2011 SOUTHEASTERN NATURALIST 10(2):199–210 A Re-description of Cambarincola bobbi Holt, 1988, a Description of a New Species of Pterodrilus, and Observations of Sympatric Species of Crayfish Worms (Annelida: Clitellata: Branchiobdellida) from the Cumberland River Watershed in Tennessee Bronwyn W. Williams1,* and Stuart R. Gelder2 Abstract - A re-description of Cambarincola bobbi Holt, 1988 is provided based on observations made during recent collections in the Cumberland River Watershed in Tennessee. The branchiobdellidan Pterodrilus robinae, a new species, is described, along with a refinement for identification of five additional Pterodrilus species. Diversity, sympatry, and range extensions of branchiobdellidans from three areas of the Cumberland River Watershed are reported. Introduction Branchiobdellidans, or crayfish worms, are freshwater ectosymbionts living primarily on astacoidean crayfish. Two or more species are frequently found on a single host, which is a characteristic reported in each of the three disjunct regions of the taxon’s Holarctic distribution (Gelder 1999). The largest diversity of branchiobdellidan taxa in the world is found in the mid-eastern region of the USA, which supports 11 of the 21 nominal genera (Gelder et al. 2002). Therefore, it is not surprising that the record of eight species representing five genera of branchiobdellidans on a single crayfish was found in Tennessee (Brinkhurst and Gelder 2001). In spite of this local diversity, many species have been recorded only from limited areas (Holt 1969, 1973; Holt and Opell 1993), which may be more a reflection of the collection sites sampled, rather than true distribution. Branchiobdellidans were identified from crayfish collected from three counties (Davidson, Overton, and Wilson) in central Tennessee as part of larger survey in the State. The focus of this paper is a re-description of an existing species and the description of a new species along with observations of additional sympatric branchiobdellidans found at each collection site. Materials and Methods Crayfish were collected by hand and dip nets from three areas of the Cumberland River Watershed in Tennessee during late April and mid-May 2010. Nine specimens of Orconectes durelli Bouchard & Bouchard were obtained from a 1Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E9. 2Department of Science and Math, University of Maine at Presque Isle, 181 Main Street, Presque Isle, ME 04769. *Corresponding author - bwwillia@ualberta.ca. 200 Southeastern Naturalist Vol. 10, No. 2 single site in Sevenmile Creek of the Mill Creek drainage, southeast of Nashville, TN (N36.09118, W086.71908). One specimen of Cambarus tenebrosus Hay was collected from a small tributary of Cedar Creek in Lebanon (N36.18833, W86.37845). Four sites were sampled in Overton County, from which between one and six crayfish of each species were taken, including specimens of Orconectes placidus (Hagen) from Morgan Creek (N36.46165, W085.41045) and Mill Creek (N36.47256, W85.42017) in Standing Stone State Park, Cambarus tenebrosus from Mill Creek in the Standing Stone State Forest (N36.46253, W085.37810), and Cambarus graysoni Faxon, C. tenebrosus, and O. placidus from Flat Creek (N36.36089, W85.43347). Live branchiobdellidans were removed from the host, prepared as wet mounts, and examined under a compound microscope using bright-field illumination (Govedich et al. 2010). Branchiobdellidans were preserved by placing them directly in 100% ethanol, and selected specimens were cleared with methyl salicylate, infiltrated with Canada balsam, and mounted on slides. Subsequent microscopical examination was carried out under differential interference contrast illumination using a Nikon microscope. Line drawings were produced with the aid of a drawing tube. Mounted specimens of Cambarincola bobbi Holt were loaned to one author (S.R. Gelder) from the National Museum of Natural History, Smithsonian Institution, Washington DC (USNM catalog numbers are given in text below) for comparison with the newly collected branchiobdellidans. Results and Discussion Although the majority of branchiobdellidan species were identified through live preparations shortly after collection, a few groups remained problematic and warranted detailed examination following permanent mounting. The morphology of specimens in the latter groups appeared to differ sufficiently from, or display characters absent in, the closest published descriptions. Subsequent examinations of permanently mounted specimens resulted in species identifications, but the problems encountered elicited a need to emend the species description of C. bobbi as well as describe a new species of Pterodrilus. In view of the latter, we present a revised diagrammatic key and comments on selected members of the genus Pterodrilus. Both