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“Smoky Bears”—Tardigrades of Great Smoky Mountains National Park
Diane R. Nelson and Paul J. Bartels

Southeastern Naturalist, Volume 6, Special Issue 1 (2007): 229–238

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1Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614-1710. 2Department of Biology, Warren Wilson College, Asheville, NC 28815. *Corresponding author - “Smoky Bears”—Tardigrades of Great Smoky Mountains National Park Diane R. Nelson1,* and Paul J. Bartels2 Abstract - As part of the All Taxa Biodiversity Inventory (ATBI) in Great Smoky Mountains National Park (GSMNP), we have collected nearly 600 samples from soil/decomposed leaf litter, lichens and mosses on trees, and stream sediment and periphyton within all 19 permanent ATBI plots, with additional samples from caves, rock lichens, seeps, and bird nests. Tardigrades have been extracted from samples using centrifugation with Ludox AMTM and mounted on individual microscope slides in Hoyer’s medium for identification under phase and DIC microscopy. Prior to our study, only three species of tardigrades had been reported from a few samples in the park. We have now examined over 9000 slides from 401 samples and recorded 73 species, 14 of which we believe may be new to science. Using EstimateS 7.5 software for each of the major tardigrade habitats, we estimate a total species richness of ≈96 limno-terrestrial species in the park. In this paper, we discuss challenges inherent in tardigrade taxonomy and the need for revisions of species groups. Introduction The Phylum Tardigrada, commonly known as “water bears,” consists of charismatic microfauna (0.05–1.2 mm) with a ubiquitous distribution in a diversity of niches in terrestrial and aquatic (both freshwater and marine) interstitial environments throughout the world. These hydrophilous metazoans are active only when there is a film of water covering their cylindrical bodies and four pairs of telescoping lobopod legs terminating in claws and/or adhesive disks. As the water evaporates, terrestrial tardigrades and some intertidal species enter a state of cryptobiosis that enhances their survival and dispersal. Long-term survival in the cryptobiotic state is probably less than a decade, rather than 100 years as previously thought (Jönsson and Bertolani 2001). Most tardigrades feed by piercing plant or animal cells with a pair of cuticular stylets and sucking out the contents with a muscular pharynx (see Kinchin 1994 and Nelson 2001, 2002 for reviews of the morphology and ecology of the phylum). Approximately 1000 species of tardigrades have been described in the literature (Guidetti and Bertolani 2005), but the total number of existing species has not even been estimated. Our knowledge of the distribution of tardigrades is based on limited collections from around the world, and the descriptions of type species range anywhere from a thorough analysis of characters with professionally produced illustrations to inadequate descriptions that are incomplete or inaccurate with poorly drawn figures. Some of the older descriptions were based on observations made only under a light 229 The Great Smoky Mountains National Park All Taxa Biodiversity Inventory: A Search for Species in Our Own Backyard 2007 Southeastern Naturalist Special Issue 1:229–238 230 Southeastern Naturalist Special Issue 1 microscope, while more recent ones have utilized observations from phase, DIC, SEM, and TEM microscopy. In addition, many type specimens from the early descriptions are lost or damaged or non-existent. Much of the taxonomy of tardigrades is in drastic need of revision of genera, species groups, and individual species. The last “complete” monograph of the phylum was by Ramazzotti and Maucci (1983), which included only 548 species. A sister group to the Phylum Arthropoda (Garey et al. 1996, 1999; Giribet et al. 1996), tardigrades are divided into two main classes, Heterotardigrada and Eutardigrada, recognized on the basis of characters of the claws, buccal (feeding) apparatus, cuticle, and eggs. A third class, Mesotardigrada, reported only once from a hot spring in Japan, is considered dubious (see Kinchin 1994, Nelson 2001, Nelson and Marley 2000, Nelson and McInnes 2002). Briefl y, the Heterotardigrada, usually found in terrestrial and marine habitats, are the “armored” tardigrades, with a thickened dorsal cuticle divided into plates, unbranched (single) claws on the four pairs of legs, separate anus and gonopore, and sensory appendages on the head (Fig. 1). The Eutardigrada, generally inhabiting freshwater and terrestrial substrates, are the “naked” tardigrades, with smooth or ornamented cuticles, paired branched (double) claws on the legs, cloaca, and a complex buccal-pharyngeal apparatus, but lacking cephalic appendages (Fig. 2). In June 2001, we initiated an inventory of the Phylum Tardigrada for the All Taxa