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Macrobiotus azzunae sp. nov. urn:lsid:zoobank.org:act: 933CCC06-F69D-49E2-AF4F-0C042D8F5C99 Figs 1–4, 5A, C, 7 Etymology The new species is dedicated in honor of Atf Azzouna, professor in the Faculty of Mathematical, Physical and Natural Sciences of Tunis and supervisor of the PhD thesis of Jamila Ben Marnissi. Type material Holotype TUNISIA • spec. of unidentified sex; North-West Tunisia, Kroumiri Mountains, Ain Soltan forest, Jendouba; 36º31′21.788″ N, 8º19′57.741″ E; 893 m a.s.l.; Apr. 2017; Marnissi leg.; moss on trunk of Quercus canariensis; UNIMORE, slide code C4218–S32. Paratypes TUNISIA • 17 specs, sex unidentified; same collection data as for holotype; UNIMORE, slide codes C4218–S2 to C4218–S7, C4218–S9, C4218–S17, C4218–S30, C4218–S31, C4218–S33 to C4218– S35 • 3 eggs; same collection data as for holotype; UNIMORE, slide codes C4218–S10, C4218–S11, C4218–S25. Type depositories The holotype (slide: C 4218– S 32), 17 paratypes (slides C 4218– S 2 to C 4218– S 7, C 4218– S 17, C 4128– S 30, C 4218– S 31, C 4218– S 33 to C 4218- S 35), 3 eggs (slides: C 4218– S 10/11/25) and two vouchers (slides SP 04 and SP 07, corresponding to specimens C 4218 V 4 and C 4218 V 7, respectively) mounted in Faure-Berlese fluid, are deposited in the Bertolani collection at the Department of Life Science, UNIMORE, Modena, Italy. Type locality NW Tunisia, Kroumrie mountains, Ain Soltan forest, Jendouba, 36º31′21.788″ N, 8º19′57.741″ E. Altitude 893 m a.s.l. Description Adult specimens Body white, transparent after mounting in Faure-Berlese, from 162.2 to 410.3 µm in length (Fig. 2A, Table 3; structures measured only in the animals more than 200 µm in length). Eye spots present, even after mounting. Cuticle smooth but with small round or oval pores, 1–1.5 µm in diameter (Fig. 2B), better visible after fixation in Carnoy and orcein staining (Fig. 3C), scattered randomly on the entire cuticle, including the dorsal surface of all legs. With SEM, pores look oval or in the shape of a seed (Fig. 3A, D) with the largest diameter of 0.7–0.8 µm. Weak cuticular granulation also present on the lateral surface of all legs and specially on legs IV (Fig. 2B, arrow). Only with SEM is it possible to define the shape of the granulation on the legs, which looks as a regular disposition of star-shaped protuberances (about 0.3 µm; Fig. 3F). Six buccal sensory lobes around the mouth, well recognizable with SEM. Mouth antero-ventral; buccal-pharyngeal apparatus of the Macrobiotus type (sensu Pilato & Binda 2010), with ventral lamina and ten small peribuccal lamellae (in the holotype, after mounting, separated from the mouth). Buccal armature, corresponding to oral cavity armature, OCA, according to Michalczyk & Kaczmarek (2003), without an anterior band of teeth visible, corresponding to the first band of teeth according to Michalczyk & Kaczmarek (2003), and to the anterior band of the buccal ring according to Guidetti et al. (2012); posterior band of teeth poorly visible, corresponding to second band of teeth, according to Michalczyk & Kaczmarek (2003), followed by three dorsal and three ventral crests, corresponding to third band of teeth according to Michalczyk & Kaczmarek (2003); the dorsal crests (Fig. 2D) are distinct transverse ridges, whereas the ventral crests (Fig. 2E) appear as two separate lateral transverse ridges and a roundish median tooth. The posterior band of teeth and the transverse ridges are part of the buccal tube, according to Guidetti et al. (2012). Buccal tube narrow; pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids, relatively short, the first longer than the second and evidently but not deeply narrowed at its middle (Fig. 2C), the second with a not particularly evident subterminal constriction. Microplacoid present. Slender claws of the hufelandi type (sensu Pilato & Binda 2010); the external claw longer than the internal one and the posterior longer than the anterior. Primary branches of each claw with distinct accessory points (Fig. 2F), a common tract of medium length (about a third of the total claw length) and an evident stalk connecting the