Minibiotus orthofasciatus, Fontoura, Paulo, Pilato, Giovanni, Lisi, Oscar & Morais, Paulo, 2009

Fontoura, Paulo, Pilato, Giovanni, Lisi, Oscar & Morais, Paulo, 2009, Tardigrades from Portugal: four new records and description of two new species, Zootaxa 2030, pp. 21-38 : 23-27

Minibiotus orthofasciatus sp. nov.

Figs. 1 , 2

Material examined: Thirty specimens and one unembryonated egg, mounted in Hoyer’s medium, collected on lichens from tree trunks at Parque Biológico, Avintes – Vila Nova de Gaia (six specimens and the egg were collected in May 2006, all the other specimens were collected in April, 2008). Four specimens were in simplex stage and 1 in molting stage.

Type repository: The holotype (slide N. 5231), a paratype (slide N. 5232) and the egg (slide N. 5233) are deposited in the collection of Binda and Pilato (Museo del Dipartimento di Biologia Animale “Marcello La Greca”, Università di Catania); the other paratypes (slides CI-1 to CI-17) in the collection of P. Fontoura (Department of Zoology and Anthropology, Faculty of Sciences, University of Porto).

Specific diagnosis: Cuticle with clearly defined eleven dorsal and eight ventral transverse bands of pores various in shape and size. Pores larger on cephalic and caudal portions of the body. Dots on the legs absent but very fine, almost invisible dots, seem to be present on all the dorsal surface of the body. Three macroplacoids (the first is the largest, second and third very similar in length) and a very minute microplacoid present. Claws Description of the holotype: Body length 186 µm, colorless, eye spots present, visible on living specimens and before slide mounting. Cuticle smooth with pores of varying size (diameter 1.0 to 2.1 µm) and shape: roundish, oval, trilobed and, rarely, quadrilobed. The pores are arranged in dorsal and ventral bands constant in number and almost constant in pore distribution ( Figs. 1A, B, C, D ).

Dorsally eleven bands are present ( Figs. 1A, B ): two cephalic ones formed by two transverse rows; three bands also formed by two rows at the level of the first three pairs of legs; three bands, single on the dorsal wall but double on the lateral walls, are present after each pair of legs; and three caudal single bands (near the single bands very rarely supplementary pores may be present). On the ventral surface of the body eight distinct bands of a few roundish pores are present ( Fig. 1D ). A few pores are also present on the legs. No granulation is visible on the legs. Extremely fine, almost invisible, dots are probably present on the dorsal surface of the body, but the presence of this granulation needs to be confirmed.

Mouth antero-ventral. Buccal armature not visible with light microscope. Rigid buccal tube 22.6 µm long, with an anterior bend ( Fig. 2B ); a posterior bend is almost absent; a thickening of the buccal tube wall is present caudal to the stylet supports where the buccal tube is 1.8 µm wide externally (pt = 8.0). Buccal tube with ventral lamina not particularly short (pt = 58.4 measured in lateral view) ( Fig. 2B ). Stylet supports inserted on the buccal tube at 66.8% of its length (pt = 66.8). Pharyngeal bulb with large triangular apophyses, three small macroplacoids ( Figs. 2 A, B ) and a very small microplacoid ( Fig. 2B ); first macroplacoid, pear shaped, and partially lying under the apophyses as in other species of the genus, 3.1 µm long (pt = 13.7); second macroplacoid 2.4 µm long (pt = 10.6); third macroplacoid 2.4 µm long (pt = 10.6). The very small microplacoid, 0.9 µm long (pt = 4.0), is difficult to see. Macroplacoid row length, excluding the microplacoid, 7.4 µm long (pt = 32.7); entire row of placoids 8.3 µm long (pt = 36.7).

Claws of hufelandi type, well developed ( Figs. 1C, D ). Primary branches of internal and external claws with well developed accessory points. External and internal claws of legs II and III 6.8 µm long (pt = 30.1) and 6.3 µm long (pt = 27.9) respectively, including accessory points. Posterior and anterior claws of fourth pair of legs 6.9 µm long (pt = 30.5) and 6.8 µm long (pt = 30.1) respectively.

Smooth lunules, better developed on the fourth pair of legs (2.5 µm longer diameter), are present. A small cuticular bar is present near the lunules on the first three pairs of legs.

The measurements of some structures of holotype and three paratypes are shown in Table 1 .

