5 47.8Claw 1 heightsExternal primary branch277.8 – 10.628.7 – 35.99.4 32.2 0.8 1.7 9.5 31.3External secondary branch265.4 – 8.620.6 – 26.66.9 23.5 0.8 1.5 7.5 24.7Internal primary branch287.2 – 10.127.1 – 34.99.0 30.8 0.9 2.1 9.1 29.9Internal secondary branch275.1 – 7.819.9 – 25.66.5 22.3 0.8 1.7 7.0 23.1Claw 2 heightsExternal primary branch288.0 – 11.230.5 – 38.59.9 34.3 1.0 1.8 10.4 34.4External secondary branch255.7 – 8.722.1 – 28.87.4 25.4 0.9 1.8 7.7 25.2Internal primary branch287.8 – 10.929.7 – 37.79.6 33.0 1.0 2.1 10.0 32.9Internal secondary branch275.0 – 8.719.6 – 26.66.9 23.8 0.9 1.9 7.1 23.2Claw 3 heightsExternal primary branch298.4 – 11.932.4 – 39.710.3 35.6 0.9 1.7 10.7 35.3External secondary branch295.9 – 9.123.1 – 29.47.6 26.2 0.9 1.8 8.3 27.3Internal primary branch277.9 – 11.130.5 – 37.99.7 33.5 1.0 2.0 9.8 32.4Internal secondary branch265.5 – 8.221.5 – 28.17.2 24.7 0.8 1.8 7.3 24.1Claw 4 heightsAnterior primary branch278.7 – 13.735.3 – 43.611.5 39.6 1.4 2.6 12.6 41.4Anterior secondary branch266.3 – 10.825.1 – 32.98.5 29.0 1.2 2.3 8.6 28.2Posterior primary branch279.7 – 14.138.0 – 46.012.2 41.7 1.2 2.6 12.7 41.8Posterior secondary branch276.8 – 10.726.0 – 34.28.7 29.6 1.1 2.5 8.9 29.5

DNA sequences

The sequenced senior specimen (V4) of Minibiotus dispositus sp. nov. is differentiated from all the other sequenced species belonging to the genus Minibiotus, as indicated by the ranges of genetic p-distances:

COI (523 bp dataset): 21.4 to 26.5% (Supp. file 4), with the most similar being unpublished sequences of M. citlalium (OP684766, OP684767) from Mexico;

ITS2 (531 bp dataset): 12.3 to 27.8% (Supp. file 5), with the most similar being M. ioculator (MT024000) from South Africa;

18S (778 bp dataset): 0.2 to 13.4% (Supp. file 6), with the most similar being M. furcatus (FJ435745) from Spain;

28S (817 bp dataset): 1.8 to 3.1% (Supp. file 7), with the most similar being Minibiotus sp. (MH079492) from Chile.

The ASAP analysis for both COI and ITS2 genes (Tables 7–8) further confirms the status of Minibiotus dispositus sp. nov. specimen V4 as a clearly distinct species from the other sequenced taxa of the genus.

The integration of the present molecular data with the morphological ones, therefore points to the validity of the erection of Minibiotus dispositus sp. nov.

Morphological differential diagnosis

Cuticular pores arranged in transverse bands are reported in many species of the genus, but the following are excluded from comparison for the reasons indicated in brackets: Minibiotus formosus Zawierucha, Dziamięcki, Jakubowska, Michalczyk & Kaczmarek, 2014, M. granatai (Pardi, 1941) and M. gumersindoi Guil & Guidetti, 2005 (they have only round/elliptical pores, lacking lobated/star-shaped ones); M. jonesorum Meyer, Lyons, Nelson & Hinton, 2011 (it lacks microplacoid and has polygonal pores, that also are very large and very densely distributed); M. pseudofurcatus (Pilato, 1972) (pores have at maximum 3 lobes/arms).

