Macrobiotus nebrodensis Pilato & Sabella & D'Urso & Lisi 2017, sp. nov.

Macrobiotus nebrodensis sp. nov.

Figs. 1, 2, 3

Type locality: Sicily, Nebrodi Mountains, Caronia, by the side of Zilio Lake, 1069 m a.s.l.; (37°57′N, 14°25′E).

Examined material: Caronia, by the side of Zilio Lake: holotype (slide No. 5708) and one egg with fully developed hatching embryo (slide No. 5708), from a moss sample collected by Mr. Angelo Zappalà, 29.5.1989.

Specific diagnosis: species of the Macrobiotus hufelandi group; colourless, cuticle smooth with circular and elliptical pores and small dots on the legs; eye spots present before mounting; bucco-pharyngeal apparatus of the Macrobiotus type; ten peribuccal lamellae present; buccal armature with three dorsal and three ventral transverse ridges and a band of small teeth in the posterior portion of the buccal cavity; pharyngeal bulb with two macroplacoids and a small microplacoid; claws of the Macrobiotus hufelandi type with accessory points and lunules; the only egg found is freely laid, ornamented and with variable shape and size of processes: most are in the form of an inverted egg-cup with a large terminal disk, but some are elongate or very elongate, tapering, with a very small terminal disk.

Description of the holotype: body length 311 µm, colourless, cuticle smooth with circular and elliptical pores and small dots on the legs; eye spots present before mounting; bucco-pharyngeal apparatus of Macrobiotus type (Figs. 1A,B); mouth terminal with ten peribuccal lamellae; a band of small teeth and three dorsal and three ventral transverse ridges are present in the caudal portion of the buccal cavity; it is not clear whether a band of anterior teeth is also present. Rigid buccal tube 32.7 µm long and 5.0 µm wide externally (pt = 15.3); stylet supports inserted on the buccal tube at 77.8 % of its length (pt = 77.8). Pharyngeal bulb (30.8 µm x 25.4 µm) with apophyses, two rod-shaped macroplacoids and a very small microplacoid (Fig. 1B). First macroplacoid, with a central narrowing, 9.3 µm long (pt = 28.4), second 5.8 µm (pt = 17.7), microplacoid, 2.2 µm long (pt = 6.7) (N.B. this is faint in the adult (Fig. 1) and was not visible in the fully developed embryo (Fig. 2)); entire placoid row 18.8 µm long (pt = 57.5), macroplacoid row 15.9 µm long (pt = 48.6).

Claws of the Macrobiotus hufelandi type (Fig.1C). The external claws are 8.7 µm long (pt = 26.6) on the first pair of legs, 9.2 µm (pt = 28.1) on the second and third pairs of legs; the anterior claws on the hind legs are 10.8 µm long (pt = 33.0). Well-developed accessory points (Fig. 1C, arrow) and smooth lunules are present. A faint cuticular bar is present near the lunules on the first three pairs of legs.

Remarks: The fully developed hatching embryo is similar to the holotype but the microplacoid is not visible.

Egg: The egg, freely laid, has very peculiar characters (Figs. 2, 3). We found only one egg, fortunately with the fully developed hatching embryo. The egg is partially open but its diameter can be measured (about 75 µm without processes). We did not measure the diameter including the processes as these structures have various heights. About 18 processes are present in the circumference and about 90 in the hemisphere. The processes are not uniform in size (Figs. 2, 3); the greater majority are in the shape of an inverted egg-cup, 4.2–4.7µm high with a terminal disc of variable diameter (3.1–5.6 µm) with jagged but not dentate margin. However, some of the processes are clearly longer (7.3–11 µm), with a variably sized terminal disc (Figs. 2 and 3C arrow a), and some are very long (Fig. 3B, arrows), for example, one is 20.6 long with a 2 µm wide terminal disc (Figs. 2 and 3C arrow b). The egg shell has a reticular design (Fig. 3C arrow c), with larger or broader mesh surrounding the process bases than the shell between processes (Fig. 3C arrow d).

Etymology: The specific name (nebrodensis = living on the Nebrodi Mountains) refers to the type locality.

Differential diagnosis: As it is well known, distinguishing between the species of the Macrobiotus hufelandi group is very difficult and careful examination of both individuals and eggs is required to ensure correct identifications. Macrobiotus nebrodensis sp. nov. is typical of the hufelandi group, and the adult is very similar to other species, but luckily we found one egg (with the fully developed embryo), which has provided unique characters that allow an immediate identification of this species from the rest of the group.

