Four new species of the Diphascon nobilei group (Eutardigrada, Hypsibiidae)

The existence of a group of species similar to Diphascon (Diphascon) nobilei is shown. These species have some common characteristics: a well‐evident drop‐shaped thickening between the buccal tube and the pharyngeal tube, pharyngeal bulb, more or less elongate, with three rod‐shaped macroplacoids and microplacoid, claws of the hind legs different from those of the first three pairs of legs in having a very wide basal portion and indented basal margin, basal spurs also present on the external claws and, in some cases, also on the internal claws of the first three pairs of legs. Four new species of this group are described: Diphascon (D.) serratum, D. (D.) nelsonae, D. (D.) platyungue, and D. (D.) hydrophilum. They differ from one another in the dimensions and shape of the claws; in some cases the difference also affects the value of the ptd index relative to the insertion point of the stylet supports, the buccal and the pharyngeal tube length or the macroplacoid length.


Introduction
Diphascon (D.) nobilei (Binda, 1969) was described for Sicilian specimens (locus typicus: Gela) and has been reported for some other Italian localities (Binda and Pilato 1971;Bertolani et al. 1977Bertolani et al. , 1987Bertolani et al. , 1995Bertolani 1982Bertolani , 1984Bertolani , 1988Bertolani , 2002Maucci 1986;Pilato et al. 1989;Bertolani and Rebecchi 1996) and for Turkey (Maucci 1975), Australia (Pilato and D'Urso 1976), Spain (Maucci and Durante Pasa 1984), Poland (Dastych 1988), and Russia (Biserov 1991). We noted that Binda (1969), in the description of the species, wrote that eyes are present, but that specimens from other localities lack them (Bertolani et al. 1977;Dastych 1988). Some authors cited the species but did not specify whether eyes were present or not. To date this difference has been attributed to individual variability; to verify this hypothesis, we examined specimens from many localities. We compared these specimens with the typical material and we noted that all these specimens were characterized in having three macroplacoids and microplacoid, claws of the hind legs different from those of the first three pairs of legs in having a very wide basal portion (particularly the posterior claws) with clearly indented basal margin, whereas they differ in many characters from the typical material. We concluded that it is justified to describe here four new species: Diphascon (D.) serratum, D. (D.) nelsonae, D. (D.) platyungue, and D. (D). hydrophilum.

Materials and methods
Besides the holotype of D. nobilei (found in a moss sample developing on sand dunes (Gela), we examined specimens from some other Italian localities and from various habitats: from mosses collected in two localities on Mt Etna: Pietra Cannone (Fornazzo) and Serra La Nave, and in two Aeolian Islands (Lipari and Panarea); from sediment of a puddle (Nebrodi Mts: Contrada Cutò), from mosses on sand dunes in pine-wood (Latium: Latina: Sabaudia); from chestnut wood leaves (Emilia: Modena: Civago); from a meadow (Marche: Sibillini Mounts: Mt Vettore: shelter Zilioli (2150-2250 m); from sediments of the Fossa stream (Emilia: Modena: Rocca Santa Maria); from sediments of the cave Grotta del Pettirosso (Trieste, Aurisina).
The holotype and some paratypes of all the new species, mounted in polyvinyl lactophenol, are deposited in the collection of Binda and Pilato (Dipartimento di Biologia Animale ''Marcello La Greca'' dell'Università di Catania); some paratypes are deposited in the collection of Roberto Bertolani (Dipartimento di Biologia Animale dell'Università di Modena e Reggio).
As stated by Pilato and Binda (1997/98), in the study of the species of the subgenus Diphascon, it is useful to use the index ptd, that is the percentage ratio between the length of a structure and the length of the buccal tube, measured from the anterior margin of the stylet sheaths to the end of the drop-shaped thickening present between the buccal tube and the pharyngeal tube. As regards the buccal tube, besides its length in mm, we used the pbf index, that is the percentage ratio between its length (measured as mentioned above) and the total length of the bucco-pharyngeal tube (pharyngeal apophyses excluded).

