Cambarus (Cambarus) hatfieldi, Loughman, Zachary J., Fagundo, Raquel A., Lau, Evan, Welsh, Stuart A. & Thoma, Roger F., 2013

Loughman, Zachary J., Fagundo, Raquel A., Lau, Evan, Welsh, Stuart A. & Thoma, Roger F., 2013, Cambarus (C.) hatfieldi, a new species of crayfish (Decapoda: Cambaridae) from the Tug Fork River Basin of Kentucky, Virginia and West Virginia, USA, Zootaxa 3750 (3), pp. 223-236 : 224-235

Cambarus (Cambarus) hatfieldi n. sp.

Figures 1–5 , Tables 1–4

Diagnosis. Body and eyes pigmented. Posterior dorsal region of rostrum concave and deflected anteriorly. Rostrum margins thickened, parallel to base of acumen. Acumen distinctly triangular with prominent dorsally deflected spiniform tubercle at terminus. Areola 2.7–4.5 (x = 3.3, n = 52, SD = 2.5) times as long as wide with 7–9 (mode = 7) punctations across narrowest point. Cervical spines absent. Mandibular, branchiostegal, and orbital regions of carapace with well-developed tubercles. Postorbital ridges short; spiniform, dorsally deflected tubercle present in juveniles and subadults; adult postorbital ridges terminating in either spiniform or truncated tubercles. Suborbital angle acutue. Antennal scale widest in middle, 0.9–2.6 (x = 1.9, n = 52, SD = 0.8) times as long as wide. Total carapace length (TCL) 1.6–2.2 (x = 1.9, n = 52, SD = 0.3) times longer than width. Form I and II males possessing hooks on ischium of third pereopods only; hook gently curved at apex, overarching basioischial joint in form I males, not reaching basioischial joint in form II males; hooks not opposed by tubercle on basis. Mesial surface of palm of chelae with single row of 5–9 (x = 7.7, n = 52, SD = 0.8) weakly developed tubercles; deep punctations found lateral to tubercles. Dorsal longitudinal ridge of dactyl consisting of several moderately-developed scattered tubercles. Dorsomedian ridge of fixed finger of propodus pronounced. Poorly defined lateral impression at the junction of the fixed finger portion of the propodus. Dactyl and fixed finger with sharp corneous tip. Form I male palm length 63.4–70.8 % (x = 67.5%, n = 12, SD = 2.3%) of palm width, form I male palm length 22.4–33.2% (x = 29.8%, n = 12, SD = 2.8%) of total propodus length; female dactyl length 52.6–62.3% (x = 58.4, n = 21, SD = 2.8%) of total propodus length.

First pleopod of form I male with short terminal elements. Central projection gently tapering distally; recurved> 90º to main shaft of gonopod, with distinct subapical notch. Mesial process directed 90° to shaft, bent cephalolaterally, inflated at its base, tapering to distinct caudal point at or slightly beyond terminance of central projection. Neither process of first pleopod projecting significantly beyond caudal margin of gonopod shaft. Annulus ventralis immovable; distinctly asymmetrical posteriorly; cephalic portion with median trough leading to strongly sculptured central fossa; exaggerated “S” bend in sinus terminating at caudal edge formed by two asymmetrical hardened ridges.

Description of holotypic male, form I. ( Figs. 1 A–C, F–I, K–L, 2; Table 1 ). Body compressed dorsoventrally ( Fig. 1 A); carapace posterior to cervical groove wider than abdomen. Total carapace length 38.9 mm; postorbital carapace length 33.0 mm. Areola 3.8 times longer than wide, 7 punctations across narrowest part ( Fig 1 G); length of areola 36.2 % of TCL (42.7 % of postorbital carapace length (PCL). Rostrum weekly excavated; margins thickened, continuous to base of acumen, parallel at midpoint and flared at base; floor of rostrum with numerous punctations. Rostrum 1.8 times longer than wide. Acumen distinctly triangular, ending in dorsally oriented corneous tip ( Fig. 1 A). Postorbital ridges developed, short, terminating in weak dorsally oriented cephalic tubercles. Suborbital angle acute, lacking tubercle ( Fig. 1 A). Cervical spine/tubercles absent. Mandibular, branchiostegal, and orbital regions of carapace punctated with well-developed tubercles; highest tubercle density in hepatic region.

