Mesobiotus helenae sp. nov.

Figures 1–4; Table 1

Type locality: New Zealand, South Island, vicinity of Karamea, Podocarpus forest, moss sample from a tree .

Material examined: Holotype and two paratypes (formerly slide No. 60(29) in Tumanov’s collection, now No. 5109 in the collection of Binda & Pilato), and one embryonated egg (formerly slide No. 60(15) in Tumanov’s collection, now No. 5108 in the collection of Binda & Pilato.

Type repository: Holotype and paratypes are preserved in the Binda & Pilato’s collection ( Museum of Department of Biological, Geological and Environmental Sciences of the University of Catania, Italy) .

Specific diagnosis: Colourless, eyes absent, cuticle smooth without dots, pores or tubercles; only a small gibbosity seems to be present laterally on the hind legs. Bucco-pharyngeal apparatus of the Macrobiotus type. Buccal armature with an anterior band of small teeth that is difficult to observe; a second, posterior row of longitudinally elongate teeth, and a posterior system of three dorsal and three ventral ridges. Claws stout with well developed accessory points; smooth lunules present. In addition, the first three pairs of legs have a faint cuticular bar present near the lunules. The eggs, freely laid, have processes with a conical basal portion abruptly continuing in a long, sharpened, terminal portion with some bubble-like structures, whereas the basal portion presents a faint, almost invisible, reticular ornamentation.

Description of the holotype. Body length 321 µm, eyes absent; cuticle smooth without tubercles, dots or pores. Only a small gibbosity seems to be present laterally on the hind legs (Figure 2C, arrows). Bucco-pharyngeal apparatus of the Macrobiotus type (Figure 1A); ten peribuccal lamellae present (Figure 1B, arrow). Buccal armature with a faint anterior band of very small teeth that is difficult to see, a second, obvious posterior band of single longitudinally elongated teeth (Figure 2A, arrow), and a posterior system of three dorsal (Figure 2A) and three ventral transverse ridges (the medio-ventral ridge in the holotype is subdivided into two transverse teeth) (Figure 2B, arrow). The rigid buccal tube is 30.6 µm long, and its external width 4.3 µm (pt = 14.1). Stylet supports inserted on the buccal tube at 75.7 percent of its length (pt = 75.7). The pharyngeal bulb has apophyses, three macroplacoids and microplacoid; as characteristic of the species in the genus Mesobiotus, the pharyngeal bulb has the macroplacoids arranged along a curved line and the microplacoid is close to the third macroplacoid (Vecchi et al. 2016) (Figure 1A). Third macroplacoid with a slight sub-terminal constriction (Figure 1A). First macroplacoid 4.9 µm long (pt = 16.0), second 4.0 µm (pt = 13.1), third 4.4 µm (pt = 14.4), microplacoid 3.0 µm (pt = 9.8), macroplacoid row 13.7 µm (pt = 44.8), entire placoid row 16.2 µm (pt = 52.9). The claws, of the hufelandi type, are short and relatively stout (Figure 1C, D); the external claws of legs from I to IV are 7.3 µm (pt = 23.9), 7.8 µm (pt = 25.5), 7.8 µm (pt = 25.5) and 9.6 µm (pt = 31.4) long, respectively. Anterior and posterior claws IV identical in shape. In all claws the primary branch have well developed accessory points (Figure 1D, arrow a). All legs without granulation. Small, smooth lunules present (Figure. 1C, arrow a), slightly larger on the hind legs (Figure 1D, arrow b). A faded cuticular thickening is present under the lunules on the first three pairs of legs (Figure 1C, arrow).

The egg, with a very evident embryo (Figure 3A), is clearly attributable to Mesobiotus helenae sp. nov. It is freely laid, spherical and ornamented (Figures 3, 4). The diameter, processes excluded, is 71.0 µm, 92.0 µm processes included. The processes (22 around the circumference) (Figure 3) are conical, 11.0 µm long, with a wider basal portion, having a diameter of 6.9–8.2 µm, continued by a abruptly thinning, long, sharpened distal portion longer than the basal portion (Figures 3, 4), with the apex only rarely bifurcated (Figure 4A, arrows). The thin terminal portion shows some bubble-like structure, whereas the basal portion has a faint reticulation almost invisible (Figure 4B). When the apex is not broken, the length of the terminal portion is up to 180% of the basal portion length. The base of each process is structured, forming a crown of dots, but the eggshell between the processes is smooth, without reticulation or dots visible when examined at x1000 phase contrast under oil immersion (Figure 4).

