2011 SOUTHEASTERN NATURALIST 10(2):211–220 First Distributional Study of Branchiobdellida (Annelida: Clitellata) in the Great Smoky Mountains National Park, North Carolina and Tennessee, USA, with a Redescription of Cambarincola holostomus Hoffman, 1963 Stuart R. Gelder1,* and Bronwyn W. Williams2 Abstract - Branchiobdellidans, or crayfish worms, are species-rich in the Appalachian area of the southeastern United States. Even so, the Great Smoky Mountains National Park (GSMNP) in the southern Appalachians has only one record of a branchiobdellidan species. As part of the All Taxa Biodiversity Inventory, we undertook a general survey of the branchiobdellidans in the GSMNP. Crayfish were collected from a total of 15 sites distributed across 11 watersheds in the GSMNP. We identified a total of 10 species of branchiobdellidans: 1 species of Bdellodrilus, 1 species of Oedipodrilus, 2 species of Pterodrilus, 1 species of Xironodrilus, and 5 species of Cambarincola. The composition and number of species per site ranged from 0 to 6, and individual branchiobdellidan species occurrence ranged from 1 to 11 sites. Our results suggest that multiple factors affect distribution and occurrence on certain hosts. Study of specimens of Cambarincola holostomus Hoffman, 1963 enabled us to add new morphological characters to the description of this species. An intensive survey of watersheds and crayfish fauna in the GSMNP is necessary to understand the multiple factors affecting species distributions and host preferences and to facilitate conservation of both the symbionts and their hosts. Introduction The Order Branchiobdellida is a small group of ectosymbiotic worms classified in the Clitellata but distinct from both the Oligochaeta and the Euhirudinea in the Phylum Annelida (Govedich et al. 2010). The taxon has a disjunct Holarctic distribution, ranging across North America, Europe, and East Asia (Gelder 1999). Branchiobdellidans, or crayfish worms, are small (0.7–10.0 mm long) and live primarily on freshwater astacoidean crayfishes (Govedich et al. 2010). The Branchiobdellida consists of 1 family, 4 subfamilies, 21 genera, and approximately 140 species (Gelder 1996a). Of the 15 genera and 96 species endemic in North America (Gelder et al. 2002), 8 genera and over 35 species have been reported from the Appalachian states of Kentucky, Virginia, Tennessee, and North Carolina, giving this area the highest taxonomic diversity of branchiobdellidans in the world. The high number of described branchiobdellidan species in the southern Appalachians also reflects the relatively large number of studies in the region, predominately initiated by the late Perry C. Holt at the Virginia Polytechnic Institute and State University. These studies have been incorporated into reviews 1Department of Science and Math, University of Maine at Presque Isle, 181 Main Street, Presque Isle, ME 04769. 2Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9. *Corresponding author - stuart.gelder@umpi.edu. 212 Southeastern Naturalist Vol. 10, No. 2 including Gelder et al. (2002), Holt (1969), and Holt and Opell (1993). Despite the attention provided to the branchiobdellidan fauna of the Appalachian region, the only reference to a branchiobdellidan in the Great Smoky Mountains National Park (GSMNP; Goodnight 1943) came from a collection made by A.R. Cahn on 9 September 1937 from Clingmans Dome; it proved to be a new species, Xironodrilus appalachius Goodnight. As part of the All Taxa Biodiversity Inventory (ATBI) initiative, partially underwritten by Discover Life in America, Inc., we undertook a survey of branchiobdellidans in riverine habitats of the GSMNP. In the process, we obtained new information on the microhabitat, range, and morphological variation of the observed species. Based on these preliminary results, future studies will focus on additional watersheds and other crayfish, such as burrowing species. Materials and Methods Sampling sites were distributed among watersheds of the GSMNP. Because of the GSMNP’s permit requirement, only the smallest number of crayfish possible were collected and sacrificed for investigation. The crayfish were collected by hand or