claw to the lunule. Lunules under all claws, smooth, larger on the hind legs (Figs 2G, 3E). Cuticular bars under claws absent. The population is dioecious (gonochoristic). Males were recognized using orcein staining, which revealed that the testis is filled with spermatozoa with a coiled head (Fig. 3G) and spermatids. No morphological secondary sexual dimorphism, such as gibbosities on legs IV in males, was identified. Eggs Eggs are laid freely, and are white, spherical or slightly oval. One egg containing a fully developed embryo showed the shape of the buccal-pharyngeal apparatus (Fig. 4A). Processes of the eggshell are in the shape of inverted goblets (Fig. 4B) with conical trunks and well-defined distal discs as large as the process bases (for measurements see Table 4). Distal discs concave, with a median small protuberance and, using PhC, with border often smooth, or sometimes slightly jagged, or slightly ragged (Fig. 4C), but never clearly jagged, serrated or dentate. Surface among processes of the hufelandi type (sensu Kaczmarek & Michalczyk 2017a), i.e., covered by a very thin grid (Fig. 4D). Meshes around the process bases slightly larger and with slightly thicker wires compared with interbasal meshes. Mesh diameter around 0.5 µm. Comparisons Macrobiotus azzunae sp. nov. has eggs with processes as inverted goblets and a reticulate eggshell between the processes. Consequently, a comparison must be done with the Macrobiotus species listed by Kaczmarek & Michalczyk (2017a) with hufelandi type eggshells, excluding the species with processes that are not like inverted goblets, and adding the species with hufelandi type chorion eggs described after that publication. The species with hufelandi type chorion eggs that do not have processes as inverted goblets are Macrobiotus acadianus (Meyer & Domingue, 2011), M. dariae Pilato & Bertolani, 2004, M. lissostomus Durante Pasa & Maucci, 1979, M. santoroi Pilato & D’Urso, 1976, and M. scoticus Stec, Morek, Gąsiorek, Blagden & Michalczyk, 2017. Moreover, M. azzunae sp. nov. has egg processes with distal discs with a smooth or slightly jagged border, therefore it differs from all the species that have clearly indented, serrated or clearly jagged distal discs, such as: Macrobiotus canaricus Stec, Krzywański & Michalczyk, 2018, M. crustulus Stec, Dudziak & Michalczyk, 2020, M. hannae Nowak & Stec, 2018 (whose egg surface is more cribriform than reticulate), M. hibiscus de Barros, 1942, M. horningi Kaczmarek & Michalczyk, 2017b (which also has very high processes), M. hufelandi C.A.S. Schultze, 1834, M. humilis Binda & Pilato, 2001, M. iharosi Pilato, Binda & Catanzaro 1991, M. joannae Pilato & Binda, 1983, M. julianae (Meyer, 2012), M. kamilae Coughlan & Stec, 2019, M. modestus Pilato & Lisi, 2009, M. noonragi s Coughlan & Stec, 2019, M. papei Stec, Kristensen & Michalczyk, 2018 (with particularly long filaments starting from the distal disc), M. paulinae Stec, Smolak, Kaczmarek & Michalczyk, 2015, M. polypiformis Roszkowska, Ostrowska, Stec, Janko & Kaczmarek, 2017 (even with cog-teeth extended to form a long, thin, hair-like and flexible filament), M. punctillus Pilato, Binda & Azzaro, 1990, M. sapiens Binda & Pilato, 1984, M. sottilei Pilato, Kiosya, Lisi & Sabella, 2012. For the shape of the egg Macrobiotus azzunae sp. nov. differs from M. rawsoni Horning, Schuster & Grigarick, 1978 because this species has only one strip of meshes around each egg process (see Kaczmarek & Michalczyk 2017b); it differs from M. serratus Bertolani, Guidi & Rebecchi, 1996 because in this species the egg surface is porous more than reticulated, with pores small and spaced from each other, and its egg processes have a large, often square, distal disc; it differs from M. seychellensis Biserov, 1994 because the distal disc of the egg processes of this species, even though not dentate, has long and very developed lobes. The remaining nine species of the hufelandi group should be compared singularly. Macrobiotus almadai Fontoura, Pilato & Lisi, 2008 Macrobiotus azzunae sp. nov. differs from M. almadai in having a posterior band of teeth in the buccal cavity visible with LM (not