While the longest processes of this new egg may in some way be similar to the egg processes of Macrobiotus kristenseni Guidetti, Peluffo, Rocha, Cesari, & Moly de Peluffo, 2013, in M. kristenseni all the processes are the same length and are not in the shape of an inverted egg-cup with a wide terminal disc; i.e. it is atypical of the usual hufelandi group egg processes. For other members of the hufelandi group, in addition to the egg characters, a careful comparison of the adult animals allows us to distinguish between Macrobiotus nebrodensis sp. nov. and other species by one or more characters.

Cuticular dots restricted to the legs separates the new species from Macrobiotus joannae and Macrobiotus punctillus; the presence of cuticular pores on the new species separates it from Macrobiotus lissostomus, Macrobotus andinus and Macrobiotus madegassus; in the buccal cavity Macrobiotus nebrodensis sp. nov. has distinctly separated transverse ridges in the caudal portion, while the dorsal ridges are fused forming a unique continuous transverse ridge in Macrobiotus diversus, Macrobiotus sapiens, Macrobiotus iharosi, Macrobiotus sottilei, Macrobiotus humilis, Macrobiotus biserovi, Macrobiotus seychellensis and Macrobiotus modestus; the absence (or not clearly visible) anterior band of teeth in the anterior portion of the buccal cavity separates the new species from Macrobiotus macrocalix, Macrobiotus persimilis, Macrobiotus hufelandi, Macrobiotus hyperboreus Biserov, 1990a, Macrobiotus trunovae, Macrobiotus martini, Macrobiotus almadai, Macrobiotus marlenae and Macrobiotus kazmierskii Kaczmarek & Michalczyk, 2009; in addition the presence of eye spots separates the new species from Macrobiotus hyperboreus; a medio-dorsal and a medio-ventral transverse ridge, present in the caudal portion of the buccal cavity of Macrobiotus nebrodensis sp. nov., are absent in Macrobiotus martini; the absence of a clearly visible tooth in the buccal tube distinguishes the new species from Macrobiotus denticulus. The pt index relative to the stylet support insertion point is higher in Macrobiotus nebrodensis sp. nov. than Macrobiotus madegassus, Macrobiotus iharosi, Macrobiotus humilis, Macrobiotus martini, Macrobiotus modestus, Macrobiotus hibiscus and Macrobiotus seychellensis (c. 78 in the new species, 67.3–69.8 in Macrobiotus modestus, c. 71 in Macrobiotus madegassus and Macrobiotus humilis, 72–75.2 in Macrobiotus iharosi, 72.9–74.3 in Macrobiotus martini, 73.5–74.6 in Macrobiotus hibiscus, 74.5–77.6 in Macrobiotus seychellensis), and lower than Macrobiotus andinus, Macrobiotus trunovae and Macrobiotus punctillus (c. 78 in the new species, 80.7–82.2 in Macrobiotus punctillus, 81–82.4 in Macrobiotus trunovae, 81–84 in Macrobiotus andinus). In Macrobiotus nebrodensis sp. nov. the first macroplacoid has a clearly visible central narrowing which separates it from Macrobiotus patagonicus, Macrobiotus vladimiri Bertolani, Biserov, Rebecchi, & Cesari, 2011 and Macrobiotus hibiscus. Lower values of the pt index relative to the microplacoid distinguishes Macrobiotus nebrodensis sp. nov. from Macrobiotus sapiens, Macrobiotus joannae, Macrobiotus trunovae, Macrobiotus almadai; Macrobiotus macrocalix. The macroplacoid row clearly longer than the mid-point of the pharyngeal bulb in the new species separates Macrobiotus sandrae, and the presence of lunules of leg IV without marginal teeth in the new species separates Macrobiotus terminalis, Macrobiotus ramoli Dastych, 2005, Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996, and Macrobiotus seychellensis. The more slender claws of Macrobiotus nebrodensis sp. nov. differentiate it from Macrobiotus rawsoni Horning, Schuster & Grigarick, 1978, Macrobiotus vladimiri, Macrobiotus terminalis and Macrobiotus polonicus; and the absence of lateral gibbosity on the legs IV from Macrobiotus polonicus.

When examining only adult animals without eggs it may prove to be very difficult to distinguish Macrobiotus nebrodensis sp. nov. from Macrobiotus personatus and Macrobiotus santoroi. However, the new species’ wider buccal tube in proportion to the body size distinguishes it from Macrobotus personatus and Macrobiotus santoroi (in the Macrobiotus nebrodensis sp. nov. holotype, 311 µm long, the index value is 15.3, while a Macrobiotus personatus paratype, 464 µm long, has a value of 13.6, and in Macrobiotus santoroi specimens, c. 400 µm long the value is c. 13). When eggs are also available for comparison there is no doubt that Macrobiotus nebrodensis sp. nov. is a bona species, completely distinct from Macrobiotus personatus and Macrobiotus santoroi.