kick-netting, with 2–5 specimens of each species per site being retained. Crayfish in the process of molting and “in berry” females were not examined, but instead were immediately returned to location of capture. Live crayfish were taken back to the laboratory for identification and examination for presence of branchiobdellidans. We could not always identify host specimens that were female, juvenile, or undescribed species. Live branchiobdellidans were placed in separate dishes after removal from the exposed surface of each live crayfish with the aid of a dissection microscope. The host was then decerebrated and disarticulated for more detailed examination. Branchiobdellidans were identified alive using a wet-mount procedure under a compound microscope (Govedich et al. 2010) and preserved in 100% ethanol for storage. Selected individuals were cleared with methyl salicylate, infiltrated with Canada balsam, and mounted on slides to museum standards. We deposited permanent slide mounts of representative branchiobdellidan species from this project in the GSMNP faunal collection under accession number GRSM-02115. Results and Discussion We sampled a total of 15 sites in 11 watersheds across the GSMNP (Fig. 1). Our collections included 5 known, 1 undescribed, and 1 unidentified species of crayfish (Table 1). Most of the crayfish were destroyed during the examination process; however, 6 representatives were placed in the Park’s museum collection (GRSM catalog numbers 113500–113505). Branchiobdellidans were found on all crayfish at all sites, with the exception of 2 Cambarus bartonii (Fabricius) (Appalachian Brook Crayfish) collected at site 4 (Fig. 1, Table 1). Based on our collections across the Appalachian region, the absence of branchiobdellidans at site 4 was unusual. No explanation could be offered for this observation, and further study is warranted. 2011 S.R. Gelder and B.W. Williams 213 We identified 10 branchiobdellidan species representing 5 genera (Table 1). The number of branchiobdellidan species per site ranged from 0 to 6, and individual species occurrence across sites ranged from 1 to 11 (Table 1). Certain species were detected throughout much of the study area (e.g., X. appalachius, Cambarincola holostomus Hoffman, Pterodrilus alcicornus Moore), whereas others appeared to be restricted in their distribution (e.g., Bdellodrilus illuminatus (Moore), Oedipodrilus anisognathus Holt, Cambarincola heterognathus Hoffman, Pterodrilus hobbsi Holt). Our data do not show a clear overall pattern of host preference, either within or among sites. Co-occurring crayfish species collected at four sites (1, 3, 7, and 12) appeared to have both host-specific and host-general branchiobdellidan species. Similarly, most branchiobdellidan species were detected on 2 or more crayfish species across all collection sites (Table 1). Overlap across host species is likely a result of shared habitat and a lack of preference for host species among most branchiobdellidan species (Gelder 2006). As branchiobdellidans do not require attachment to a host for individual survival, their time on the substratum or in physical contact with additional hosts (e.g., through direct crayfish-crayfish interaction) provides ample opportunity to pass from one host to another. Because of the preliminary nature of our survey, we were unable to sample burrowing crayfish species and species found in high-altitude headwater habitats. It is probable that, given the ecological isolation of these host species, additional branchiobdellidan species are present in the GSMNP. An intensive survey of crayfish in a variety of habitats within watersheds in the GSMNP is still needed. We deposited slide-mounted representatives of the branchiobdellidan species collected, except C. heterognathus and O. anisognathus, in the museum under GRSM catalog numbers 113506–113558. Figure 1. Map of the Great Smoky Mountains National Park in North Carolina and Tennessee, showing the collection sites and watersheds: 1 = Abrams Creek, 2 and 3 = Middle Prong Little River, 4 = West Prong Little Pigeon River, 5 = Le Conte Creek, 6 = Little Pigeon River, 7 = Cosby Creek, 8 = Big Creek, 9 and 10 = Cataloochee Creek, 11 and 12 = Oconaluftee River, 13 and 14 = Hazel Creek, and 15 = Twentymile Creek. The insert shows the Park’s location in southeastern USA. 214 Southeastern Naturalist Vol. 10, No. 2 Table 1. Distribution of branchiobdellidans in the Great Smoky Mountains National Park, NC and TN, with respect to their watershed, location in latitude (Lat.) and longitude (Long.), and crayfish host(s). Crayfish (n, number examined): C.