visible in M. almadai), and distal disc with a jagged margin instead of very small teeth as in M. almadai. Macrobiotus canaricus Stec, Krzywański & Michalczyk, 2018 With LM the margin of the distal disc of M. azzunae sp. nov., never dentate in this species, looks similar to that of M. canaricus, but the SEM images of the eggs of the latter species evidence the presence of an almost dentate disc. Moreover, the peribasal meshes of the eggshell are larger than interbasal ones in the new species while they do not differ from the interbasal ones in M. canaricus; regarding the animals there are differences in the buccal armature: in M. azzunae sp. nov. the posterior band of teeth is visible with LM (even though poorly) and the three dorsal crests are distinct transverse ridges, while in M. canaricus the posterior band of teeth is visible only with SEM and with LM the dorsal teeth form a transversal ridge weakly divided into three teeth. Macrobiotus madegassus Maucci, 1993 The new species differs from M. madegassus by the presence of the eye spots (absent in M. madegassus), pores on the cuticle (absent in M. madegassus), presence in the buccal armature of posterior band of teeth, even though weak (fully absent in M. madegassus), buccal tube much larger (pt of the holotypes 15.9 vs 7), insertion of the stylet supports on the buccal tube much more posterior (pt of the holotypes 76.1 vs 68), first and second macroplacoid longer (pt of the holotypes 25.5 and 18.1 vs 21.3 and 12.0), lunules on the hind legs without kerning (crenate in M. madegassus), eggshell processes with distal disc as large as the base (similar range 3.2–5.2 for both measurements) with respect to that of M. madegassus (disc vs base: 4.3–5.4 vs 2.3–2.6). Macrobiotus martini Bartels , Pilato, Lisi & Nelson, 2009 The cuticular pores in M. azzunae sp. nov. are much smaller than those of M. martini; the distal disc of the egg processes in M. azzunae sp. nov. has a diameter similar to that of the process base, while in M. martini the distal disc is much narrower than the base. Macrobiotus nebrodensis Pilato, Sabella, D’Urso & Lisi, 2017 Macrobiotus azzunae sp. nov. differs from M. nebrodensis by the absence of the cuticular bar near the lunules on the first three pairs of legs (a faint bar is present in M. nebrodensis). The egg processes of M. azzuane sp. nov. are in higher number on the circumference (29–33) with respect to those of M. nebrodensis (18). In the latter species there are some egg processes very high (up to 20.6 µm), while in the new species process height and shape are more uniform. The difference in the eggshell between meshes around the process base and the others is much less evident in M. azzunae sp. nov. than in M. nebrodensis. Macrobiotus personatus Biserov, 1990 The new species differs from M. personatus by the posterior band of the buccal armature less evident, the presence of a clear constriction in the first macroplacoid (Fig. 5A), in the paratype of M. personatus examined by us barely identifiable (Fig. 6A) and, according to Biserov (1990) usually absent in the type material of that species. The pores on the cuticle of M. azzunae sp. nov. are small, approximately 1 µm in diameter, while in M. personatus they are strongly elliptic and about 3 µm in length (Fig. 6B). Lunules on leg IV are always smooth in M. azzunae sp. nov., sometimes indented in M personatus. With respect to the eggs of M. personatus (Fig. 6C–D), the egg processes of M. azzunae sp. nov. (Figs 4C–D, 5C) are clearly shorter, 5.4 ± 0.6 vs 9.5 ± 0.5 (range 4.2–6.4 vs 9–10.5) and with a narrower base and distal disc (both 3.2–5.2 vs 7–10.5 and 7–9 respectively). Males are present in the new species, while in M. personatus only females were found (Biserov 1990), suggesting parthenogenesis in that species. Macrobiotus sandrae Bertolani & Rebecchi, 1993 The new species differs from M. sandrae for the eggshell shape, with thinner wires of the reticulum and meshes around the processes larger than the inter-process meshes in M. azzunae sp. nov. (Fig. 5C), all meshes similar in size in M. sandrae (Fig. 5D). Figure 5C–D also show a difference in the process base