C. bobbi and the new Pterodrilus species were found to live in sympatry with additional species, which are reported below. Re-description of Cambarincola bobbi Holt, 1988 (Fig. 1A—G) Brief description. Total length about 2.0 mm, head tubular, width about equal segment 1, body slim terete, dorsal ridges absent; dorsal projections absent, supernumerary muscles absent; lateral paired lobes segments 8 and 9 absent; dorsal lip four lobes, lateral lobes two pairs, ventral lip median emargination; oral papillae present; jaws similar size, shape triangular, teeth median larger, laterals small, dental formula 5/5; pharyngeal sulcus one pair; glandular atrium thick tubular, curved, length 0.7x segment diameter, deferent lobes absent; prostate gland tubular slim, ends mid-glandular atrium, differentiated, ental bulb present; muscular atrium tubular, length 0.3x segment diameter; bursa elongate 2011 B.W. Williams and S.R. Gelder 201 pyriform, length 0.4x segment diameter, penial sheath ental 0.4 of bursa, penis ovoid, protrusible; spermatheca club-shaped, length 1.0x segment diameter, duct shape tubular thick, length 0.5x organ, bulb shape ovoid, length 0.5x organ, ental process absent. Variations. The only variation reported by Holt (1988) was the “extent of reflexion of the spermiducal gland” (= glandular atrium) which can be best explained as a preparation artifact rather than actual variability. The triangular jaws in specimens from Virginia do not extend laterally as far as those from Tennessee (Fig. 1D—E). The male genitalia ranges from almost completely filling segment 6 in the type description to filling about half of segment 6 (Fig. 1G); component organs vary proportionately in size. The bursa varies from ovoid to pyriform. Apparent variations in the length of the spermatheca are due to folding. Microhabitat. On the cephalothorax. Host. Cambarus bartonii bartonii Fabricius, C. tenebrosus. Distribution. Overton and Wilson counties, TN; Augusta, Rockingham, and Shenandoah counties, VA. Figure 1. Cambarincola bobbi. A. lateral view of a whole specimen (redrawn and modifi ed from Holt, 1988), scale bar = 0.2 mm; B. dorsal view of Tennessee specimen, scale bar = 0.2 mm; C. ventral view of peristomium, scale bar = 40 μm; D. oblique view of jaws, teeth shown as they appear on pharyngeal surface (dorsal over lower jaw, teeth pointing posteriorly, USNM 99584), bar = 20 μm; E. dorso-ventral and F. lateral views of jaws in Tennessee specimens, scale bar = 20 μm; G. dorsal view of spermatheca and male genitalia in the left sides of segments 5 and 6, respectively (b = bursa; eb = ental bulb; ga = glandular atrium; ma = muscular atrium; pg = prostate gland; sb = spermathecal bulb; sd = spermathecal duct), scale bar = 40 μm. 202 Southeastern Naturalist Vol. 10, No. 2 Note. The gut contents in the NMNH specimens included diatom frustules and small amounts of detritus. In addition to debris already noted, specimens from Tennessee contained clay and silt particles, an oligochaete, head of a chironomid larva, and fibers that appeared to be muscles due to their birefringence. Justification for accepting variations. Dorsal ridges and supernumary muscles are absent from the material examined by Holt (1988) (Fig. 1A). As Holt routinely preserved specimens in 70% ethanol with formalin (Holt 1960:169), the appearance of raised major annuli or ridges in our Tennessee specimens (Fig. 1B) is likely to have been caused by general body contraction due to preservation in 100% ethanol. The peristomium consists of four lobes on the dorsal lip, a median emargination on the ventral lip, and two pairs of small lateral lobes; the latter are not always easy to resolve (Fig. 1C). Holt (1988:795) recorded only four dorsal lobes with some distortion that caused the ventral lip to appear to have two lobes. Details on the jaws were limited to a lateral view (Fig. 1D of Holt 1988), and a dental formula of 5/5. The shape of the jaws was revealed to be triangular only after examination of a paratype (USNM 101500) and other specimens (USNM 99584) prepared by Holt (Fig. 1D; S.R. Gelder, pers. observ.). These data are consistent with additional details provided in Figure 1E–F. The male genitalia in the type description were shown (Fig. 1B of Holt 1988) as nearly filling the lateral area of segment 6. Although such an aspect was observed in one of the Tennessee specimens, two others appeared to fill only half the segment (Fig. 1G), with the component organs being of proportional size. A similar size difference was used to justify the separation of Cambarincola osceola Hoffman from Cambarincola vitreus Ellis (Hoffman 1963). However, this view has subsequently been considered questionable, and it has been proposed that C. osceola and C. vitreus are conspecific (Holt and Opell 1993:265). The length of the