= Cambarus; O. = Orconectes. Branchiobdellidans: B.i. = Bdellodrilus illuminatus, C.f. = Cambarincola fallax, C.he. = C. heterognathus, C.ho. = C. holostomus, C.i. = C. ingens, C.p. = C. philadelphicus, O.a. = Oedipodrilus anisognathus, P.al. = Pterodrilus alcicornus, P.h. = P. hobbsi, and X.a. = Xironodrilus appalachius; “X” = presence and “-” = absence of a species. Branchiobdellidan species Site Watershed Lat. (°N) Long. (°W) Crayfish host (n) B.i. C.f. C.he. C.ho. C.i. C.p. O.a. P.a. P.h. X.a. 1 Abrams Creek 35.60620 83.93433 C. longirostris (2) - - X - - - - - - - O. forceps (2) - - X X - - X - - - O. erichsonianus (3) - - - - - X X - - - 2 Middle Prong Little River 35.61794 83.67017 C. bartonii (3) - - X X X - - X X X 3 Middle Prong Little River 35.66765 83.71470 C. bartonii (1) - - - X - - - - X X C. longirostris (1) - - - X - - - - X X 4 West Prong Little Pigeon River 35.62422 83.41685 C. bartonii (2) - - - - - - - - - - 5 Le Conte Creek 35.68673 83.50110 C. bartonii (5) X - - X X - - X - X 6 Little Pigeon River 35.72647 83.40141 C. longirostris (4) X X - - X - - - - X 7 Cosby Creek 35.78340 83.21808 C. bartonii (3) - X - X X - - X - X C. longirostris (2) - X - - X - - X X X 8 Big Creek 35.75078 83.10898 C. sp. - Cataloochee morph (2) - - - X X - - X - - 9 Cataloochee Creek 35.61637 83.12038 C. sp. - Cataloochee morph (3) - X - X X - - X - X 10 Cataloochee Creek 35.62864 83.09403 C. sp. - Cataloochee morph (4) - - - X - - - - - - 11 Oconaluftee River 35.59855 83.41326 C. bartonii (2) - - - - - X - - - X 12 Oconaluftee River 35.55650 83.31410 C. bartonii (3) - - - - - X - - - X C. reburrus (1) - - - - - X - - - X 13 Hazel Creek 35.49395 83.68852 C. bartonii (3) X - - - - - - X - X 14 Hazel Creek 35.47397 83.72209 Unknown females (2) - - - - - - - - - X 15 Twentymile Creek 35.46770 83.87635 C. bartonii (2) - X - - X - - X - X 2011 S.R. Gelder and B.W. Williams 215 Observations on microhabitat and distribution of branchiobdellidan species The opportunity to observe branchiobdellidans on live hosts prior to their removal and identification enabled previously unreported details on microhabitat and variation in morphological characters to be made. Observations of several species in the GSMNP extended their recorded range. Xironodrilus appalachius was fairly ubiquitous across the GSMNP, occurring at 11 of our 15 collection sites (Table 1). Populations of X. appalachius were observed on anterior areas of the host’s cephalothorax, including the chelipeds and 2nd and 3rd walking legs. This microhabitat is identical to that occupied by Xironogiton instabilis (Moore), a species not found in the GSMNP, but common throughout the Appalachian Mountain region, from Virginia north to New Brunswick, Canada (Gelder et al. 2009). The dorso-ventrally flattened body and behavior are also similar in the 2 species, suggesting potential adaptations to microhabitat. These 2 species are not presently known to co-exist. A study of their reactions to each other in an in vitro exposure would help discern whether this is a simple geographical separation or a more complex behavioral one. Specimens of Cambarincola heterognathus, estimated at 20 or more individuals per host, were observed to tightly cluster around the eyes and in the expiratory water channel between the carapace and the mouth parts. Such a density of worms and their cocoons would likely reduce the respiratory flow and cause some distress to the host if the worms were to remain in that position for an extended period of time. Interestingly, tight clustering behavior was observed of worms transferred to a dish a short time after removal from the host. Although the condition of the gills of the hosts from which specimens of C. heterognathus were removed was