A new species of Calohypsibius (Phylum Tardigrada, Eutardigrada) from Roan Mountain, Tennessee-North Carolina, U.S.A

Zoological Journal of the Linnean Society (1996), 116: 167–174. With 3 figures

Tardigrade biology. Edited by S. J. McInnes and D. B. Norman

A new species of Calohypsibius (Phylum Tardigrada, Eutardigrada) from Roan Mountain, Tennessee-North Carolina, U.S.A.

DIANE R. NELSON Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee 37614, U.S.A. AND KAREN L. McGLOTHLIN Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634, U.S.A. The ecological distributions of tardigrade species from six sites on Roan Mountain, Tennessee-North Carolina, were determined in a study begun by D. R. Nelson in 1971. Three new species of eutardigrades were discovered in Nelson’s study, but remained undescribed. A follow-up to that study was initiated by K.L. McGlothlin in 1988 to determine the long-term stability of tardigrade populations at two of Nelson’s original collection sites. Two of Nelson’s three new eutardigrade species were rediscovered by McGlothlin, and one new Pseudechiniscus species was found. The description of one of the eutardigrades, Hypsibius roanensis, was published previously. The remaining eutardigrade found during both studies, a new species of Calohypsibius, is described here: Calohypsibius schusteri sp. nov. ©1996 The Linnean Society of London

ADDITIONAL KEY WORDS: — Parachela – Calohypsibiidae – taxonomy – systematics – morphology – Calohypsibius schusteri – eutardigrade. CONTENTS Introduction . . . . . . . Material and methods . . . . Taxonomic account . . . . . Calohypsibius schusteri sp. Discussion . . . . . . . . References . . . . . . . .

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INTRODUCTION

Located in the Southern Appalachians on the border between Tennessee and North Carolina, Roan Mountain is the highest cross range between the Unaka 0024–4082/96/010167 + 08 $18.00/0

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Mountains and the Blue Ridge Mountains. Twenty-one species of tardigrades were recorded from epiphytic mosses on beech trees on Roan Mountain by Nelson (1973, 1975). McGlothlin (1990) recollected at two of Nelson’ six sites and found 18 of the same species and three rare species. A new species of Hypsibius found in both studies was described by Nelson & McGlothlin (1993). Two other new species were also present on Roan Mountain, a species of Pseudechiniscus found only by McGlothlin and a species of Calohypsibius reported in both studies. The Calohypsibius species is described here.

MATERIAL AND METHODS

Epiphytes were collected 08 September 1988 from four beech trees (Fagus grandifolia) at 1524 m and 1654 m, respectively, on the north-facing slope of Roan Mountain, Carter County, Tennessee. These were collection sites 5N and 6N in Nelson’s original study (1973, 1975). Fifty-six samples were taken: 27 from 5N and 29 from 6N and the new species was present in two of them. The mosses were soaked in tap water to activate the tardigrades. After the moss was removed, the top layer of water in the beaker was decanted, and the tardigrades were killed by the addition of boiling water. The contents of the beaker were rinsed successively through two sieves (No. 18, 1.0 mm; and No. 325, 0.045 mm). The material remaining on the fine-mesh sieve (No. 325) was washed into a labelled jar with 95% ethanol. Later, each sample was poured into a gridded petri dish and searched under a darkfield stereomicroscope at 25 3 . With an Irwin loop, tardigrades were transferred to a labelled vial containing 95% ethanol. Specimens were mounted in Hoyer’s mounting medium on separate microscope slides and later ringed with epoxy paint to seal the preparation. Identifications were made using a differential interference contrast (DIC)-phase contrast microscope with oil immersion. Measurements were made with a calibrated ocular micrometer, and photographs were taken with Kodak T-Max 100 film. Scanning electron micrographs of Calohypsibius from Nelson’s study (1973) were also used in preparing the description.

TAXONOMIC ACCOUNT

Class Eutardigrada Marcus 1927 Order Parachela Schuster, Nelson, Grigarick, & Christenberry 1980 Family Calohypsibiidae Pilato 1969 Genus Calohypsibius Thulin 1928 Calohypsibius schusteri sp. nov. Hypsibius (Hypsibius?) schusteri Nelson (manuscript name in a Ph.D. thesis) Hypsibius “b”: Nelson, 1975 (Figs 1–3) Description. Holotype (USNM # 264076). McGlothlin (1990): collection number 5N01E. Length 137 µm. Eye spots absent. Cuticle colourless, with distinct sculpture. Irregular, rhomboidal, knob-like tubercles cover the entire dorsal and lateral cuticle

ROAN MOUNTAIN CALOHYPSIBIUS

B

A

C

Figure 1. Calohypsibius schusteri sp. nov. A, lateral view, drawn from scanning electron micrograph. Scale bar = 10 µm. B, buccopharyngeal apparatus. Scale bar = 5 µm. C, claws on leg IV, drawn from scanning electron micrograph. Scale bar = 3 µm.