diameter, narrower in M. azzunae sp. nov. With regard to the animals, M. azzunae sp. nov. differs from M. sandrae by a constriction of the first macroplacoid much more pronounced (Fig. 5A; it is hardly visible in M. sandrae; Fig. 5B). Moreover, in animals of similar size the posterior band of the buccal armature is just less evident in the new species, and lunules on the hind legs are without hint of teeth (but teeth, present in the holotype of M. sandrae, are often difficult to identify in other specimens of that species). Macrobiotus terminalis Bertolani & Rebecchi, 1993 Macrobiotus azzunae sp. nov. differs from M. terminalis for the absence of granulation on the cuticle (noted only in the redescription of M. terminalis; see Cesari et al. 2011), for the absence of teeth on the lunules, especially evident on the hind legs of M. terminalis, and for the presence of males, absent in M. terminalis (see redescription by Cesari et al. 2011). Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011 With respect to M. vladimiri, animals of M. azzunae sp. nov. reach a shorter length (up to 410.3 µm vs 515.1 µm), in M. azzunae sp. nov. the posterior band of teeth of the buccal armature is less evident and the lunules on the hind legs are not indented. In M. azzunae sp. nov. the egg diameter without processes (64.7–80.6 µm) is less than that of the eggs of M. vladimiri (89.9–92.0 µm); the processes are shorter (4.2–6.4 µm in the new species vs 6.5–8 µm in M. vladimiri). In the new species the base process diameter is narrower (3.2–5.2 µm) than in M. vladimiri (5.1–7.3 µm), the distal disc is weakly or not jagged (clearly jagged in M. terminalis). In M. azzunae sp. nov. males are present, while they are absent in M. vladimiri. Genetic distances The ranges of uncorrected genetic p-distances between M. azzunae sp. nov. and the other species of the M. hufelandi group (Supp. file 7, Supp. file 8, Supp. file 9, Supp. file 10), are as follows: 18S 0.1–5.6%, with the most similar being M. sandrae from Germany (present paper) 28S 0.1%, with the only available M. vladimiri from Spain (FJ435751 –5) ITS-2 7.7–32.2%, with the most similar being Macrobiotus vladimiri (MN888347) from Finland COI 6.3–25.6%, with the most similar being Macrobiotus sandrae (HQ876574, HQ876577, HQ876578, HQ876579, HQ876581) from Germany The COI dataset is the most complete and informative for species delimitation investigation. Both phylogenetic reconstructions on the COI dataset resulted in the same topology, and thus the ML tree was utilized for the PTP analysis (Fig. 7, left), which shows 14 putative species clusters: M. crustulus, M.hannae, M. cf. recens, M.canaricus, M.hufelandi, M. cf. hufelandi sp.1, M. terminalis, M. cf. terminalis, M. wandae, M. macrocalix, M. cf. macrocalix, M. vladimiri, M. sandrae and M. azzunae sp. nov. This subdivision is further validated by both the ABGD and the haplotype network analysis (Fig. 7, centre and right). Present molecular data therefore confirms the validity of the erection of M. azzunae sp. nov., Published as part of Marnissi, Jamila Ben, Cesari, Michele, Rebecchi, Lorena & Bertolani, Roberto, 2021, Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. 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Macrobiotus Hufelandii animal e crustaceorum classe novum, reviviscendi post diuturnam asphyxia et ariditaten potens, etc. C. Curths, Berlin.","Pilato G., Binda M. G. & Catanzaro R. 1991. Remarks on some tardigrades of the African fauna with the description of three new species of Macrobiotus Schultze 1834. Tropical Zoology 4: 167 - 178. https: // doi. org / 10.1080 / 03946975.1991.10539487","Pilato G. & Lisi O. 2009. Tardigrades of the Seychelles Islands, with the description of three new species. Zootaxa 2124: 1 - 20. https: // doi. org / 10.5281 / zenodo. 188178","Pilato G., Binda M. G. & Azzaro M. 1990. Tardigradi di Terra del Fuoco e Magallanes. III. Macrobiotus punctillus, nuova specie di Macrobiotidae del gruppo hufelandi. Animalia 17: 123 - 129.","Pilato G., Kiosya Ye., Lisi O. & Sabella G. 2012. New records of Eutardigrada from Belarus with the description of three new species. Zootaxa 3179: 39 - 60. https: // doi. org / 10.11646 / zootaxa. 3179.1.2 Pilato G., Sabella G., D'Urso V. & Lisi O. 2017. Macrobiotus nebrodensis and Adropion vexatum, two new species of Eutardigrada (Tardigrada, Parachela) from Sicily. Zootaxa 4362 (2): 267 - 279. https: // doi. org / 10.11646 / zootaxa. 4362.2.6","Biserov V. I. 1990. On the revision of the Macrobiotus genus. The subgenus Macrobiotus s. str. is a new taxonomic status of the hufelandi group (Tardigrada, Macrobiotidae). Communication 2. Zoologicheskij Zhurnal 69: 38 - 50. [In Russian.]"]}