Biodiversity Inventory (ATBI) (see www.discoverlifeinamerica. org) in the Great Smoky Mountains National Park (GSMNP) (Sharkey 2001). Previously, Riggin (1962) had reported only three species identified from the park (Indian Gap at 1615 m, Sevier County–Swain County, TN–NC): Diphascon pingue (Marcus), Macrobiotus harmsworthi Murray, and Minibiotus intermedius (Plate). We reported our preliminary results for our first two field seasons (Bartels and Nelson 2006) using species accumulation curves and seven species richness estimates (EstimateS) to assess tardigrade species richness in the GSMNP (Colwell 1997, Colwell and Coddington 1994). Based on a subset of our analyzed samples (60 samples, 1510 identified tardigrades, 41 species), we predicted a range of 47 to 76 species, with generally similar species richness in soil, lichen, moss, and stream habitats (Bartels and Nelson 2006). We also stated that we expected the number of tardigrade species, as well as state records, to increase as more specimens were examined from our samples. We compared the tardigrade fauna of the GSMNP with that previously reported from Roan Mountain, TN–NC (Guidetti 1998; Guidetti et al. 1999; Maucci 1987; Nelson 1975; Nelson and McGlothlin 1993, 1996). Two other multihabitat inventories in Italy (Bertolani and Rebecchi 1996) and Poland (Dastych 1980) have indicated similar diversity, although it is difficult to compare studies without sufficient information on sampling effort. In this paper, we list the species identified thus far and discuss problems associated with making definitive determinations of species at this time. Methods During five field seasons (2001–2005), we collected in all 19 of the ATBI plots designated to represent all major biological communities within the 2007 D.R. Nelson and P.J. Bartels 231 Figure 1. A. SEM of the heterotardigrade Echiniscus mauccii, lateral view. B. SEM of the heterotardigrade Echiniscus mauccii, frontal view. 232 Southeastern Naturalist Special Issue 1 Park. Approximately 600 samples were collected from the major habitats of limno-terrestrial tardigrades: mosses and lichens on trees, soil/decomposed leaf litter, and sediment and periphyton in streams. Additional samples were collected from mosses and lichens on rocks as well as some samples from caves, seeps, and bird nests (see Bartel’s and Nelson 2006 for details on collection.) Environmental factors (altitude, slope, aspect, land cover, geology, soil type, and moisture index) were recorded at the time of collection or obtained from the GSMNP GIS database. Tardigrades were extracted from samples using centrifugation with Ludox AM™, individually mounted on microscope slides in Hoyer’s medium, and studied with phase-contrast and DIC microscopy (Bartels and Nelson 2006). The seven species richness estimates were re-calculated with EstimateS 7.5 software (Colwell 1997) for each of the major tardigrade habitats, using Figure 2. A. SEM of the eutardigrade Macrobiotus tonollii, lateral view. B. SEM of the eutardigrade Macrobiotus tonollii, frontal view. 2007 D.R. Nelson and P.J. Bartels 233 6220 identifiable specimens, rather than the 1334 from our previous study (Bartels and Nelson 2006). Results As of June 2006, we have examined 9125 slides from a total of 401 samples. These samples included 103 stream samples from 13 streams, 86 tree lichen samples, 116 tree moss samples, 66 soil/decomposed leaf-litter samples, and 30 miscellaneous samples. Of these slides, we have identified 8133 tardigrades to species. The total species list for the Park is now 73 species in 22 genera and 6 families (Table 1; see the current list as it is updated at Fourteen of these species are believed to be new to science and will be described in subsequent papers. We have now established 70 new records for the Park (Table 1). Including the new species in each state, we have found 34 new state records for North Carolina and 38 for Tennessee (Table 1), since the GSMNP lies along the border of two states. Table 1 also indicates which species were found in the various habitats sampled, as of June 2006. Based on our more extensive dataset (compared with Bartels and Nelson 2006), we have now increased our species richness estimates to range from 86–105 species in moss, lichen, soil, and stream habitats. Although none of the seven estimators in EstimateS worked best for all habitats, we used the best estimator for each habitat to predict a total species richness of ≈96 tardigrade species in the GSMNP (Bartels and Nelson 2007). Discussion Since the publication of the monograph on the Tardigrada by Ramazzotti and Maucci (1983), several new taxa have been added or previous taxa amended as revisions in the systematics of the phylum have been published (Guidetti and Bertolani 2005). The discovery of new taxonomic criteria (Schuster et al. 1980), the evaluation of intraspecific variability (Pilato 1972, 1982, 1987), and the use of