usually poor, with excessive scarring, we observed varying degrees of host gill damage at all collection sites, irrespective of the presence of C. heterognathus or the gill-dwelling branchiobdellidans, B. illuminatus and O. anisognathus. According to Holt (1973:96), “It is possible that C. heterognathus is at least a facultative inhabitant of the gill chambers of its hosts and, if it is so, the fact that its Floridian host is a burrowing crayfish would aid its survival.” Our observations of the microhabitat of C. heterognathus support the initial part of the above statement, but Holt (1973) provided no reasons or observations to support the second part. Regrettably, this speculative and unsupported statement by Holt could easily be taken as fact and used to argue a close functional relationship between gill-dwelling branchiobdellidans and burrowing crayfish. Hoffman (1963:365) stated that in C. heterognathus, “... the spermatheca offers very good specific characters, particularly its length and pronounced median constriction which are evident.” However, these features were not found in the specimens examined from the GSMNP nor in the holotype (S.R. Gelder, pers. observ.). The spermatheca is approximately half the diameter of body segment 6, with a tubular duct and an ovoid bulb, each approximately the same length. The species has been reported in an area ranging from West Virginia and Virginia to Florida (Gelder et al. 2002) on a number of Cambarus species. Accordingly, our observations fall within the known distribution and host affinities. 216 Southeastern Naturalist Vol. 10, No. 2 Cambarincola ingens Hoffman appears to have a somewhat restricted range in North Carolina, Tennessee, and West Virginia. It is relatively large, about 10 mm long when live, and easily recognizable on a host. We observed C. ingens primarily on the anterior abdomen and infrequently on the dorsal surfaces of the host’s carapace. Collections of this species in the GSMNP extend its reported distribution slightly farther south. Oedipodrilus anisognathus has been reported only from eastern Tennessee; thus, its presence in the Abrams Creek watershed (Site 1, Fig. 1) is not surprising. Specimens were recovered from the gill chamber and the inner surface of the carapace, providing the first evidence of the species’ microhabitat. Holt’s (1988:800) conjecture that O. anisognathus was a gill-dweller was based solely on its external appearance. The two pterodrilid species, Pterodrilus alcicornus and P. hobbsi, have been previously described from the general vicinity of the southern Appalachian Mountains. It is not uncommon to find two sympatric species of Pterodrilus on the same host (Gelder 1996b). The gill-dwelling Bdellodrilus illuminatus is well known, if sporadic in its reported distribution along the Appalachian chain, from southeastern Canada to South Carolina and Georgia (Gelder et al. 2002). Therefore, the presence of this species in the GSMNP was predictable. Bdellodrilus illuminatus was observed at 3 sites and in low densities of 1–3 worms per host. In addition, B. illuminatus was not found on all hosts when present at a site. This irregular distribution, both range-wide and across hosts at a single site, and low density is consistent with our prior collections of the species. One notable exception was the observation of 39 B. illuminatus worms and 96 cocoons on a single Cambarus bartonii host from New Brunswick, Canada (Gelder et al. 2009). The general body-roaming Cambarincola fallax Hoffman was observed at 4 of the 15 collection sites (Table 1). The distribution of C. fallax is similar to that of B. illuminatus, ranging along the length of the Appalachian chain. Cambarincola philadelphicus (Leidy) appeared to be restricted to the Oconaluftee River drainage in the GSMNP (Table 1). Although this is the most commonly occurring species east of the Mississippi River in the USA, the species description is unfortunately one of the least precise. As Hoffman (1963) noted, it will require a thorough range-wide re-examination to establish whether C. philadelphicus is a single species or if it consists of multiple morphologically similar species. We