Pseudechiniscus is the second most species-rich genus in Heterotardigrada and in the family Echiniscidae. However, previous studies have pointed out polyphyly and heterogeneity in this taxon. The recent erection of the genus Acanthechiniscus was another step in making Pseudechiniscus monophyletic, but species identification is still problematic. The present investigation aims at clarifying biodiversity and taxonomy of Pseudechiniscus taxa, with a special focus on species pertaining to the so-called 'suillus-facettalis group', by using an integrated approach of morphological and molecular investigations. The analysis of sequences from specimens sampled in Europe and Asia confirms the monophyly of the genus Pseudechiniscus. Inside the genus, two main evolutionary lineages are recognizable: the P. novaezeelandiae lineage and the P. suillus-facettalis group lineage. Inside the P. suillusfacettalis group, COI molecular data points out a very high variability between sampled localities, but in some cases also among specimens sampled in the same locality (up to 33.3% p-distance). The integrated approach to the study of Pseudechiniscus allows confirmation of its monophyly and highlights the relationships in the taxon, pointing to its global distribution.

RICHTERSIIDAE FAM. NOV. Description: Double claws Y-shaped, with the two branches forming an evident common tract of variable length. Large teeth on all lunules. Buccal tube with ventral lamina and a cuticular thick on the anterior, dorsal wall of the buccal tube (which can form a large apophysis). Absence of transverse crests in the buccal armature. Two macroplacoids in the pharynx. Cuticular pores (at least in a phase of the life cycle). Eggs laid freely with cuticular processes on their surface. Type genus. Richtersius Composition. Richtersius, Diaforobiotus gen. nov., Adorybiotus (provisionally, see Remarks). Remarks. Based on molecular data, Richtersius, Diaforobiotus gen. nov., and Adorybiotus do not belong to Macrobiotidae, but the phylogenetic relationship of Adorybiotus with the other genera of the families Richtersiidae fam. nov. and Murrayidae needs to be clarified with further molecular data. Based on several morphological affinities with Richtersius and Diaforobiotus gen. nov. (i.e. a cuticular thickening on the anterior dorsal wall of the buccal tube, large teeth on all lunules, absence of transverse crests in the buccal armature, two macroplacoids in the pharynx, and presence of cuticular pores), we place Adorybiotus in this family., Published as part of Guidetti, Roberto, Rebecchi, Lorena, Bertolani, Roberto, Jonsson, Kjell Ingemar, Kristensen, Reinhardt M��bjerg & Cesari, Michele, 2016, Morphological and molecular analyses on Richtersius (Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea, pp. 834-845 in Zoological Journal of the Linnean Society 178 (4) on page 843, DOI: 10.1111/zoj.12428, http://zenodo.org/record/5369103

DIAFOROBIOTUS GEN. NOV. Description: Peribuccal lamellae (ten) and ventral lamina present. A dorsal thickening present in the anterior portion of the buccal tube, in conjunction with a large tooth on the internal surface of the tube. Some strong, scattered round teeth present posterior to the second band of teeth of the buccal armature present. Type species. Macrobiotus islandicus Richters, 1904. Etymology. Diaforobiotus, from diaforos (Greek) = different, different from all other macrobiotoid genera. Composition. Diaforobiotus islandicus (Richters, 1904)., Published as part of Guidetti, Roberto, Rebecchi, Lorena, Bertolani, Roberto, Jonsson, Kjell Ingemar, Kristensen, Reinhardt M��bjerg & Cesari, Michele, 2016, Morphological and molecular analyses on Richtersius (Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea, pp. 834-845 in Zoological Journal of the Linnean Society 178 (4) on page 843, DOI: 10.1111/zoj.12428, http://zenodo.org/record/5369103