Etymology: The first author dedicates this species to his wife Helen.

Remarks: The paratypes are similar to the holotype in both qualitative and quantitative characters (Table 1).

Differential diagnosis: The new species was compared with species of the Mesobiotus genus having similar the metric values for the bucco-pharyngeal apparatus, the shape and size of the claws, and characters of the eggs (shape and size of the processes, their ornamentation more evident on the terminal portion, and smooth eggshell between the processes). The most similar species are: Mesobiotus australis (Pilato & D’Urso, 1976), Mesobiotus baltatus (McInnes, 1991), Mesobiotus blocki (Dastych, 1984), Mesobiotus krynauwi (Dastych & Harris, 1995), Mesobiotus rigidus (Pilato & Lisi, 2006), Mesobiotus wuzhishanensis (Yin, Wang & Li, 2011), and Mesobiotus pseudoblocki Rozskowska, Stec, Ciobanu & Kaczmarek, 2016 . In the following comparisons, we have not included the presence of a small, lateral gibbosity on legs IV observed in the new species as we have only three specimens and because this structure may have passed unnoticed in the description of other species.

Mesobiotus helenae sp. nov. differs from Mesobiotus australis (recorded from Australia) in lacking eyes; in having a buccal armature of the harmsworthi - type with a band of well-developed elongated triangular teeth (Figure 2A) while Mesobiotus australis has a buccal armature of the echinogenitus - type without such elongated teeth but with a posterior band of small teeth (Figure 5A, arrow). The egg processes in the new species are longer with a more flexible terminal portion, clearly ornamented, and the basal portions with a faint reticular ornamentation (Figures 3 and 4); while in Mesobiotus australis the ornamentation of the processes is less evident (Figure 5B). The eggshell between the processes of the new species is smooth, whereas it appears dotted in Mesobiotus australis (Figure 5B, arrows).

The new species differs from Mesobiotus baltatus (reported from Spain) in lacking eyes; in having shorter and less slender claws (Figures 1C, D and 6A). The eggs processes are more numerous around the circumference (about 22 in Mesobiotus helenae sp. nov., about 12 in Mesobiotus baltatus), are shorter (about 11 µm long in the new species, 14–18 µm in Mesobiotus baltatus), and have a different shape, with a long and thin distal portion (Figures 3, 4 in this paper, and figure 9c, d, e in McInnes, 1991). The basal portion of the egg processes has a faint, almost invisible, reticular ornamentation (Figure 4B, arrows), which is very evident in Mesobiotus baltatus, and each process in the new species has a very obvious basal crown of dots, which is not present in Mesobiotus baltatus .

Mesobiotus helenae sp. nov. differs from Mesobiotus blocki (reported from Antarctica) in lacking eyes, and unpigmented (some specimens of Mesobiotus blocki have sparse brown pigmentation). The claws of the new species have small, smooth lunules slightly larger on leg IV, whereas the lunules of leg IV in Mesobiotus blocki have tiny, irregular teeth. The egg processes of Mesobiotus helenae sp. nov. with a faint reticular ornamentation, compared with the sparsely distributed small dots (pores?) of Mesobiotus blocki . The base of the processes in the new species has an obvious basal crown of dots, which in Mesobiotus blocki is described as “finger-like appendices” averaging 7–10 around the base.

Mesobiotus helenae sp. nov. differs from Mesobiotus krynauwi (recorded from Antarctica) in lacking eyes and cuticular pores, having a buccal armature of the harmsworthi - type ( Mesobiotus krynauwi has a buccal armature of the echinogenitus - type), in having less slender claws (Figure 1C, D in this paper, and Figures 6, 7 of Dastych & Harris, 1995). The new species has smaller eggs (of diameter ~ 71 µm without processes and ~ 92 µm including, whereas in Mesobiotus krynauwi it is 98–115 µm and 130–145 µm, respectively). Egg processes are more widely spaced and with a shorter basal diameter in the new species (6.9–8.8 µm, vs with 9–15 µm in Mesobiotus krynauwi). The basal portion of the Mesobiotus helenae sp. nov. egg processes have a faint, almost invisible reticular ornamentation (Figure 4B), which is clearly visible in Mesobiotus krynauwi (Figures 6B), and the base of each process has a very visible crown of dots (Figures 3B, and 4A,B) that is absent in Mesobiotus krinauwi (Figures 6B).