glandular atrium of C. bobbi varies from 1.0x segmental diameter to 0.6x and is directly related to the size of the male genitalia. Holt (1988:794) described the bursa as oval and the penial sheath as occupying its ental half. Our specimens show a pyriform bursa with a slightly shorter ental penial sheath of 0.3x to 0.4x the bursa’s length. The penis was described as “membranous” (Holt 1988), further elaborated as “distinctly less muscular … with a few strands (? muscular)”, but Fig. 1C of Holt (1988) did not include actual details. In the Tennessee specimens, the penis was observed to be subspherical, composed of a few interstitial spaces between fibers parallel to the central penial duct, rather than the usual dense, conical Cambarincola arrangement. Whether the subspherical penis allows the internal fibers to separate rather than being compressed into a conical configuration is not known; our observations agree with the above quoted statement made by Holt. All reports of the relative length of the spermatheca are consistent; however, the duct was not as thick nor the bulb as large in the Tennessee specimens relative to what was reported in the type description. The thickness of the duct can be explained by contraction caused by the preserving ethanol, whereas the bulb varies with the volume of sperm it contains (Fig. 2G). As a result of all of the above factors, the preceding brief description was emended from Holt (1988). 2011 B.W. Williams and S.R. Gelder 203 Initially, Holt recognized a spherical penis with fibers and interstitial spaces which led him to tentatively label three slides with multiple specimens, “Oedipodrilus bobbi”. Unfortunately the label names were not replaced by the subsequent and different published name, and have since become part of the NMNH catalog (USNM 99560, 99584, 99595). Therefore, this name is considered nomina nuda (ITZN 1999:glossary p. 111); a Latin term for “names without a description.” As a result, it does not represent a valid species. The USNM specimens were collected from Augusta and Rockingham counties, VA, despite an inadvertent statement that C. bobbi was only known from the type locality in Shenandoah County, VA (Holt 1988). Pterodrilus robinae, new species (Fig. 2A–D) Type specimens. The holotype (NBM-007151) and three paratypes (NBM- 007152, NBM-007153, NBM-007154) were deposited in the New Brunswick Museum, Saint John, NB, Canada; three additional paratypes were retained by the authors. Specimens were obtained from Orconectes durelli taken from Sevenmile Creek in the Mill Creek system of the Cumberland River Watershed (N36.09118, W086.71908) on the property of Ms. Robin M. Evans by David Ian Withers on 5 May 2010, and David Ian Withers and Hannah Sophia Withers on 16 May 2010. Diagnosis. Length about 0.73 mm, head width slightly wider than segment 1, body slim pyriform, segments distinct, prominent transverse ridges segments 3 through 8, one pair unbranched digitiform appendages segments 4 and 5, five unbranched appendages segment 8, supernumery muscles present; dorsal lip smooth, lateral lobes absent, ventral lip median emargination; oral papillae present; jaws small, subrectangular, teeth small, dental formula 3/2; pharyngeal sulci one pair; glandular atrium ovoid, length 0.5x segmental diameter, deferent lobes absent; Figure 2. Pterodrilus robinae new species. A. drawing of the holotype, scale bar = 0.1 mm; B. ventral view of the jaws, dorsal over ventral, with teeth pointing posteriorly from a paratype; scale bar = 16 μm; C. male reproductive system in segment 6 (left) and spermatheca in segment 5 (right) in the holotype, with a double-headed arrow showing the observed variation in the start of the differentiated (vacuolar) epithelial cells of the prostate gland in the type series (organs as labeled in Fig. 1), scale bar = 30 μm; D. spermatheca in a paratype showing a thickened duct and enlarged bulb compared to that in the holotype; same scale as C. 204 Southeastern Naturalist Vol. 10, No. 2 prostate arising third from ectal end, terminates subterminal, differentiated, ental bulb absent; muscular atrium slim terete, length 0.2x segment diameter; bursa pyriform, length 0.6x segment diameter, penial sheath ental third, penis cone-shaped, protrusible; spermatheca length 1.2x segment diameter, duct tubular, length 0.6x organ, bulb tubular, length 3x organ, ental process 0.1. Etymology. Named for Ms. Robin M. Evans, who kindly granted permission to collect crayfish on her property. The species epithet is derived from Robin according to the International Code of Zoological Nomenclature (ICZN) ( ITZN 1999:Art. 31.1.2). Description. The anterior nephridial pore opens dorso-medially on body segment 3, the vasa deferentia enter the glandular atrium entally, and the prostate gland arises from the ectal third of