TEM, SEM, and improved imaging techniques (Grigarick et al. 1973, Kristensen 1987, Rebecchi 2001) have resulted in revisions of some families, genera, species, and species groups. During our preliminary identifications, specimens were often categorized as belonging to “species groups” when the identification of the precise species was not readily discernible. This may have been due to (1) the condition or orientation of the specimen, (2) the quality of our phase/DIC microscope and thus our inability to see specific characters, (3) the lack of eggs, and/or (4) the quality of the original species descriptions, including taxonomic criteria, illustrations, and measurements. Comparisons of specimens with hard-to-locate type material (types are often missing or non-existent) and original descriptions in the older literature make the problem of identification of species more difficult. Difficulties arise in species identification especially in the eutardigrades due to “the group’s homogeneity” and differentiation based on few “minute characteristics” (Kinchin 1994). Quantitative measurements may vary due to (1) the fl exibility and orientation of structures, such as the buccal-pharyngeal apparatus, 234 Southeastern Naturalist Special Issue 1 Table 1. Tardigrade species list for GSMNP, with habitat associations and range extensions, as of June 2006. * = new state record, ** = new USA record, *** = new North America record, based on Bateman and Collins(2001), Boeckner et al. (2006), Guidetti and Bertolani (2005), Kaczmarek and Michalczyk (2003), and McInnes (1994). “Other” = bird nests, rock moss, and rock lichen. Tree Tree Family Species State moss lichen Soil Streams Other Echiniscidae Bryodelphax n. sp. TN*** X Echiniscus horningi TN/NC X X X E. mauccii TN/NC X X X X E. mosaicus TN*/NC* X X E. perviridis NC* X E. virginicus TN*/NC* X X X E. viridis group TN* X Hypechiniscus gladiator TN/NC X X X X Pseudechiniscus brevimontanus TN/NC* X X X X P. n. sp. TN***/NC*** X X X P. suillus group TN/NC X X X X X Calohypsibiidae Calohypsibius schusteri TN/NC X X X Eohypsibiidae Amphibolus cf. smreczinskii NC** X A. cf. weglarskae TN** X Hypsibiidae Astatumen trinacriae TN/NC X X X X Diphascon belgicae TN*/NC* X D. cf. carolae TN***/NC*** X X X D. cf. ramazzottii TN X D. granifer TN*** X X D. higginsi TN/NC* X X X X X D. nobilei TN/NC* X X D. nodulosum TN*/NC* X X X D. patanei TN***/NC*** X X X X D. pingue TN/NC X X X X D. scoticum TN/NC X X X X X Doryphoribius cf zappalai TN*** X X D. n. sp. TN*** X X D. sp. 1 TN* X Doryphoribius sp 2 TN* X Hypsibius convergens TN/NC* X X X X X H. dujardini TN/NC* X X X H. roanensis NC* X X X Isohypsibius cf basalovoi TN***/NC*** X X I. cf brevispinosus TN***/NC*** X X I. cf deconincki TN*** X I. granulifer TN/NC X X I. lunulatus TN/NC X X X X I. n. sp. 1 TN***/NC*** X X X I. n. sp. 2 TN***/NC*** X X I. n. sp. 3 TN***/NC*** X X I. n. sp. 4 TN***/NC*** X I. n. sp. 5 TN***/NC*** X X I. sattleri TN/NC* X X I. tuberculatus group TN* X X X X X Itaquascon n. sp. TN***/NC*** X X Mesocrista cf. spitzbergensis TN/NC* X X Platicrista angustata TN/NC X X X 2007 D.R. Nelson and P.J. Bartels 235 Table 1, continued. Tree Tree Family Species State moss lichen Soil Streams Other P. horribilis TN***/NC*** X X Pseudobiotus n. sp. NC*** X Ramazzottius baumanni TN* X X R. oberhaeuseri group TN* X X X X Thulinius augusti NC* X T. ruffoi NC*** X T. stephaniae TN*** X Macrobiotidae Dactylobiotus cf. grandipes TN/NC* X Macrobiotus cf. crenulatus TN**/NC** X X M. cf. echinogenitus NC* X M. cf. liviae TN***/NC*** X X X M. harmsworthi TN/NC X X X X M. hufelandi group sp. 1 TN/NC X X X X X M. hufelandi group sp. 2 TN/NC X X M. richtersi TN*/NC X X X X M. n. sp. 8 TN***/NC*** X X M. n. sp. 9 TN*** X M. tonollii TN/NC X X X X Minibiotus intermedius group TN/NC X X X M. lazzaroi TN**/NC** X X M. pustulatus TN/NC X X X Murrayon cf hastatus TN* X M. cf. pullari TN X M. n. sp. TN*** X M. stellatus TN X Milnesiidae Milnesium n. sp. TN***/NC*** X X X X X (2) the deformation of structures by coverslip pressure, (3) differences in mounting media, or (4) variation in the measurements used by individual investigators. Pigmentation in the cuticle and eyespots may vary considerably over time and with various mounting media and therefore may be unreliable for identification of some species. In the echiniscid heterotardigrades, the pattern on the dorsal cuticular plates is of great importance (Kristensen 1987), but difficult to describe verbally, especially if the observer is using different types of microscopy (light vs. phase or DIC or SEM) and various levels of focus. Many of the earlier descriptions are particularly confusing in this regard. One example of a species group is the Macrobiotus hufelandi complex, a group of species similar to Macrobiotus hufelandi Schultze, the type species for the genus. Ramazzotti and Maucci (1983) referred to this species as being the “most common