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and extend onto the ventral surface. Larger tubercles are in 10 transverse rows with smaller tubercles between rows and on all legs. Tops of tubercles appear star-shaped when viewed with phase microscopy. The height of the tubercles increases in size posteriorly, with caudal tubercles about 2.0–2.5 µm in height and about 2.0 µm in width at the top of the tubercle. Buccal tube narrow, 17.5 µm long by 1.0 µm wide.

Figure 2. Scanning electron micrographs of Calohypsibius schusteri sp. nov. A, lateral view. B, detail of cuticular sculpture between legs III and IV.

ROAN MOUNTAIN CALOHYPSIBIUS

Figure 3. Scanning electron micrographs of Calohypsibius schusteri sp. nov. A, peribuccal papulae surrounding mouth opening. B, ventral view, legs IV and cloacal opening.

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Wall of buccal tube thickened posterior to insertion of stylet supports. Distance from end of buccal tube to insertion of stylet supports 7.5 µm, approximately 45% of the length of buccal tube. Spherical pharyngeal bulb (16 µm 3 15 µm) with welldeveloped apophyses and two granular macroplacoids; placoids approximately equal in length, 1.5 µm. Length of placoid row from apophysis to end of second macroplacoid, 4.5 µm. Microplacoid and septulum absent. Two double claws on each leg Calohypsibius-type, with the primary branch of the external claw rigidly attached to the secondary branch; claw sequence 2-1-2-1. On legs I–III, distance from base of external claw to tip of primary branch about 3.5–4.0 µm. Claws on legs IV slightly larger, but not accurately measurable on holotype. Angle between primary and secondary branch approximately 44 degrees. Primary and secondary branches of external claws with widely separated tips, 4.5 µm apart. Collected from moss (Dicranum sp.) on beech tree (Fagus grandifolia), 08 September 1988, site 5N (1524 m, north-facing slope of Roan Mountain, Carter County, Tennessee). Paratypes. McGlothlin (1990): Collection number 5N04B. Length 145 µm. Sculpture on cuticle as in holotype. Simplex stage. External claw on leg IV 5 µm from base to tip of primary branch. Collected from moss (Dicranum sp.) on beech tree (Fagus grandifolia), 8 September 1988, site 5N (1524 m, north-facing slope of Roan Mountain). Nelson (1973): Collection number 6S08F. Nelson (1973, 1975) previously found the species in moss on beech trees at the same site (site 5N, collection number 5N06C) and at site 6S (1654 m) (collection numbers 6S07A and 6S08F) on the southfacing slope of Roan Mountain, Mitchell County, North Carolina. However, these eight paratypes were mounted in a non-permanent mounting medium (Turtox CMC-9), and 10–12 individuals of different species were mounted on each slide, making identification more difficult. One specimen from site 6S (collection number 6S08F) was mounted in Hoyer’s on a separate slide and is suitable as a paratype. All paratypes deposited in the Nelson collection, East Tennessee State University, Johnson City, Tennessee, U.S.A. Type locality. North-facing slope at 1524 m (station 5N, Nelson, 1973) on Roan Mountain, Carter County, Tennessee, U.S.A. Collected from moss (Dicranum sp.) from beech tree (Fagus grandifolia), 08 September 1988. Etymology. This species is named for the late Robert O. Schuster, University of California-Davis, for his pioneer work on North American tardigrades, and for his constant friendship, encouragement, and assistance.

DISCUSSION

The family Calohypsibiidae, erected by Pilato (1969), includes the type genus Calohypsibius Thulin 1928 as well as the genera Haplohexapodibius Pilato & Beasley 1987, Haplomacrobiotus May 1948, Hexapodibius Pilato 1969, and Parhexapodibius Pilato 1969 (Pilato, 1989). Calohypsibius is the only known genus of eutardigrades with six distinct peribuccal papulae, which are low profile peribuccal structures that are visible only with scanning electron microscopy (Fig. 3). Of the six papulae, four are located around the mouth opening and two are slightly larger and more dorsolateral in position. In contrast, the genus Minibiotus has ten papulae, all similar in size and shape (Schuster et al., 1980). The mouth ring of Ramazzottius has not been clearly