Abdomen supraequal in length to carapace, pleura rounded cephaloventrally, angled distoventrally. Lateral margin of terga angulate; lateral margin of second pleura deeply furrowed. Cephalic section of telson with 2 large spines in each caudolateral corner. Proximal podomere of uropod with distal spine on mesial lobe; mesial ramus of uropod with median ridge ending distally in distomedian spine not overreaching margin of ramus; laterodistal spine pronounced. Distal margin of proximal segment of lateral ramus of right uropod having 10 immovable, small spines and 1 lateral, large, movable spine.

Cephalomedian lobe of epistome subtriangular, zygoma moderately arched ( Fig. 1 K); cephalolateral margins thickened, forming sharp angle at junction with endostyle ( Fig. 1 K). Body of epistome possessing prominent cephalomedian fovea. Antennal scale broadest in middle ( Fig. 1 F); lateral margin thickened, terminating in large corneous spine; setiferous. Right antennal scale 5.5 mm long, 2.5 mm wide ( Fig. 1 F). Tip of right antenna reaching middle of telson when adpressed.

Mesial surface of right chela palm with single row of 9 tubercles ( Fig. 1 H), length 69.5 % of width; depth 8.6 mm, ventral surface with 0 subpalmar tubercles. Dorsal longitudinal ridge of dactyl developed, mesial margin possessing moderate equal-sized tubercles ( Fig. 1 F). Dorsomedian ridge of fixed finger of propodus pronounced. Poorly defined lateral impression at junction of fixed finger with the propodus. Dactyl and fixed finger of propodus with sharp, corneous tip.

Carpus with prominent dorsal furrow ( Fig. 1 H) and 6 weak dorsomesial tubercles; rest of surface with some setiferous punctations; mesial margin with large, procurved spine at midlength, subtended by reduced proximal spine. Distodorsal surface of merus with 8 spiniform tubercles; ventrolateral ridge with 3 small spines and large, corneous distal spine; ventrolateral margin of ischium with 2 small, spiniform tubercles. Carapace depth less than width. Hook on ischium of third pereopods only ( Fig. 1 L.); hook gently curved at apex, overarching basioischial joint, not opposed by tubercle on basis. Form I gonopod as described in diagnosis ( Fig. 1 C–D); tip reaching anterior margin of fourth caudomesial boss.

Description of allotypic female. ( Fig. 1 J, Table 1 ). Differing from holotype in following respects; carapace depth less than carapace width (15.8 and 20.2 mm, respectively); TCL 37.7 mm, PCL 32.0 mm. Areola 37.1% of TCL (43.8% of PCL), 3.7 times as long as wide. Rostrum 1.3 times longer than wide. Abdomen length 39.9 mm; abdomen width 45% of abdomen length. Mesial surface of palm of chelae with single row of 8 weak tubercles. Palm length (8.6 mm) 66.1% of palm width (13.0 mm); depth of palm 8.0 mm. Antennal scale 5.9 mm long, 2.7 mm wide. All measurements and counts from right chela. Annulus ventralis as described in diagnosis ( Fig. 1 J); width of postannular sclerite 33.0% total width of annulus ventralis; first pleopods uniramous, reaching central region of annulus ventralis when abdomen flexed.

Description of morphotypic male, form II. ( Fig. 1 D–E, Table 1 ). Differing from holotype in the following respects: TCL 26.0 mm and PCL 21.8 mm. Areola length 35.3% of TCL (42.2% of PCL), 5.1 times longer than wide. Rostrum margins subparallel to base of acumen; rostrum 1.9 times as long as wide. Abdomen 14.0 mm long. Mesial row of tubercles on palm of chela with 8 tubercles. Palm length (6.2 mm) 69.7% of palm width (8.9 mm). All measurements and counts from right chela. Antennal scale 2.2 mm long, 0.9 mm wide. Gonopods 23.4% of TCL length. Central projection with complete apex rounded ( Fig. 1 D–E). Mesial process bluntly tapered, bulbous at base. Hook on ischium of third pereopod small, not reaching basioischial joint.