Thulinius cf. saltursus Sampling localities: Five specimens from sediment and periphyton in Caldwell Fork, North Carolina (813 m asl, N 35 �� 37.5813, W 83 �� 5.3610) and four specimens in sediment from Cataloochee Creek, North Carolina (788 m asl, N 35 �� 38.2405, W 83 �� 4.9303). The specimens were compared with the type material of both Thulinius saltursus (Schuster, Toftner & Grigarick, 1978) (a species previously attributed to Isohypsibius in Schuster et al. 1978) and Thulinius ruffoi (Bertolani, 1981) and look similar to both species. The buccal armature is typical of the genus and similarly the macroplacoids are rod-shaped and there are no microplacoids. The basal portions on all claws are normally developed and slender. A lunule appears to be present only at the base of the external (posterior) claw (as in T. saltursus, whereas T. ruffoi has lunules on all claws), but it is not always possible to determine the presence or absence of a lunule. In any case, currently this is the only character that distinguishes the two species and for this reason we tentatively attribute our specimens from North Carolina to T. cf. saltursus., Published as part of Bertolani, Roberto, Bartels, Paul J., Guidetti, Roberto, Cesari, Michele & Nelson, Diane R., 2014, Aquatic tardigrades in the Great Smoky Mountains National Park, North Carolina and Tennessee, U. S. A., with the description of a new species of Thulinius (Tardigrada, Isohypsibiidae), pp. 524-536 in Zootaxa 3764 (5) on pages 527-528, DOI: 10.11646/zootaxa.3764.5.2, http://zenodo.org/record/230310, {"references":["Schuster, R. O., Toftner, E. C. & Grigarick, A. A. (1978) Tardigrada of Pope Beach, Lake Tahoe, California. The Wasmann Journal of Biology, 35, 115 - 136."]}

Thulinius augusti (Murray, 1907) Sampling localities. Palmer Creek, Caldwell Fork, Rough Fork, and Cataloochee Creek in North Carolina, and Crying Creek in Tennessee. Predominantly in sediment but rarely in periphyton, this is one of the most abundant and widespread aquatic species in our collection, second only to H. cf. dujardini. Remarks. The specimens collected look very similar to the type material. In particular, the peculiar crown of marked sub-lobes that surrounds the mouth is evident (Fig. 2 A). Another similarity is the long, thin basal tract of the claws, in particular the basal tract of the external claw (Figs 2 C, 2 D). In the type material the absence of lunules is cited, whereas sometimes lunules are visible in the American specimens (Fig. 3 D). A transverse bar exists under the claws of the first three pairs of legs, which is not in contact with the claws, but at least in some specimens it is clearly separated into two parts (Figs 2 C, 3 C), which is different from the type material that has only a single bar. The other characters (posterior band of teeth followed by a single row of large teeth, buccal tube relatively wide, long pharyngeal apophyses and rod-shaped macroplacoids without microplacoid) are common to all the species of the genus known to date. Thulinius augusti has been cited several times as Macrobiotus augusti, Hypsibius augusti, Isohypsibius augusti and Pseudobiotus augusti, and has been considered cosmopolitan (McInnes 1994), but in most cases these were misidentifications as documented by Bertolani et al. (1999). Therefore, the only definite citations of the species are the type locality (Scotland) and those here in the GSMNP. Apart from the presence of thin lunules present only in some fresh American specimens, the morphologies of the Scottish and American material correspond. We want to emphasize the difficulty in identifying these structures when they are thin and buried in the leg. We believe that molecular comparisons would be definitive. We did successfully sequence a portion of the 18 S rDNA gene for three GSMNP specimens (slides C 3180 V 1, V 2, V 3; Table 3; voucher