the glandular atrium; all three characters confirm the species as a member of the genus Pterodrilus. Seven fixed adult specimens averaged 0.73 mm in length, ranging from 0.58 to 0.83 mm long. Live specimens measured approximately 1.1 mm in length. The head is slightly wider than segment 1 and distinct from the slim pyriform body. Segments 3 through 8 have prominent transverse ridges across the dorsal and lateral surfaces, and all ridges contain supernumary muscles. Segments 4 and 5 each support one pair of dorsal unbranched, digitiform appendages separated medially. Segment 8 displays a pronounced dorso-lateral ridge on which five unbranched digitiform dorsal appendages appear in a fan-like arrangement (Fig. 2A). The peristomium consists of a dorsal lip with a smooth margin, no lateral lobes, and a ventral lip with a median emargination. Oral papillae surround the mouth. The jaws are small, with a width less than 0.2x the head’s diameter, and with the dorsal slightly larger than the ventral. Both jaws are subrectangular, slightly concaved with sharply pointed teeth and a dental formula of 3/2 (Fig. 2B). The lateral margins of each jaw have a “shoulder” where an additional tooth would be expected. However no specimens were found with a dental formula of 5/4, and there was no evidence that a lateral tooth had fractured from the jaw base. The pharynx has one pair of sulci. The male reproductive organs in segment 6 seen from the lateral aspect extend across most of the segment (Fig. 2C). The glandular atrium is ovoid to sacculate and with a length of approximately 0.6x the diameter of the segment. The prostate gland is tubular, arises from the ectal third of the glandular atrium, then ends approximately terminal to it. The prostate gland is lined with highly vacuolated or “differentiated” cells. However, in two of the seven specimens examined, differentiated cells started about 0.2x in from the ectal end of the prostate gland as shown in Figure 3 with a double-headed arrow. An ental bulb is absent. The muscular atrium is terete, with a length of 0.3x segment diameter. The bursa is pyriform and about 0.6x the segment diameter in length. The ental third of the bursa forms the penial sheath, which surrounds the conical, protrusible penis. The spermatheca in segment 5 has a length approximately 1.2x that of the segment’s diameter. The duct is 0.6x the total length of the organ, with the bulb varying from tubular to subspherical and 0.3x in length (Fig. 2C–D). An ental process is present. 2011 B.W. Williams and S.R. Gelder 205 Variations. The only observed variations are in the start of the differentiated cells in the prostate gland and the shape of the spermathecal bulb, which is a reflection of the amount of sperm held in the organ. Microhabitat. Specimens were observed on the cephalothorax region and chelae. Individuals appeared to prefer clefts and depressions along the edge of the carapace and joints at the base of the chelae. Distribution. Specimens known only from the type location. Host. Orconectes durelli. Other branchiobdellidans on the host. Cambarincola philadelphicus (Leidy). Justification of the new species. Pterodrilus robinae differs from all other species in the genus with its dental formula of 3/2 rather than 5/4. As a result, a dental formula of 5/4 (Gelder 1996a) is no longer a generic character. Five digitiform appendages on the dorso-lateral ridge of segment 8 are common to five species: Pterodrilus robinae, Pterodrilus distichus Moore, P. mexicanus Ellis, P. cedrus Holt, and P. hobbsi Holt. However, P. robinae and P. distichus are the only species with pairs of unbranched dorsal digitiform appendages anterior to segment 8; while these appendages extend from segments 2 to 7 on P. distichus (Fig. 3A), they are restricted to segments 4 and 5 on the new species (Fig. 2A, 3B). Relative lengths of the various male organs are used extensively in defining branchiobdellidan species. In P. distichus, the prostate gland ends subterminal to the glandular atrium, while in P. robinae the ental tips of both glands end equally. When viewed in lateral aspect, the male genitalia of P. robinae nearly fill segment 6, whereas only about half the segment is filled in P. distichus. Remarks. The dorsal ridges and supernumary muscles are not easy to see when live specimens extend themselves or when preserved specimens curl. All of the specimens collected had large amounts of detritus with a few ciliates adhering to their dorsal and lateral surfaces. One ciliate is attached to the dorsal surface of segment 4 on the holotype and was initially thought to be one of the dorsal digitiform appendages. The detritus had the same composition as that on the surface of the crayfish host and consisted of a mixture of clay particles, live diatoms, and unidentifiable organic-appearing