One unembryonated egg has been found with the described specimens. It is an egg very similar to those of some other species of Minibiotus ( M. intermedius , M. floriparus and M. poricinctus ) with screw-like processes each surrounded by a membranous hood ( Figs. 2C, D, E ); therefore we think that it may be attributed to the only species of Minibiotus genus found in the sample (other species collected in the same sample were Milnesium tardigradum Doyère, 1840 ; Diphascon pingue ; Macrobiotus hufelandi Schultze, 1834 ; Macrobiotus recens Cuénot, 1932 of which the eggs were also present).

The diameter is 54.0 µm including the processes, 48.0 µm excluding them; the processes ( Fig. 2C ) are 4.2–4.5 µm in height with a base diameter of 2.3–2.7 µm and a top diameter of 3.3–3.6 µm. The processes are 6.4–6.9 µm apart. About 24 processes are present around the circumference and about 60 in the hemisphere. The base of the membranous hood of each process is connected to the surrounding ones and, as a consequence, the egg shell appears areolated ( Figs. 2D, E ); generally six polygonal areolae are present around each process, and their margins appear formed by many dots ( Fig. 2E ).

Etymology: The name orthofasciatus refers to the very regular straight (ortho) disposition of the bands (fasciatus) of cuticular pores.

Differential diagnosis: The presence of very fine cuticular dots has to be confirmed; therefore this character is not taken into consideration in the following comparisons.

The distribution of the cuticular pores in Minibiotus orthofasciatus sp. nov. is almost identical to that of Macrobiotus striatus Mihelčič, 1949 , which probably has to be transferred into the genus Minibiotus as hypothesized also by Guidetti et al. (2007), but it differs from this species in many characters: colorless ( M. striatus is yellow), eye spots present, cuticular pores of various size and shape, first macroplacoid longer than the third, a small microplacoid present, different egg.

The distribution of the cuticular pores of the new species is also similar to that of Minibiotus pustulatus ( Ramazzotti, 1959) but the new species differs from this species in having smaller caudal pores (in M. pustulatus the diameter is 6–7 µm), eye spots present and different egg.

Nine other species of Minibiotus have three macroplacoids and cuticular pores arranged in distinct bands. One of them, M. gumersindoi , has pores with a distribution similar to that of Minibiotus orthofasciatus sp. nov., but the new species differs from it in also having trilobed and quadrilobed pores, the stylet supports inserted on the buccal tube in a more caudal position (pt = about 67 in the new species, 50–57.5, according to Guil & Guidetti (2005) and confirmed by us, in M. gumersindoi ), longer ventral lamina, slightly larger macroplacoids and smaller microplacoid.

The new species differs from the remaining eight species with three macroplacoids and microplacoid in having a different pore distribution. In addition it differs from M. poricinctus and M. keppelensis in also having trilobed and quadrilobed pores, in lacking granulation on the legs, in having the stylet supports inserted on the buccal tube in a more caudal position (pt = about 67 in Minibiotus orthofasciatus sp. nov., about 60 in M. poricinctus and about 61 in M. keppelensis ) and in having longer ventral lamina (pt = about 58 in Minibiotus orthofasciatus sp. nov., about 36 in M. poricinctus and about 40 in M. keppelesis ).

Minibiotus orthofasciatus sp. nov. differs from M. bisoctus and M. weglarskae in lacking granulation on the legs and in having the stylet supports inserted on the buccal tube in a more caudal position (pt = about 67 in the new species, about 60 in M. bisoctus and 54.5–59.6 in M. weglarskae ) and in having longer ventral lamina (pt = about 58 in M. orthofasciatus , about 42 in M. bisoctus ); in the description of M. weglarskae the authors did not indicate the length of the ventral lamina, but they wrote that it is “very short”, and their figures 7, 14, and 15 show that this structure is clearly shorter than in M. orthofasciatus sp. nov. In addition M. orthofasciatus sp. nov. differs from M. bisoctus in having eye spots, and from M. weglarskae in having a small cuticular thickened bar near the lunules on the first three pairs of legs.

The new species differs from Minibiotus furcatus and Minibiotus eichorni Michalczyk & Kaczmarek, 2004 , in lacking granulation on the legs and in having a smaller microplacoid. From M. furcatus also in having narrower buccal tube (unfortunately, due to a qui pro quo, Binda and Pilato, in a paper of 1992, wrote that the pt value relative to the buccal tube in M. furcatus is 7.94; we noted the mistake, and we found a value 11.2).