Results
As with D. (D.) nobilei, all the new species considered have the typical characters of the genus Diphascon and the subgenus Diphascon, i.e. claws of Hypsibius type, buccopharyngeal apparatus of Diphascon type, buccal tube without ventral lamina and with apophyses for the insertion of the stylet muscles in the shape of semilunar hook symmetrical with respect to the frontal plane, pharyngeal tube with a spiral thickening, evident dropshaped thickening between the buccal tube and the pharyngeal tube, peribuccal lamellae and peribuccal papulae absent. As mentioned above, they are also characterized in having three macroplacoids and microplacoid, claws of the hind legs different from those of the first three pairs of legs in having a very wide basal portion (particularly the posterior claws) with clearly indented basal margin. Diphascon (Diphascon) nobilei (Binda, 1969) ( Figure 1; Table I)
The description of the species is confirmed but we noted the presence of granules of pigment also in the position of the eyes. We suspect that in the description of Binda (1969) these granules were mistaken for eyes. The holotype is mounted in polyvinyl lactophenol and therefore it is not possible to solve this problem, it is therefore necessary to find other specimens.
In the description, Binda (1969) wrote that one accessory point is present on the main branches of the claws; we noted that the orientation of the claws makes it difficult to see the accessory points, but on one claw two accessory points seem to be present. The claws have wide branches and, as a consequence, they appear stout ( Figure 1B, C); on the hind legs the basal portion of the claws is wide with well-visible basal spines; smaller basal spines are present on the claws (both external and internal) of the first three pairs of legs ( Figure 1B).
In Figure 1A the bucco-pharyngeal apparatus is shown. In Table I the dimensions of some structures of the holotype are indicated.

Description of the holotype
Body length 217 mm, colourless, cuticle smooth without pearls; eyes seem to be absent. The specimens were mounted in polyvinyl lactophenol more than 30 years ago, and no specification about the presence of eyes is indicated on the label. a The pharyngeal tube is slightly stretched and therefore the buccal tube, as %, appears slightly shorter.
The bucco-pharyngeal apparatus is shown in Figure 2A. The bucco-pharyngeal tube, measured from the anterior margin of the stylet sheaths to the base of the pharyngeal apophyses, is 49.9 mm in length; the buccal tube, measured from the anterior margin of the stylet sheaths to the end of the drop-shaped thickening is 21.6 mm in length (pbf543.3) and 2.5 mm wide (ptd511.6). The stylet supports are inserted on the buccal tube at 67.2% of its length (ptd567.2). The pharyngeal bulb has apophyses, three rod-shaped macroplacoids and microplacoid; the septulum is absent. The entire placoid row is 18.5 mm in length (ptd585.6) including the microplacoid, 16.5 mm (ptd576.4) excluding it; the first macroplacoid is 4.9 mm long (ptd522.7), the second 4.6 mm (ptd521.3), the third 6.9 mm (ptd531.9), the microplacoid 1.8 mm (ptd58.3).
The claws ( Figure 2B, C) have wide branches and as a consequence they appear stout. The claws of the hind legs (particularly the posterior claws) have an enlarged basal portion medially and laterally prolonged in a cuticular thickening and basal spines ( Figure 2C). Basal spines are also present on the external claws of the first three pairs of legs (the basal margin of the internal claws is smooth). Well-developed accessory points are present on the main branches of all claws. The internal claws are 6.4 mm long (ptd529.6) on the second and third pair of legs, the external claws of the same pairs of legs are 8.6 mm long (ptd539.8); on the hind legs the anterior claws are 7.3 mm long (ptd533.8) and the posterior claws 9.2 mm (ptd542.6). On the first three pairs of legs a cuticular bar is present near the base of the internal claws. Eggs not found. The paratype is similar to the holotype; in Table I the dimensions of some structures of the holotype and of the known paratype are indicated.