The new species differs from Mesobiotus rigidus (recorded from New Zealand) in having more numerous egg processes (22 around the circumference in Mesobiotus helenae sp. nov. and 12 in Mesobiotus rigidus). The egg processes in Mesobiotus helenae sp. nov. are smaller (height 11.0 µm and base width 6.9–8.2 µm vs 15.2–16.2 µm and 14.5–15.2 µm, respectively, in Mesobiotus rigidus). The apices of the egg processes of the new species are flexible and rarely bifurcated (Figures 3 and 4), whereas in Mesobiotus rigidus they are rigid and never subdivided (Figure 7A,B). The basal portions of the egg processes in Mesobiotus helenae sp. nov. have a faint, almost invisible reticular ornamentation (Figure 4B), which is clearly visible in Mesobiotus rigidus (Figures 7A,B). In the new species the eggshell between the basal dots of the processes is smooth, whereas in Mesobiotus rigidus stripes are visible, which seem to continue from the basal dots (Figure 7B, arrows).

The new species differs from Mesobiotus wuzhishanensis (recorded from China) in lacking eyes, in having less slender claws, and lunules on legs IV not indented. There are smaller egg processes in Mesobiotus helenae sp. nov. (which are ~ 11 µm long with a basal diameter 6.9–8.2 µm, and 18.5 µm long with basal diameter 10.6 µm long in Mesobiotus wuzhishanensis), and a higher number of processes around the egg circumference (~ 22 in the new species and ~ 16 in Mesobiotus wuzhishanensis). The egg processes in the new species are more widely spaced and have the difficult to see faint reticulation at the basal portion, but which is more visible in Mesobiotus wuzhishanensis (Figure 4B arrows, arrows, in this paper and Figure 5c of Yin et al. 2011).

Mesobiotus helenae sp. nov. is very similar to Mesobiotus pseudoblocki (reported from Argentina) but differs in lacking eyes, having the stylet supports inserted on the buccal tube wall in a more caudal position (pt = 75.7–75.9 vs 71.5–75.2 in Mesobiotus pseudoblocki) (Figures 1A and 7A), and claws with a longer and more slender terminal portion on both branches; particularly the secondary (Figure 1C, D, and Figure 7B). In the new species the basal portion of the egg processes abruptly continues in a terminal long, sharpened portion, whereas in Mesobiotus pseudoblocki the processes thin gradually, appearing stouter (Figures 3, 4, in this paper and Figures 9–11 of Roszkowska et al. 2016). In addition, the egg processes of the new species are rarely bifurcated at the apices, whereas in Mesobiotus pseudoblocki the number of bifurcated processes is clearly higher, and some have short terminal filaments (Figure 11 of Roszkowska et al. 2016). In Mesobiotus helenae sp. nov. the basal portion of the egg processes has a faint, almost invisible reticulation (Figure 4B, arrows), whereas in Mesobiotus pseudoblocki the reticulation is quite evident. The basal dots of the egg processes in Mesobiotus helenae sp. nov. are not elongated like those in Mesobiotus pseudoblocki, which were described as “finger-like structures” (see Figure 4B in this paper, and Figure 10 of Roszkowska et al. 2016).

Conclusions: The history of the description of Mesobiotus helenae sp. nov. is unique. Tumanov (2004) first identified examples as Macrobiotus cf. coronatus . Pilato, Binda & Lisi (2006), studying slides on loan from the Horning et al. (1978) collection, Museum of New Zealand, Te Papa Tongareva, examined two specimens of “ Macrobiotus harmsworthi coronatus ” from North Island and one egg from South Island, and noticed that they were similar to those collected in South Island and attributed by Tumanov (2004) to Macrobiotus cf. coronatus . As a consequence, Pilato et al. (2006) also identified the specimens exactly as Macrobiotus cf. coronatus . Today we suspect that the specimens and egg examined in 2006 might belong to Mesobiotus helenae sp. nov. Further analysis of specimens identified as “ Macrobiotus harmsworthi coronatus ” in the Horning et al. (1978) collection, Museum of New Zealand, Te Papa Tongareva, would be required for confirmation. If this hypothesis is confirmed, Mesobiotus helenae sp. nov. would be present on both the North Island and South Island of New Zealand.

The new species is very similar to Mesobiotus pseudoblocki recorded in southern Argentina, south of the Rio Negro (Nahuel Huapi National Park), i.e. in an area with a biogeographic affinity with the Antarctica, and Mesobiotus blocki reported from Enderby Land, Continental Antarctica. This confirms that a careful study of the tardigrade species may also be significant in discussing biogeographic problems.