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viewed with scanning electron microscopy, and therefore the presence of peribuccal papulae is undetermined. Four species of Calohypsibius have been described: Calohypsibius caelatus Pilato, Claxton & Binda 1989, Calohypsibius ornatus (Richters 1900), Calohypsibius placophorus (Da Cunha, 1943), and Calohypsibius verrucosus (Richters 1900). Varieties of Calohypsibius ornatus, based on variation in the cuticular ornamentation, were discussed by Pilato, Claxton & Binda (1989). After examining specimens of Calohypsibius ornatus caelatus Marcus 1928, Calohypsibius ornatus carpaticus Bartos 1940, and the ‘typical’ form, they concluded that Calohypsibius caelatus (Marcus 1928) was a valid species. The revised description states that C. caelatus has two anterior elliptical sense organs and a thickened buccal tube wall posterior to the stylet supports (Pilato et al., 1989). Both characters are also present in the genus Ramazzottius (Binda & Pilato, 1986). In our specimens of C. schusteri, however, the elliptical sense organs are not readily discernible. Calohypsibius schusteri is easily separated from the other species in the genus by its colourless, distinctly sculptured cuticle. The ornamentation consists of irregular rhomboidal tubercles that appear star-shaped on the surface under phase microscopy. In C. ornatus, with many described varieties, cuticular sculpturing consists of transverse rows of spines alternating with small rounded papillae. Pilato et al. (1989) described C. caelatus with 12 bands of larger tubercles alternating with smaller ones. In contrast, C. placophorus has dorsal flattened plates of various sizes arranged symmetrically along the median line, interspersed with small papillae. The similar species C. verrucosus has numerous tubercles that may merge to form protruding dorsal transverse bands or small plates. Further analysis of the variability of these species is required. Calohypsibius schusteri can be separated from other eutardigrades with cuticular sculpturing by the structure of the claws. Although Calohypsibius shares some characters with the genus Ramazzottius (Binda & Pilato, 1986), the oberhaeuseri-type claw in Ramazzottius easily distinguishes the two genera. The claws of Isohypsibius and Hypsibius have a flexible junction between the primary and secondary branches; whereas, those of Calohypsibius are rigid at the junction (Ramazzotti & Maucci, 1983).

REFERENCES Binda MG, Pilato G. 1986. Ramazzottius, nuovo genere di Eutardigrada (Hypsibiidae). Animalia 13: 159–166. Bartos E. 1940. Uber die Variation der Art Hypsibius ornatus Richt. (Tardigrada). Zoologische Jahrbuecher Abteilung fuer Systematic Jena 73: 369–384. Cunha AX da. 1943. Un Tardigrade nouveau du Portugal: Hypsibius placophorus sp. n. Memorias e Estudos do Museu Zoologico da Universidade de Coimbra No. 143: 1–4. McGlothlin KL. 1990. Long-term stability of tardigrade populations on Roan Mountain, Tennessee. Unpublished Master’s Thesis, East Tennessee State University, Johnson City. Nelson DR. 1973. Distribution of tardigrades on Roan Mountain, Tennessee-North Carolina. Ph.D. Dissertation, University of Tennessee, Knoxville. Nelson DR. 1975. Ecological distribution of tardigrades on Roan Mountain, Tennessee-North Carolina. Memorie dell’Istituto Italiano di Idrobiologia 32(Suppl): 225–276. Nelson DR, McGlothlin KL. 1993. A new species of Hypsibius (Phylum Tardigrada) from Roan Mountain, Tennessee, U.S.A. Transactions of the American Microscopical Society 112(2): 140–144. Pilato G. 1969. Schema per una nuova sistemazione delle famiglie e dei generi degli Eutardigrada. Bollettino delle Sedute dell’Accademia Gioenia di Scienze Naturali in Catania, Serie IV, 10(2): 181–193. Pilato G. 1989. Phylogenesis and systematic arrangement of the family Calohypsibiidae Pilato, 1969 (Eutardigrada). Zeitschrift fuer Zoologische Systematik und Evolutionsforschung 27: 8–13.

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Pilato G, Claxton S, Binda MG. 1989. Tardigrades from Australia. II. The evaluation of Calohypsibius ornatus (Richters, 1900) caelatus (Marcus, 1928) as a valid species and description of Minibiotus fallax n. sp. Animalia 16: 21–27. Ramazzotti G, Maucci W. 1983. Il phylum Tardigrada. Memorie dell’Istituto Italiano di Idrobiologia 41: 1–1012. Richters F. 1900. Beitrage zur Kenntnis der Fauna der Umgebung von Frankfurt a. M. Berichte Senckenberg. Naturforschenden Gesellschaft Frankfurt a. M. 1900: 21–44. Schuster RO, Nelson DR, Grigarick AA, Christenberry D. 1980. Systematic criteria of the Eutardigrada. Transactions of the American Microscopical Society 99(3): 284–303.