Etymology
The name serratum refers to the indentation of the basal margin of the claws.

Remarks
Diphascon serratum sp. n. differs from D. nobilei in having a longer bucco-pharyngeal tube with respect to the body length, stylet supports inserted on the buccal tube in a more caudal position (Table I), longer placoids both with respect to the body size and to the buccal tube length (Table I), shorter claws with less-evident basal spines (absent on the internal claws of the first three pairs of legs) (Figures 1, 2).

Material examined
Emilia: Modena: Civago: eight specimens: holotype (slide N. 4928) and paratypes in chestnut wood leaves; Aeolian Island: Lipari (one specimen in a moss) sample, Panarea (two specimens in a moss sample).

Description of the holotype
Body length 231 mm, colourless, eyes absent, cuticle smooth without pearls. Buccopharyngeal apparatus is shown in Figure 3A. The bucco-pharyngeal tube is 46.4 mm long; the rigid buccal tube 21 mm long (pbf545.2) and 2 mm wide (ptd59.5). The stylet supports are inserted on the bucal tube at 63.7% of its length (ptd563.7). The pharyngeal bulb (29 mm 6 18.6 mm) has small apophyses, three rod-shaped macroplacoids and microplacoid, the septulum is absent. The first macroplacoid is 4.3 mm long (ptd520.5), the second 4.0 mm (ptd519), the third 6.5 mm (ptd530.9), microplacoid 1.6 mm (ptd57.6); the entire placoid row 17.7 mm in length (ptd584.3) including the microplacoid, 15.5 mm (ptd573.8) excluding it. The claws ( Figure 3B, C) have accessory points on the main branches; the claws of the hind legs ( Figure 3C) have the basal portion enlarged with the basal margin indented and prolonged in one medial and one lateral thickening. The branches are normally developed and not particularly wide. On the first three pairs of legs ( Figure 3B) the external claws have a slightly developed basal indentation; the basal margin of the internal claws is smooth.
The internal claws are 7 mm long (ptd533.3) on the second and third pairs of legs, the external claws of the same pairs of legs are 10.5 mm long (ptd550); on the hind legs the anterior claws are 7.9 mm long (ptd537.6) and the posterior claws 11.5 mm (ptd554.8).
A cuticular bar is present near the internal claws on the first three pairs of legs. Two exuviae with three and four smooth eggs were found.
The paratypes are similar to the holotype. In Table I the dimensions of some structures of the smallest and of the largest measured specimens are indicated.

Etymology
The species is named nelsonae in honour of the tardigradologist, our dear friend, Diane Nelson (Department of Biological Sciences, East Tennessee University, Johnson City).

Remarks
Diphascon nelsonae sp. n. differs from D. nobilei in the following features: entire placoid row, and particularly the second and third macroplacoid, longer both with respect to the body length and to the buccal tube length (Table I), claws more slender with longer secondary branches, claw basal spines less evident (absent in the internal claws of the first three pairs of legs).
The new species differs from D. serratum in having the support of the stylet inserted on the buccal tube in a more cephalic position (Table I), claws longer (Table I) and more slender with longer secondary branches, anterior and posterior claws of the hind legs are more different in shape and size from one another (Table I; Figures 2, 3). Diphascon (D.) platyungue sp. n. (Figure 4; Table I)