For differential diagnosis, we here compare M. dispositus sp. nov. with the species sharing the presence of smooth cuticle, multilobated pores (3–5 arms/lobes) and three macroplacoids plus evident microplacoid. However, considering the paucity of 5-armed pores recognisable under PCM (only 0–2 per specimen, and only caudally or on legs) in the new species, we excluded from comparison the species with very numerous, evident star-shaped pores (5 arms or more) clearly observable under PCM on all the body (thus excluding: M. citlalium Dueñas-Cedillo & García-Román, 2020, M. claxtonae Rossi, Claps & Ardohain, 2009, M. constellatus Michalczyk & Kaczmarek, 2003, M. pentannulatus Londoño, Daza, Lisi & Quiroga, 2017, M. pseudostellarus Roszkowska, Stec, Ciobanu & Kaczmarek, 2016, M. sidereus Pilato, Binda & Lisi, 2003).

Lastly, we decided not to limit the comparison with species clearly having a pore pattern (described or at least well visible in the description illustrations), because such a character may not have been noticed or reported in some past descriptions; in this way, M. aculeatus Murray, 1910 and M. vinciguerrae Binda & Pilato, 1992 are included, also considering that the old illustrations available may not be perfectly accurate.

All that considered, Minibiotus dispositus sp. nov. is to be compared with: M. aculeatus (Murray, 1910); M. bernhardi Schuster, 2021; M. bisoctus (Horning, Schuster & Gregarick, 1978); M. eichhorni Michalczyk & Kaczmarek, 2004; M. ethelae Claxton, 1998; M.furcatus (Ehrenberg, 1859); M. harrylewisi Meyer & Hinton, 2009; M. lazzaroi (Maucci, 1986); M. orthofasciatus Fontoura, Pilato, Lisi & Morais, 2009; M. pustulatus (Ramazzotti, 1959); M. vinciguerrae Binda & Pilato, 1992; M. weglarskae Michalczyk, Kaczmarek & Claxton, 2005; M. xavieri Fontoura, Pilato, Morais & Lisi, 2009 .

For correct morphometric comparisons, considering the allometry of some characters of the new species, we compared the morphometric characters in the present differential diagnosis taking into account the body size (available from the literature) of each compared species. Our young specimens had body sizes of up to about 156 µm while our senior specimens had body lengths starting from about 180 µm; most of the compared species had a body length starting from at least 200 µm; thus, we compared the morphometry of these species with that of our senior specimens; only two species, M. orthofasciatus and M. weglarskae, had body lengths starting from less than 180 µm (from 138 µm and 166 µm, respectively) but exceeding 200 µm in the maximum value: in this case we joined together our morphometric ranges of young and senior specimens for comparison (no compared species had a body length range compatible only with our young specimens).

Minibiotus dispositus sp. nov. specifically differs from the various compared species as follows.

Minibiotus aculeatus (according to the description, drawings and measurements by Claxton 1998), reported from the type locality in Australia, and from New Zealand: this species (also) has star-shaped pores (even 6-armed) and should have no pore bands; judging from the drawings, however, it is not clear how many such star-shaped pores are common and spread, or whether the bands are surely absent (the drawings may be schematic or not sufficiently accurate). Minibiotus dispositus sp. nov. and M. aculeatus differ in smallest pore size (smallest 0.9 µm in the new species vs 0.5 µm in M. aculeatus); the absence of three pairs of soft conical spines on the dorsal cuticle in connection with the segment bearing legs II–III–IV (such spines present in M. aculeatus); claws in M. dispositus less robust than in M. aculeatus . Different characteristics of the egg: granulated chorion in the new species (smooth in M. aculeatus); diameter without/with processes larger 55.6–82.1 / 77.2–110 µm (54 / 65 µm in M. aculeatus); 29–34 (24–30 in M. aculeatus) processes around the circumference; process height 13.0–17.0 µm, and with 1.3–3.4 µm process base distance (process height 9–11 µm, 3–4 µm base distance in M. aculeatus); base of each process smooth in the new species, indented in M. aculeatus .

Minibiotus bernhardi, reported only from a few localities in Germany: multilobate pores very common in M. dispositus sp. nov. vs scarce in M. bernhardi . Nine dorsal bands of pores in the new species vs ten (total band number of both species includes cephalic and caudal bands but excluding legs IV); in particular, M. dispositus has on all the dorsum of the hind segment, including legs IV, a continuous, large, caudal group of pores with no bands or rows distinguishable, while in M. bernhardi three separate bands are distinguishable. Different number of ventral pore bands: seven in the new species vs eight in M. bernhardi . Totally different egg since M. bernhardi produces eggs of the intermedius - type.