observed C. holostomus at several sites (Table 1). These specimens showed that a number of morphological characters were missing from the original species description. Accordingly, we provide the following redescription. Redescription of Cambarincola holostomus Hoffman, 1963 (Fig. 2A–C) Brief description. Length 1.6 to 2.3 mm, head terete, width greater than segment 1, body tubular, segments distinct; dorsal ridges present; dorsal projections absent; supernumerary muscles present; lateral paired lobes segments 8 and 9 small; dorsal lip, 4 lobes, lateral lobes 1 pair; ventral lip, median emargination; oral papillae present; jaws, similar size, shape triangular, median teeth large, lateral teeth smaller, dental formula 5/5; pharyngeal sulcus one pair; glandular 2011 S.R. Gelder and B.W. Williams 217 atrium tubular, length 0.5x segment diameter, deferent lobes absent; prostate gland tubular, slim, ends 0.9x segment diameter over glandular atrium, differentiated, ental bulb present; muscular atrium terete, length 0.1x segment diameter; bursa ovoid elongate, length 0.4x segment diameter; penial sheath ental, 0.3x bursa length; penis protrusible; spermatheca tubular, length 1.2x segment diameter, duct shape tubular and length 0.7x organ, bulb tubular, dilated slightly, length 0.3x organ, ental process absent. Variations. No variations were reported in the type description; however, the body shape ranges from slim to wide tubular, dorsal ridges are low but pronounced when the body contracts. The peristomial lobes vary in their degree of prominence, with an extreme case appearing as an oral ring. The dental formula of 5/5 has not been found to vary. The prostatic ental bulb is obscure in some of the paratypes, but usually it is prominent. The spermatheca is longer than previously reported, and the tapered ental end of the bulb is not present in all specimens; however, no ental process was evident. Microhabitat. Tucked into the articulation folds of the walking legs, maxillipeds and chelae of the host crayfish. Hosts. Cambarus bartonii, Cambarus longirostris Faxon, Cambarus longulus Girard, Orconectes forceps (Faxon), and Orconectes erichsonianus (Faxon). Distribution. Alleghany, Augusta, Craig, Highland, and Rockingham counties, VA; Blount, Cocke, and Sevier counties, TN, and Haywood County, NC (Great Smoky Mountains National Park). Justification for accepting variations. Hoffman (1963:Fig. 61) drew a paratype and described the peristomium as “... distinctly flared, its margin entire, without any trace of division into dorsal and ventral halves …”; other specimens in the series do not show this extreme peristomial deformation and show slight indications of dorsal lobes. The jaws were reported to have a 3/3 dental formula. Although no shape was mentioned, Hoffman (1963:Figs. 65, 66) did show them as being triangular. These figures, along with drawings of a pair of triangular jaws and 3/3 dentition from a dorso-ventral aspect, appeared in Figure 2. Cambarincola holostomus. A. lateral view of a whole specimen, scale bar = 0.2 mm; B. dorsal view of jaws (dorsal, top; ventral, bottom; teeth pointing posteriorly), scale bar = 30 μm; C. lateral view of male genitalia (left, segment 10) and spermatheca (right, segment 9), scale bar = 50 μm. 218 Southeastern Naturalist Vol. 10, No. 2 Holt and Opell (1993:Figs. 85 and 86). Specimens of this taxon loaned from the National Museum of Natural History (NMNH) – Smithsonian Institution and examined by S.R. Gelder (pers. observ.), including the holotype (USNM 29946), have a 5/5 dental formula (Fig. 2B). The large spermatheca and relative lengths of the duct and bulb are important characteristics in identifying this species. However, the lengths of the two structures were not differentiated by Hoffman (1963:Fig. 64), but were shown in Holt and Opell (1993:Fig. 84); the latter figure agrees with our observations. Note. The deformed peristomium reported and shown by Hoffman (1963) is a preservation artifact. Unfortunately, he did not recognize the deformity and stated, “conceivably the peristomium [in this form] … represents … a more efficient holdfast device than the dissected and lobed mouth of