Minibiotus orthofasciatus sp. nov. differs from Minibiotus granatai ( Pardi, 1941) and M. ethelae in having the cuticle of the caudal portion not different (not more thickened) from that of other parts of the body, and without gibbosities. It also differs from M. ethelae , M. furcatus , M. keppelensis and M. weglarskae in having a different egg. The eggs of M. bisoctus , M. gumersindoi and M. eichorni are not known. The egg found with the new species and, according to us attributable to it, is similar to that of M. intermedius , M. floriparus , M. weglarskae and M. poricinctus but it differs from them in some details. It differs from the egg of M. intermedius because it has processes larger and less numerous (Table 2); in addition the egg shell is areolated and small dots are present where the membranous hoods reach the egg shell. Also the egg of M. poricinctus has dots at the base of the membranous hoods, but the egg attributed to the new species has smaller and more numerous processes (Table 2). The egg attributed to Minibiotus orthofasciatus sp. nov. differs from those of M. floriparus in having areolated egg shell with dots where the membranous hoods reach the egg shell; in addition it is smaller, with smaller processes and, in particular, with smaller terminal portion (Table 2) where a ring of pores well evident in M. floriparus , is almost invisible. The egg attributed to the new species differs from those of M. weglarskae in having areolated egg shell with dots where the membranous hoods reach the egg shell; in addition it has smaller processes with smaller terminal portion (Table 2) where a ring of pores, well evident in M. weglarskae , is almost invisible.

TABLE 2. Measurements (in µm) and other characteristics of the egg of Minibiotus orthofasciatus sp. nov., Minibiotus intermedius , Minibiotus poricinctus , Minibiotus floriparus and Minibiotus weglarskae .

M. orthofasciatus M. intermedius M. poricinctus M. floriparus M. weglarskae

Diameter processes excluded 48 40–45 49 62 40

Diameter processes included 54 45–52 60 70 50

Processes around circumference 24 about 30 18–20 20–22 24

Processes in hemisphere about 60 about 130 50 60–75 about 50 Processes height 4.2–4.5 2.5–3.5 6.5–7.0 5.5–6.0 5.0–5.5 Basal diameter 2.3–2.7 0.5–1.5 1.5 2–3 1.6–2.0 Terminal portion diameter 3.3–3.6 2.5–3.5 5.5 6–7 5.0–5.5 Egg shell areolation + - - - -

Basal dots + - + - -

Distal pores very small very small? evident evident

Figures

FIGURE 1. A – D, Minibiotus orthofasciatus sp. nov.: A, anterior portion of the body of a paratype, the dorsal bands of pores are visible; B, postero-dorsal bands of pores of a paratype; C, details of second and third leg pores of a paratype; D, posterior portion of the body of a paratype, the ventral bands of pores and the claws are visible (scale bars = 10 µm).

FIGURE 2. A – E, Minibiotus orthofasciatus sp. nov.: A, bucco-pharyngeal apparatus of a paratype, the thickening of the buccal tube wall is shown; B, bucco-pharyngeal apparatus of the holotype; C, egg, arrows indicates the egg processes; D, areolated design of the egg shell formed by the connections of the membranous hood of the processes (arrows); E, polygonal areolae around processes with dots on their margins are shown (scale bars = 10 µm).

Tables

TABLE 1. Measurements (in µm) and pt values of some structures of the holotype, and three paratypes, the smallest and largest ones included, of Minibiotus orthofasciatus sp. nov.

	smallest paratype holotype	paratype	largest paratype
Body	138.0 186.0	186.0	189.0
Buccal tube	19.9 22.6	21.4	21.2
Buccal tube external width pt	1.5 1.8 7.5 8.0	1.7 7.9	1.6 7.5
Stylet support insertion pt	13.4 15.1 67.3 66.8	14.5 67.8	14.1 66.5
Ventral lamina pt	11.0 13.2 55.3 58.4	? -	? -
First macropalcoid pt	1.9 3.1 9.5 13.7	2.8 13.1	? -
Second macroplacoid pt	1.5 2.4 7.5 10.6	2.3 10.7	1.8 8.5
Third macroplacoid pt	1.9 2.4 9.5 10.6	2.3 10.7	2.2 10.4
Microplacoid pt	? 0.9 - 4.0	0.7 3.3	? -
Placoid row pt	? 8.3 - 36.7	7.6 35.5	? -
Macroplacoid row pt	6.1 7.4 30.7 32.7	7.3 34.1	6.7 31.6
External claw III pt	6.3 6.8 31.7 30.1	6.6 30.8	6.6 31.1
Internal claw III pt	? 6.3 - 27.9	6.3 29.4	? -
Anterior claw IV pt	? 6.8 - 30.1	6.5 30.4	? -
Posterior claw IV pt	6.6 6.9 33.2 30.5	6.7 31.3	? -