Thulinius augusti (Murray, 1907)

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), Zootaxa 3764 (5), pp. 524-536 : 528-533

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, 2D). 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, 3C), 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 the18S rDNA gene for three GSMNP specimens (slides C3180 V1, V2, V3; Table 3 ; voucher images in Fig. 4 ) from Cataloochee Creek (825 m asl, N 35° 37.881; 83° W 05.294), GenBank accession numbers KF360230 - 2, and a portion of cox1 gene for one specimen of them V3 ( KF360229 ; 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 18S rDNA sequences, T. augusti clearly differs from T. stephaniae by a K2P distance of 2.7–3.0% (p-distance: 2.7–2.9%) ( Table 3 ). A specimen of T. augusti showed a cox1 haplotype that differs from that of T. stephaniae by a K2P distance of 31.9% (p-distance: 25.9%) and from that of Thulinius sp. by a K2P distance of 28.2% (p-distance: 23.4%). T. stephaniae and Thulinius sp. differ by a K2P distance 28.0% (pdistance: 23.2%) ( Table 4 ). These results for cox1 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 µm 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 µm 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 µm 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.

Figures

FIGURE 2. Thulinius augusti. A. Mouth with sub-lobes (arrow). B. Claws II. C. Claws II, cuticular bars under claws (arrows). D. Claws III, cuticular bars under claws (arrow). (PhC; bars = 10 µm)

FIGURE 3. Thulinius augusti: hologenophores in vivo; A. C 3180 V 1: placoids and bulb apophysis. B. C 3180 V 3: buccalpharyngeal apparatus. C. C 3180 V 2: claws II, cuticular bars under claws (arrows). D. C 3180 V 3 claws IV, with lunules (arrow). E. C 3180 V 3: peribuccal sub-lobes (arrow). (DIC; bars = 10 µm)

FIGURE 4. Thulinius romanoi sp. nov. A. Habitus of holotype. B. Buccal-pharyngeal apparatus. C. Cuticular sculpturing. (PhC; bars: A = 50 µm, B-C = 20 µm)

FIGURE 5. Thulinius romanoi sp. nov. A. Claws II. B. Claws III, cuticular bars under claws (arrows). C. Claws IV, cuticular bars are absent. (PhC; bars = 10 µm).

Tables

TABLE 3. Genetic distances for the rDNA 18 S gene computed among specimens of the Thulinius genus (below the diagonal, Kimura 2 - parameters distance; above the diagonal, p-distance).

		1	2		3	4		5		6		7		8
1	AF056023 Thulinius stephaniae			0.002	0.005		0.002		0.005		0.027		0.027		0.027
2	GQ925698 Thulinius stephaniae clone 3	0.002			0.005		0.000		0.005		0.027		0.027		0.027
3	GQ925699 Thulinius stephaniae clone 4	0.005		0.005			0.005		0.000		0.029		0.029		0.029
4	GQ925700 Thulinius stephaniae clone 7	0.002		0.000	0.005				0.005		0.027		0.027		0.027
5	GQ925701 Thulinius stephaniae clone 8	0.005		0.005	0.000		0.005				0.029		0.029		0.029
6	MOD: BRT: C3180 V1	0.027		0.027	0.030		0.027		0.030				0.000		0.000
7	MOD: BRT: C3180 V2	0.027		0.027	0.030		0.027		0.030		0.000				0.000
8	MOD: BRT: C3180 V3	0.027		0.027	0.030		0.027		0.030		0.000		0.000

TABLE 4. Genetic distances for the cox 1 gene computed among specimens of the Thulinius genus (below the diagonal, Kimura 2 - parameters distance; above the diagonal, p-distance).

	1	2	3
1 EF620417 Thulinius stephaniae Ts- Sinai-1		0.232	0.259
2 HM600784 Thulinius sp. DVL-2010	0.280		0.234
3 MOD: BRT: C3180 V3	0.319	0.282
Thulinius romanoi sp. nov.