Description of the holotype
Body length 194 mm, colourless, eyes absent, cuticle smooth without pearls. Buccopharyngeal apparatus shown in Figure 4A. The bucco-pharyngeal tube, measured as indicated in the description of D. serratum, is 45.2 mm long; the rigid buccal tube 20.8 mm long (pbf546) and 1.7 mm wide (ptd58.2). The stylet supports are inserted on the bucal tube at 62.9% of its length (ptd562.9). The pharyngeal bulb (27.4 mm 6 19 mm) has small apophyses, three rod-shaped macroplacoids and microplacoid, the septulum is absent. The entire placoid row is 16.1 mm in length (ptd577.4) including the microplacoid, 14.1 mm (ptd567.8) excluding it. The first macroplacoid is 3.8 mm in length (ptd518.3), the second 3.6 mm (ptd517.3), the third 5.7 mm (ptd527.4), microplacoid 1.7 mm (ptd58.2). The claws ( Figure 4B-D) have accessory points on the main branches. The claws of the hind legs ( Figure 4C), particularly of the posterior claws, have the basal portion extremely enlarged; their branches are short; the indented basal margin of both internal and external claws appear prolonged in one medial and one lateral thickening. On the first three pairs of legs ( Figure 4B, D) the external claws have an indented basal margin, the internal claws have a smooth basal margin. The internal claws are 6.1 mm long (ptd529.3) on the second and third pairs of legs, the external claws of the same pairs of legs are not measurable due to their unfavourable orientation (in other specimens the value of the ptd index relative to these claws is 36-39.3); on the hind legs the anterior claws are 6.5 mm long (ptd531.2) and the posterior claws 8.5 mm long (ptd540.9). A cuticular bar is present near the internal claws on the first three pairs of legs.
Eggs not found. The paratypes are similar to the holotype. In Table I the dimensions of some structures of the smallest and of the largest measured specimens are indicated.

Etymology
The name platyungue refers to the shape of the basal portion of the hind legs.

Remarks
Diphascon platyungue sp. n. differs from D. nobilei in having claws shorter with respect to the buccal tube length (Table I), claws of the hind legs with a wider basal portion, internal claws on the first three pairs of legs without basal spines.
The new species differs from D. serratum in having the stylet supports inserted on the buccal tube in a more cephalic position (Table I), narrower buccal tube, shorter placoids (Table I), claws of the hind legs with a wider basal portion.
The new species differs from D. nelsonae in the following features: slightly longer buccal tube and, as a consequence, lower values of the ptd index relative to placoid row and to the claw length (Table I); claws shorter and less slender with shorter secondary branches, claws of the hind legs with a clearly larger basal portion (Figures 3, 4).

Material examined
Modena: Rocca Santa Maria: one specimen in sediments of the Torrente Fossa (holotype, slide N. 4927); Modena: Levizzano: three specimens in sediments of the Guerro stream; Trieste: Aurisina: seven specimens in sediments of the cave Grotta del Pettirosso.

Description of the holotype
Body length 307 mm, colourless, eyes absent, cuticle smooth without pearls. Buccopharyngeal apparatus is shown in Figure 5A. The bucco-pharyngeal tube is 49.4 mm long; the rigid buccal tube 23.3 mm long (pbf547.2) and 2.2 mm wide (ptd59.4). The stylet supports are inserted on the bucal tube at 63.1% of its length (ptd563.1). The pharyngeal bulb (32.7 mm 6 24 mm) has small apophyses, three rod-shaped macroplacoids and microplacoid, the septulum is absent. The first macroplacoid is 4.9 mm long (ptd521.0), the second 4.6 mm (ptd519.7), the third 5.8 mm (ptd524.9), microplacoid 1.7 mm (ptd57.3); the entire placoid row 18.1 mm long (ptd577.7) including the microplacoid, 15.8 mm (ptd567.8) excluding it. The claws ( Figure 5B-E) with accessory points on the main branches; the external claws are long and slender. The claws of the hind legs ( Figure 5E) have the basal portion enlarged with the basal margin indented and prolonged in one medial and one lateral thickening. The external claws on the first three pairs of legs ( Figure 5C, D) have an indented basal margin. They are 16.1 mm in length (ptd569.1) on the second and third pair of legs; the internal claws are not measurable due their unfavourable orientation; we measured them in a specimen having the buccal tube 24.5 mm long where they are 9 mm long (ptd536.7); on the hind legs the anterior claws are 10.4 mm in length (ptd544.6) and the posterior claws 15.8 mm (ptd567.8). A cuticular bar is present near the internal claws on the first three pairs of legs. Eggs not found.
The paratypes are similar to the holotype. In Table I the dimensions of some structures of the smallest and of the largest measured specimens are indicated. The name hydrophilum refers to the fact that the species was found in water.