Minibiotus bisoctus (according to the description, drawings and measurements by Claxton 1998) reported only from the type locality in New Zealand: presence of pores on the ventral cuticle (absent in M. bisoctus); eyes present in the new species (absent in M. bisoctus); pt of the ventral lamina [45.1–53.6] (42.0 in M. bisoctus); stylet supports inserted more posteriorly, pt [65.0–71.4] (more anteriorly 60.3 in M. bisoctus); pt of macroplacoid row [32.7–39.4] (31.0 in M. bisoctus); legs IV posterior primary branches longer in M. dispositus sp. nov. (pt [38.0–46.0] vs 31.0 in M. bisoctus); leg granulation around the claws absent (present in M. bisoctus).

Minibiotus eichhorni reported only from the type locality in Perú: clear pattern of cuticular pores arranged in rows within the bands, while no rows are visible in M. eichhorni; the authors of M. eichhorni did not mention any pore arrangement in rows within the bands; but if any, the rows seems to be no more than 3 per band (see Michalczyk & Kaczmarek 2004: figs 1–2), while M. dispositus sp. nov. has 2–5 welldisposed rows per band; a clear pattern of ventral pores disposed in 8 rows while randomly distributed pores in M. eichhorni (“Round and oval pores present over whole cuticle, however in lower density on ventral cuticle” according to Michalczyk & Kaczmarek 2004); granulations around the claws on all legs absent (present on all legs in M. eichhorni). Morphometric differences regard partially overlapping morphometric ranges that were statistically tested (Table 9; Supp. file 8).

Minibiotus ethelae reported only from the type locality in Australia and from South Africa: dorsal pattern of cuticular pores distinct from ventral one in the new species, including different number between dorsal and ventral rows, while “9 bands around the body” in M. ethelae according to Claxton (1998); cuticle not thickened around the pores of the caudal region (while thickened in M. ethelae); evident microplacoid in M. dispositus sp. nov., while “small, indistinct” in M. ethelae (Claxton 1998); absence of the refractive zone at base of the claws (present in M. ethelae). The eggs of the two species are very similar, both morphologically and morphometrically; however, two possible differences can be singled out: the distance between the egg processes is 1.3–3.4 µm in the new species, while Claxton (1998) reported a distance of “about 5 µm”; this may be confirmed by the slightly higher average number of processes on the egg hemisphere in M. dispositus (135–177) with respect to M. ethelae (120–160).

Minibiotus furcatus (according to the redescription by Binda & Pilato 1992) reported from the type locality in Europe (Monte Rosa, Italian Alps), the Americas, South Africa and India: shorter ventral lamina (pt [45.1–53.7] in M. dispositus sp. nov. vs 62.0 in M. furcatus); smooth lunules of all legs, while slightly indented on legs IV of M. furcatus . Different egg characteristics: orange color (colorless or yellowish in M. furcatus), process height 13.0–17.0 µm (5–6 µm in M. furcatus), granulated chorion (smooth chorion in M. furcatus).

Minibiotus harrylewisi reported only from the type locality in South Africa: morphometric differences regarding partially overlapping morphometric ranges but statistically tested (Table 9; Supp. file 8). Different egg characteristics: long cones, 13.0–17.0 µm high, uniformly tapering from the base, with granulated chorion in the new species, while M. harrylewisi has shorter cones (7.6–12.8 µm high) with bulbous base, with smooth chorion.