material. The external debris on the branchiobdellidans did not interfere with locomotion. Observations of gut contents showed that the branchiobdellidans had ingested the detritus already described, but in varying amounts and composition. Differences in the observed detrital composition in the gut were likely a result of digestion, with only indigestible residues remaining as visible components. Identification and distribution of selected Pterodrilus species Excluding monotypic genera in North America, Ceratodrilus and Pterodrilus are unique in that their species can be identified using only external characters. Pterodrilus species identifications are based on presence or absence of dorsal segmental ridges caused by supernumerary muscles, and the presence and sequence of dorsal segmental appendages or their absence. Species descriptions are based on preserved specimens where the ridges are enhanced due to the contraction of underlying muscles. Therefore, while the ridges are 206 Southeastern Naturalist Vol. 10, No. 2 still visible in live specimens, they are not as obvious. Consequently, the five Pterodrilus species anticipated in the Cumberland River Watershed are illustrated in Figure 3, with the addition of the new species described during the present study. Digitiform appendages on segments 2 to 7 are found on P. distichus (Fig. 3A), but only on segments 4 and 5 on P. robinae (Fig. 3B). Dorsal appendages are absent in P. annulatus Gelder and P. choritonamus Holt, with dorsal ridges prominent on segments 2 to 7 in the former (Fig. 3C) and absent in the latter species (Fig. 3E). The five digitiform appendages on the segment 8 ridge of P. cedrus (Fig. 3D) may be partially obscured or folded, resulting in specimens being misidentified as P. choritonamus. The five appendages on segment 8 in P. hobbsi are fan-like (Fig. 3F), but often appear more digitiform and similar to the segment 8 appendages of P. cedrus. Consequently, it is important to establish the presence or absence of dorsal ridges as a secondary character to distinguish between the two species; if ridges are found, the specimen is P. cedrus, whereas it is P. hobbsi if dorsal ridges are absent. The new species, P. robinae, was found only at a single location, although there is every reason to expect a wider distribution as additional sites are sampled. The genus Pterodrilus is relatively widespread, ranging from New York State and Michigan into northern Georgia. The most widespread species is P. distichus, extending from the northernmost edge of the pterodrilid distribution to Kentucky, with the remaining species forming generally separate groups from Arkansas-Missouri to the Appalachian Mountains in Virginia–North Carolina and Georgia. In a restricted review of mid-Eastern States, Holt (1969) identifi ed three species—P. alcicornus Moore, P. hobbsi, and P. simondsi Holt—in Figure 3. Drawing of the dorso-lateral surfaces of the body segments of five species of Pterodrilus: A. P. distichus, B. P. robinae, C. P. annulatus, D. P. cedrus, E. P. choritonamus, and F. P. hobbsi; scale bar 0.2 mm (redrawn and modified from Gelder 1996b, except B). 2011 B.W. Williams and S.R. Gelder 207 the Appalachian Mountains ranging from Kentucky–Virginia to Georgia–North Carolina, and three species—P. cedrus, P. hobbsi, and P. choritonamus—in a relatively small area around Overton County, TN. The presence of P. hobbsi in two apparently disjunct areas indicates that it might be more widely distributed than current records indicate. Pterodrilus cedrus was also recorded in Missouri (Holt 1968), suggesting a distribution similar to that of P. annulatus, which has also been reported in Missouri and central Tennessee (Gelder 1996a). Additional pterodrilid diversity is also expected from the mid-eastern region of the USA. The Tennessean P. robinae was compared to a limited description of an undescribed species of Pterodrilus referred to in Holt (1968:32) from Union County, GA and Cherokee County, NC. Significant differences indicate that Holt’s specimens remain undescribed. Observations on sympatry and distribution of branchiobdellidans Only two branchiobdellidan species, C. philadelphicus and the newly described P. robinae, were found on specimens of O. durelli from Sevenmile Creek near Nashville. Cambarincola bobbi was the only branchiobdellidan species found on Cambarus tenebrosus in an unnamed tributary to Cedar Creek in Lebanon. In contrast, the species diversity observed among the four Overton County collection sites totalled ten branchiobdellidan species from five genera, although the number of branchiobdellidan species per host species and site ranged from one to six. Two of the Overton County sites were located in Standing Stone State Park (SSSP), namely Morgan Creek at the head of the southeast arm of Standing