fallax” (Hoffman, 1963: 360). This statement indicates that Hoffman followed Holt’s opinion in believing, incorrectly, that the usual anterior attachment site in branchiobdellidans is the mouth, as in leeches, and not the correct location which is the adhesive site on the ventral surface of the ventral lip (Moore 1895, Weigl 1994). Two slides from Hoffman’s (1963) study and the Holt collection (United States National Museum; USNM 36769, 37287, 37292, 37404, and 37409) were erroneously entered into the NMNH Catalog as being from Florida. However, these collection-site details were repeated later in the catalog, but cited for Virginia. This error was not noticed when the North American distribution of branchiobdellidans was being prepared, thus they were included, incorrectly, as being from Florida (Gelder et al. 2002). Sympatry. This species was found on hosts in “... rather small, swift, mountain brooks and is sympatric with both C. fallax and C. philadelphicus” according to Hoffman (1963). In their detailed study of the Mountain Region, VA, Hobbs et al. (1967:58) found C. holostomus to be sympatric with the two branchiobdellidans mentioned above plus Ankyrodrilus koronaeus Holt, B. illuminatus, Cambarincola branchiophilus Holt, C. ingens, P. alcicornus, Xironodrilus formosus Ellis, and Xironogiton instabilis. Observed gut contents. The contents of the stomach and intestine in a total of 16 mounted specimens from sites 1–3 and 5–10 were examined. In most specimens, the stomach was full and the intestine less than half full. Stomach contents consisted of rotifer-like bodies, an oligochaete that was only recognizable because of the chaetae, a protozoan test, and diatom frustules ranging in length from just less than the segment diameter to a tenth that size. Most frequently, the lumen was filled with dissociated ovoid and tubular bodies in a granular, amorphous mass varying in color from dark to light brown with occasional clay mineral particles. The intestines rarely contained any amorphous material, and only then in the anterior portion. However, one or two small masses of mineral particles, diatom frustules and fragments, and other debris were usually observed, these being the indigestible residue of the diet. The best that can be inferred from these observations is that C. holostomus is an opportunistic omnivore, ingesting small invertebrates, protozoans, diatoms, and organic debris with no apparent dietary preference. 2011 S.R. Gelder and B.W. Williams 219 Conclusions We have established a baseline for understanding branchiobdellidan diversity and distribution in the GSMNP. Further studies are needed on high-altitude and burrowing crayfish and of crayfish in other watersheds in the Park. Better knowledge of this diversity and distribution will assist in the development of management plans needed both to preserve the endemic fauna of the Park and to protect their populations from invasive species known to occur in adjacent areas. Acknowledgments Funding for this study was provided in part by a research grant from Discover Life in America, Inc., and publication costs were met in part by funds from the University of Maine at Presque Isle to S.R. Gelder and from Alberta Innervates, Canada to B.W. Williams. We would like to thank the staff at Discover Life in America office in Gatlinburg, Chuck Cooper, and Chuck Parker for their assistance. Guidance and support from members of the National Park Service, Great Smoky Mountains National Park, ensured that the collecting aspect was a success, particularly, Keith Langdon, Paul Super, Becky Nichols, Matt Kulp, Adriean Major, and Susan J. Simpson. All fieldwork in the GSMNP was conducted under permit GRSM-2010-SCI-0004 issued by the National Park Service. Literature Cited Gelder, S.R. 1996a. A review of the taxonomic nomenclature and a checklist to species of the Branchiobdellae (Annelida: Clitellata). Proceedings of the Biological Society of Washington 109:653–663. Gelder, S.R. 1996b. Description of a new branchiobdellidans species, with observations on three other species, and a key to the genus Pterodrilus (Annelida: Clitellata). 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