images in Fig. 4) from Cataloochee Creek (825 m asl, N 35 �� 37.881; 83 �� W 05.294), GenBank accession numbers KF 360230 - 2, and a portion of cox 1 gene for one specimen of them V 3 (KF 360229; Table 4), but we were unable to find fresh material in the type locality for comparison. This molecular information would be very useful for establishing whether T. augusti has a widespread geographic distribution or is composed of cryptic species with more limited distribution. Our data allowed us to compare the molecular analysis of three species of Thulinius: T. augusti (found in this GSMNP study), T. stephaniae (found in two locations in Sinai, Egypt) and Thulinius sp. (found in an unidentified location) (Tables 3, 4). Regarding the 18 S rDNA sequences, T. augusti clearly differs from T. stephaniae by a K 2 P distance of 2.7 ���3.0% (p-distance: 2.7���2.9 %) (Table 3). A specimen of T. augusti showed a cox 1 haplotype that differs from that of T. stephaniae by a K 2 P distance of 31.9 % (p-distance: 25.9 %) and from that of Thulinius sp. by a K 2 P distance of 28.2 % (p-distance: 23.4 %). T. stephaniae and Thulinius sp. differ by a K 2 P distance 28.0% (pdistance: 23.2 %) (Table 4). These results for cox 1 distance values are in line with the values that separate different tardigrade species (Cesari et al. 2009; Bertolani et al. 2011; Cesari et al. 2011). Type locality. Stream sediment in Road Prong, Tennessee (145 m asl, N 35 �� 36.5113, W 83 �� 27.5637). One paratype was found in sediments from the same site at Road Prong, and another was found at Kephart Prong, North Carolina (1075 m asl, N 35 �� 36.6226, W 83 �� 22.0946). Type material. Holotype (slide number 23 Mar08- 1-4) and paratypes (slide numbers 23 Mar08- 1 -3, 9 May08- 11-16) are in the collection of Roberto Bertolani at the Department of Life Sciences, University of Modena and Reggio Emilia. Coll. Margaret Phillips, 23 -Mar-08 and 9 -May-08. Diagnosis. Large body size (328.7���542.3 ��m in length), body shape sometimes suggesting a fat piglet (Fig. 4 A). Cuticle sculptured on all surfaces (dorsally, ventrally and on all legs) with polygonal mesh (Fig. 4 C) forming an extended roughness where the cuticle is contracted. Eyes not observed in the mounted material. Peribuccal lamellae present. Lobes around the mouth not subdivided. Buccal armature with only the posterior band of small teeth followed by a line of large rounded teeth (Fig. 4 B). Large straight buccal tube. Large pharyngeal apophyses developed longitudinally; three long rod-shaped macroplacoids; no microplacoid. The curvatures of the placoid rows resemble a Grecian urn (Fig. 4 B). Small stylet furcae (Fig. 4 B). Large claws of Isohypsibius type (Figs 5 A��� C), the external claw more slender than the internal. Primary branch of the internal claw with a distinct dorsal knob. Accessory points on the primary branches present. Basal portions of all claws slender, wider in their distal portion in the internal claw (Figs 5 A���B). Lunules probably absent on all legs (Figs 5 A���C), or present only on the external or posterior claw but very reduced. Long cuticular bars, subdivided into two parts, present under the claws on the first three pairs of legs (Figs 5 A���B). Eggs unknown. Description of the holotype: (for measurements of the sclerified structures see Table 2). Adult 357.6 ��m in length. Cuticle sculptured, with polygonal mesh dorsally, ventrally and on all legs. Eyes not observed in the mounted specimen. Peribuccal lamellae present but the number was not detectable. Buccal armature composed of only the posterior band of small teeth, followed by a line of large rounded teeth (see also taxonomic remarks) (Fig. 4 B). Buccal tube with thin walls and with long stylet supports inserted at 62.9 % of its length. Pharynx apophyses 5.5 ��m in length. Three macroplacoids present, first is the longest, the second is the shortest. The distance between second and third macroplacoid larger than between the first and second macroplacoid. Microplacoid absent. Double-claws of Isohypsibius type. Primary branch of the internal claw with a distinct dorsal knob; proximal portion of the internal