Stone Lake and Mill Creek below the Standing Stone Lake dam at the head of the northwest arm. An additional collection site in Mill Creek was located 4 km southeast and upstream of the Park’s lake in Standing Stone State Forest (SSSF). The fourth Overton County site in Flat Creek was approximately 14 km south of Standing Stone Lake and separate from the Park’s drainage. Cambarincola bobbi was found at Mill Creek in SSSF on Cambarus tenebrosus and at Flat Creek on C. tenebrosus and C. graysoni. Branchiobdellidan species found in sympatry with Cambarincola bobbi were, respectively, Ankyrodrilus legaeus Holt, P. choritonamus, Bdellodrilus illuminatus (Moore) and Xironodrilus formosus Ellis at Mill Creek; Ankyrodrilus koronaeus Holt, A. legaeus, Cambarincola holti Hoffman, P. cedrus, and P. hobbsi on Cambarus tenebrosus hosts at Flat Creek; and A. legaeus, Cambarincola branchiophilus Holt, C. holti, and P. cedrus on Cambarus graysoni hosts at Flat Creek. Orconectes placidus specimens were collected from three sites and carried P. cedrus, P. hobbsi, and X. formosus at Morgan Creek; P. cedrus at Mill Creek below the dam; and A. legaeus and P. cedrus at Flat Creek. If the collection from the Cedar Creek tributary near Lebanon is ignored, a lower diversity of branchiobdellidans was observed on Orconectes hosts, ranging from one to three, relative to the four to six branchiobdellidan species found on Cambarus hosts examined during this study. However, the Lebanon collection with one branchiobdellidan species on C. tenebrosus illustrates that ectosymbiont sympatry is complex and a result of many factors. 208 Southeastern Naturalist Vol. 10, No. 2 The occurrence of branchiobdellidan species reported in our Overton County collections present interesting qualitative results which are independent of the host’s genus. Four species, A. koronaeus, B. illuminatus, C. branchiophilus, and P. choritonamus, were only observed at a single site and on a single host. None of these species are limited in distribution to the collection area. Indeed, the known range of B. illuminatus extends from New Brunswick, Canada to Alabama, USA. In contrast to our single host and site observations, P. cedrus was found on all hosts at three of the four sampled sites, and A. legaeus occurred on all host species examined from Flat Creek and Mill Creek (SSSF). Both P. cedrus and A. legaeus are endemic to the mid-eastern region of the USA. Similar observations on ectosymbiont- host diversity, including B. illuminatus on four different host crayfish, were reported from the Mountain Lake region, VA, by Hobbs et al. (1967). These data refute the idea of wide-spread host-specificity as periodically suggested (Brown and Creed 2004, McManus 1960). The assertion of host-specificity also ignores the successful adoption of exotic crayfish by native branchiobdellidans in Europe (Ďuriš et al. 2006, Gelder 2006). The most unusual host-branchiobdellidan symbiosis reported to date involves Cambarincola mesochoreus Hoffman and Cambarincola pamelae Holt, usually found on Procambarus clarkii Girard, adopting Callinectes sapidus Rathbun (Blue Crab) as hosts in estuarine habitats in the southeastern USA (Overstreet 1983) and Chesapeake Bay, MD (Gelder and Messick 2006). The distribution of branchiobdellidan species in North America (Gelder et al. 2002) is a result of both general sampling, like Perry C. Holt’s circum-USA journey (P.C. Holt, Virginia Polytechnic Institute and State University, Blacksburg, VA, pers. comm.), as well as collections within more restricted or disjunct areas. The discovery of C. holti in Overton County, TN (Gelder 1996b) showed that the species was not restricted to its type location in Kentucky as previously thought. Similarly, prior to this study, C. bobbi was only known from a restricted area of northern Virginia. As additional studies are conducted, the distribution of branchiobdellidan species will be further emended and the extent of species sympatry better understood. Acknowledgments The authors thank David Ian Withers and Hannah Sophia Withers for their supply of crayfish, and Ms. Robin M. Evans for permission to collect on her property. We are grateful for support from the Tennessee Department of Environment and Conservation - Natural Heritage Program, Tennessee Wildlife Resources Agency, and the Tennessee Department of Agriculture, Forestry Division, for providing permission to collect crayfish, and for the help and hospitality while at Standing Stone State Park. We would also like to extend great appreciation to Dr. and Mrs. Ralph O. Brinkhurst for their support and generosity. Literature Cited Brinkhurst, R.O., and S.R. Gelder. 2001. Annelida: Oligochaeta and Branchiobdellida. Pp. 431–463, In J.H. Thorpe and F. Covitch (Eds.). 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