Minibiotus lazzaroi, reported only for few Italian localities: the species was compared with senior specimens of M. dispositus sp. nov. as the reported body length of M. lazzaroi was “up to 420 μm” and no differences were reported between young and senior specimens by Maucci (1986). Roundish (PCM), elliptical and multilobate (up to 5 lobes) pores in the new species vs triangular, trapezoidal or rhomboidal pores in M. lazzaroi; pores having a wide dimensional range (from 0.9 to 3.1 µm) in M. dispositus vs pores of almost the same size on all the body (from 2 to 2.2 µm) in M. lazzaroi; 9 bands of pores vs 8 bands of pores (according to the drawing in the original description) in M. lazzaroi (band number of both species including cephalic and caudal bands). Totally different egg characteristics, since the new species has eggs with very slender, smooth, conical processes (process height 13.0–17.0 μm, process base width 2.0–4.5 μm) tapering apically, and a granulated chorion without ridges, while M. lazzaroi has a peculiar egg with very wide, reticulated, trunco-conical processes (process height 8–12 µm, process base width 28–30 µm) with jagged apical portion and irregular ridges on the chorion surface.

Minibiotus orthofasciatus reported only from the type locality in Portugal: cuticular pores arranged in 10 dorsal bands in the new species, while 11 in M. orthofasciatus; shorter ventral lamina (pt [45.1–53.7] in M. dispositus sp. nov. vs [55.3–58.4] in M. orthofasciatus). Totally different egg characteristics since the new species has eggs with conical processes and a granulated chorion not covered by a membrane including the processes, while M. orthofasciatus has eggs of the intermedius group, i.e., with screw-like processes joined by a membrane covering a non-granulated egg chorion.

Minibiotus pustulatus reported from the type locality in Italia, and from Chile: eye-spots present in the new species (absent in M. pustulatus); dorsal and leg pores occur to be multilobate (usually 3–4 lobes/arms) in the new species, while subcircular to triangular or polygonal, but with no lobes/arms, in M. pustulatus; pores are also smaller in the new species (about 1–2 µm diameter), while very large (4–7 µm) in M. pustulatus). Minibiotus pustulatus was not described as having a pore distribution pattern, but but if it had the number of possible pore rows (according to the original drawing) is clearly far lower than in the new species.

Minibiotus vinciguerrae, only recorded from Antarctica, by the pore shape and size: in M. vinciguerrae many pores are elliptical, reaching the size of 2.1 µm (largest pore size for the species), while few pores are triangular/trilobate or (even more rarely) quadrangular/quadrilobate, in any case of smaller or equal size to the elliptical pores; in the new species, instead, elliptical pores are rare and small (around 1 µm), while the triangular/trilobate and quadrangular/quadrilobate are common and bigger (up to 3.1 µm). The new species has shorter ventral lamina (pt [45.1–53.7] vs [58–60] in M. vinciguerrae); macroplacoid length sequence 3≤ 2<1 in the new species vs 2<3 <1 in M. vinciguerrae; robust claws (very slender in M. vinciguerrae); different details of the egg morphology: the egg processes have only sometimes bifurcated end (very commonly in M. vinciguerrae), have smooth base margin (jagged/irregular in M. vinciguerrae), and are higher (13.0–17.0 µm vs 8.17 µm in M. vinciguerrae), closer to one-another (distance between processes 1.3–3.4 µm vs 5 µm in M. vinciguerrae) and more numerous on the egg circumference (29–34 vs 26 in M. vinciguerrae).

Minibiotus weglarskae reported from the type locality in Mongolia: stylet supports inserted in more posterior position in the new species (pt [62.9–72.9] vs [54.5–59.6] in M. weglarskae); longer claws in the new species (higher pt indices), see Table 9 for statistical significance of differences in overlapping pt ranges of claw heights and other metric characters. Leg granulation absent in the new species (present in M. weglarskae). Totally different egg characteristics since the new species has eggs with conical processes and a granulated chorion not covered by a membrane including the processes, while M. weglarskae has eggs of the intermedius group, i.e., with screw-like processes joined by a membrane covering a non-granulated egg chorion.

Minibiotus xavieri reported from the type locality in Portugal: multilobate dorsal/leg pores with 3–4 lobes/arms, sometimes 5, in the new species, while only trilobate in M. xavieri; pt of ventral lamina length [45.1–53.7] in the new species (vs [55.2–57.4] in M. xavieri), additional morphometric differences regard partially overlapping morphometric ranges statistically tested (Table 9; Supp. file 8). Egg processes in the circumference far more numerous in the new species (29–34), while only 20–23 in M. xavieri .