claw wider than the primary branch of the external claw. Accessory points on the primary branch of the external claw as long as the branch and turned downwards (Fig. 5 A), the accessory points of the primary branch of the internal claw shorter than the branch. Basal portions of all claws slender, with a septum dividing the primary branch from the rest of the claw. The base of the external claw with a drop-shaped cuticular internal expansion (lunule?), the base of internal claw expanded with lateral filaments. Two long cuticular bars, subdivided into two parts, present under the claws on each of the first three pairs of legs. Differential diagnosis. Thulinius romanoi sp. nov. differs from all other Thulinius species by the unique sculptured cuticle. It is similar to T. augusti and T. stephaniae by the absence of lunules, but the first species has a very thin claw basal tract (in addition to the peculiar presence of subdivided lobes), and the latter has a stronger basal tract and a very marked transverse bar at the base of the claws on the first three pairs of legs. In addition to the sculptured cuticle, T. romanoi sp. nov. differs from T. saltursus and all other Thulinius species by the absence, or not evident, lunule at the base of each external claw and by having a more robust basal tract. A key to the species of Thulinius, excluding T. romanoi sp. nov., can be found in Kaczmarek et al. (2010). Etymology. This species is named after the tardigradologist, and our friend and colleague, the late Frank Romano, whose enthusiasm for tardigrades was infectious. He and his students attended several of the International Symposia on Tardigrades during his tenure as Professor and Chairman of the Department of Biology at Jacksonville State University, Alabama, USA., Published as part of Bertolani, Roberto, Bartels, Paul J., Guidetti, Roberto, Cesari, Michele & Nelson, Diane R., 2014, Aquatic tardigrades in the Great Smoky Mountains National Park, North Carolina and Tennessee, U. S. A., with the description of a new species of Thulinius (Tardigrada, Isohypsibiidae), pp. 524-536 in Zootaxa 3764 (5) on pages 528-533, DOI: 10.11646/zootaxa.3764.5.2, http://zenodo.org/record/230310, {"references":["McInnes, S. 1994. Zoogeographic distribution of terrestrial / freshwater tardigrades from current literature. Journal of Natural History, 28, 257 - 352. http: // dx. doi. org / 10.1080 / 00222939400770131","Bertolani, R., Marley, N. J. & Nelson, D. R. (1999) Re-description of the genus Thulinia (Eutardigrada: Hypsibiidae) and of Thulinia augusti (Murray, 1907) comb. n. Zoologischer Anzeiger, 238, 139 - 145.","Cesari, M., Bertolani, R., Rebecchi, L. & Guidetti, R. (2009) DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Molecular Ecology Resources, 9, 699 - 706. http: // dx. doi. org / 10.1111 / j. 1755 - 0998.2009.02538. x","Kaczmarek, L, Bertolani, R. & Nedzynska-Stygar, M. (2010) Thulinius saltursus comb. nov.: a new systematic position for Isohypsibius saltursus Schuster, Toftner & Grigarick, 1978 (Eutardigrada: Hypsibiidae) and a key for the genus Thulinius. Zootaxa, 2483, 58 - 64."]}

As part of the All Taxa Biodiversity Inventory (http://www.dlia.org), an extensive survey of tardigrades has been conducted in the Great Smoky Mountains National Park (GSMNP) in Tennessee and North Carolina, U.S.A., by Bartels and Nelson. Freshwater tardigrades include three species in the aquatic genus Thulinius (Eutardigrada, Isohypsibiidae). A new species, Thulinius romanoi, described from stream sediment, is distinguished from all other congeners by having a sculptured cuticle. In addition, the presence of Thulinius augusti (Murray, 1907) was verified by combined morphological and molecular analysis, and nine specimens of a third species, Thulinius cf. saltursus, were also found. Thulinius augusti is a new record for the United States. Thulinius saltursus (Schuster, ToftnerGrigarick, 1978) was previously recorded in California and Ohio, but our specimens vary slightly in morphology. The list of tardigrades from streams in the GSMNP was updated to a total of 44 species, 22 of which were predominantly or exclusively aquatic.