Remarks
Diphascon hydrophilum sp. n. differs from D. nobilei in the following features: stylet supports inserted on the buccal tube in a more caudal position (Table I); slightly higher value of the pbf index relative to the buccal tube length (Table I); lower value of the ptd index relative to the placoid row length as a consequence of the shortness of the third macroplacoid (Table  I); longer and slender claws (Figures 1, 5; Table I); the internal claws on the first three pairs of legs without basal spines.
The new species differs from D. serratum in having a slightly higher value of the pbf index relative to the buccal tube length (Table I); stylet supports inserted on the buccal tube in a more cephalic position (Table I), shorter third macroplacoid, lower value of the ptd index relative to the placoid row length (Table I), longer and more slender claws with longer secondary branches (Figures 2, 5).
The new species differs from D. nelsonae in having a slightly higher value of the pbf index relative to the buccal tube length (Table I), lower value of the ptd index relative to the placoid row length as a consequence of the shortness of the third macroplacoid (Table I), longer and more slender claws (Figures 3, 5; Table I).
The new species differs from D. platyungue in having a slightly shorter bucco-pharyngeal tube (both the buccal tube and the pharyngeal tube are shorter in relation to the body length) (Table I), shorter third macroplacoid, claws very different in size and shape (the claws of D. hydrophilum are longer and more slender, and those of the fourth pair of legs have the basal portion less enlarged) (Table I; Figures 4, 5).

Conclusions
It seems necessary to check the specific diagnosis regarding specimens lacking eyes that in the past were attributed to Diphascon nobilei, both from Italy and other geographic areas. The Italian fauna has been enriched by four species, one of which is found in freshwater.
As a consequence of our statements one can conclude that a group nobilei can be recognized within the genus Diphascon, subgenus Diphascon. To this group five species can be surely attributed to date: Diphascon nobilei, D. serratum sp. n., D. nelsonae sp. n., D. platyungue sp. n., and D. hydrophilum sp. n. Some other species of the subfamily Itaquasconinae are similar to them as regards the shape of the claws of the hind legs and the marginal indentation of these claws: D. (D.) birklehofi Schuster, 1999, D. (Adropion) higginsi (Binda, 1971), D. (Adropion) greveni Dastych, 1984, Platicrista angustata (Murray, 1905 (Pilato 1973); others have the claws of the hind legs (particularly the posterior claws) with an enlarged base but with a smooth basal margin, e.g. D. (Adropion) scoticum Murray, 1905 (Pilato 1974(Pilato , 1975, or have the basal portion of that claw not enlarged but with an indented margin, e.g. D. (Adropion) mirabile Dastych, 1984. But all these species differ due to relevant characters from those of the nobilei group: D. ) higginsi a very small, flat thickening is present, very difficult to see; P. angustata has apophyses for the insertion of the stylet muscles in the shape of flat ridge stylet furcae. Due to these differences D. higginsi, D. greveni, D. mirabilei, and D. scoticum are attributed to a subgenus (Adropion) different from that (Diphascon) to which the nobilei group is ascribed, and P. angustata is attributed to a different genus, and we consider this systematic arrangement justified. In conclusion, the presence of hind claws with an enlarged basal portion, together with their basal indentation, can be noted in species of different subgenera or also of different genera, and therefore we think that this particular shape of the hind claws in some cases can be due to evolutionary convergence. Diphascon (D.) birklehofi differs from the species of the nobilei group only in the presence of the septulum; the presence or absence of the septulum is a variable character within the genus, but we think it opportune to speak of ''group of species'' only when some species appear very similar to each other and differ only in some details. Based on this criterion, we think it opportune not to consider D. birklehofi as certainly belonging to the nobilei group.