tardigrade” with a cosmopolitan distribution, although variations in specimens identified as M. hufelandi have long been recognized. Variations in the egg shell surface and processes of M. hufelandi presented other problems (Grigarick et al. 1973). Since this species lays ornamented eggs freely, outside the body of the female, it is necessary to find embryonate eggs which have a buccal-pharyngeal apparatus sufficiently developed in order to correlate the eggs, with their specific inverted egg-cup ornamentations, with adults having similar buccal-pharyngeal structures. For example, 236 Southeastern Naturalist Special Issue 1 a number of morphologically similar species in the M. hufelandi complex have been described based on characters of the eggs and adults by Bertolani and Rebecchi (1993), as well as many other authors in recent years. Our slides of the M. hufelandi group are currently being analyzed by Prof. Dr. Bertolani (University of Modena and Reggio Emilia, Italy) and Prof. Dr. Pilato (University of Catania, Italy), world-renowned authorities on this group. Other genera that lay ornamented eggs useful (or essential) for identification are Minibiotus, Ramazzottius, Amphibolus, Murrayon, and Dactylobiotus. Often these eggs are very small (≈50 μm) and frequently overlooked (or absent) or misidentified when tardigrades are extracted from the samples. Claxton (1998) revised the genus Minibiotus and the type species, Minibiotus intermedius (Plate), and she described 11 new species of Minibiotus from Australia, which may have been identified incorrectly as the “cosmopolitan” Minibiotus intermedius based on older, imprecise descriptions. Dr. Claxton is now analyzing our collection of the Minibiotus intermedius group. Characters associated with both the eggs (Bertolani et al. 1996) and sperm (Rebecchi 2001) have phylogenetic significance in tardigrades and should be considered in systematic revisions. Milnesium was long considered a cosmopolitan monotypic genus with Milnesium tardigradum Doyère designated as the type species; however, since 1990, additional species have been described, and the entire genus is in need of revision (Nigel Marley, University of Plymouth, Plymouth, UK, pers. comm.). Our specimens from GSMNP differ sufficiently from new material collected from the type locality in France and will be described as a new species in a separate paper. Ultimately, as taxonomic issues are resolved and our database is completed, we will determine the total number of species of limno-terrestrial species in the GSMNP. We will evaluate the effectiveness of the seven species-richness estimators (EstimateS) and compare biodiversity within the GSMNP with that of other large-scale studies. Our long-term goal is a multivariate ecological analysis of the environmental factors that contribute to tardigrade diversity and distribution. We are also developing a key to the species of tardigrades in the GSMNP, including photographs of the species and illustrations of all the characters and character states. This key will be available on the Internet. Acknowledgments Partial funding for this work was provided by Discover Life in America and The Environmental Leadership Center of Warren Wilson College. Gilbert Hale has been our chief “bear hunter” and slide-maker. Over 20 undergraduate students from Warren Wilson have assisted in the lab and with data processing. We appreciate the assistance of our tardigrade colleagues with some species identifications. We also are grateful for the comments of the reviewers. Literature Cited Bartels, P.J., and D.R. Nelson. 2006. A large-scale, multihabitat inventory of the Phylum Tardigrada in the Great Smoky Mountains National Park, USA: A preliminary report. Hydrobiologia 558:111–118. 2007 D.R. Nelson and P.J. Bartels 237 Bartels, P.J., and D.R. Nelson. 2007. 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The bryofauna of remote coastal Labrador: Including a review of current Canadian records. Zootaxa 1105:1–16. Claxton, S.K. 1998. A revision of the genus Minibiotus (Tardigrada: Macrobiotidae) with descriptions of eleven new species from Australia. Records of the Australian Museum 50:125–160. Colwell, R.K. 1997. EstimateS: Statistical estimation of species richness and shared species from samples. Version 6. Available online at estimates. Accessed 29 March 2006. Colwell, R.K., and J.A. Coddington. 1994. Estimating terrestrial biodiversity through extrapolation. Philosophical Transactions of the Royal Society (Series B) 345:101–118. Dastych, H. 1980. Tardigrades from the Tatra National Park. Polska Akademia Nauk Zaklad Zoologii Systematycznej i Doswiadczalnej. Monografie Fauny Polski. Tom 9. 232 pp. Garey, J.R., M. Krotec, D.R. Nelson, and J. Brooks. 1996. Molecular analysis supports a tardigrade-arthropod association. Invertebrate Biology 115:79–88. Garey, J.R., D.R. Nelson, L.Y. 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