Size. Form I male (n = 12) TCL size range 19.0– 40.1 mm (PCL 23.5–34.7 mm), mean TCL of 33.5 mm. Form II male (n = 19) mean TCL 32.3 mm, size range 18.4–36.6 mm (PCL 18.5–31.7 mm). Non-ovigerous female (n = 21) mean TCL 34.3 mm, size range 26.6–42.8 mm (PCL 21.9–36.5 mm). Ovigerous female (n = 4) TCL size range 26.1–39.5 mm, mean TCL of 32.8 mm. The largest specimen examined was a form I male with a TCL of 40.9 mm (PCL 34.7 mm).

Color. Carapace ground color orange-brown to pink-brown; posterior and anterior margins of carapace dark. Hepatic and antennal region of carapace punctuated with yellow, white, or cream tubercles. Postorbital ridge reddish to orange-brown. Rostrum margins and acumen distinctly orange to red-orange. Cephalic section of carapace immediately anterior to and including cervical groove black; mandibular abductor scars ranging from black to brown. Lateral margin of antennal scale gray-blue to gray; body of antennal scale gray to blue-gray. Antennal flagellum and antennules blue-gray, with olivaceous hue; dorsal surface of lamellae green-brown to bluebrown; ventral surface tan to olivaceous. Dorsal surface of chelae green, olive green to brown-orange with green highlights, olivaceous to orange mottling; mesial surface of dactyl tubercles amber, orange or red-brown. Denticles on opposable surfaces of fingers yellow, white, or tan. Ventral surface of chelae orange or orange-brown. Dorsal surface of carpus brown, olivaceous or green-brown; occasionally orange; region adjacent to and including furrow olivaceous orange to green; carpus spine orange. Merus orange-brown, green-brown, or olivaceous brown. Podomeres of pereopods light blue, blue-green, or blue-gray; joints of pereopod podomeres white. Dorsal and dorsolateral surface of abdomen olivaceous, green-brown or brown; tergal margins brown, reddish brown or crimson red. Uropods green-brown, with olivaceous tint; margins gray to brown. Ventral surface of abdomen and carapace tan. Dorsal ridge of form I gonopod central projection amber; body of central projection, gonopod, and mesial process tan. Form II gonopod and all associated processes cream. Cephalic portion of annulus ventralis pink to pink-cream; ridge of fossa pink; caudal region of annulus ventralis ranges from pink to cream colored.

Type locality. Mate Creek at CR 6 crossing in Red Jacket, Mingo County, West Virginia (37.64807º N, - 82.13524º W). The holotype, allotype, and morphotype were all collected mid channel in a riffle underneath large slab boulders in 0.75 m of water. Mate Creek ranged between 9.0–15.0 m wide, and 0.3–1.0 m deep when the typeseries was collected. Stream substrate was composed primarily of gravel, cobbles, boulders and slabs. Anthropogenic impacts included siltation, channelization, and bank erosion. Coal fines were also prevalent in situations with sands and gravels. Cambarus hatfieldi also was associated with coarse woody debris snags and leaf packs. The holotype, morphotype, and allotype were collected on 20 Mar 2013 by K. R. Loughman, C. Z. Loughman, and ZJL.

Disposition of types. The holotype, allotype, and morphotype are deposited in the National Museum of Natural History (USNM), Smithsonian Institution, Washington, D. C. (catalogue numbers USNM 122861 122862, 122863, respectively). Paratypes are deposited in the following museums and collections: Carnegie Museum of Natural History, Pittsburgh, PA (CMNH 3829.1, 3829.2), and West Liberty University Astacology Collection, West Liberty, WV (WLU 2000).

Range and specimens examined. Cambarus hatfieldi appears to be endemic to the Tug Fork River system and its associated tributaries in Kentucky, Virginia and West Virginia. Kentucky’s population occurs in Pike and Martin counties, with substantial populations occurring in Blackberry and Pond Creeks ( Fig. 3 ). Virginia populations are confined to the Dry Fork watershed in Tazewell Co. West Virginia’s populations are limited to McDowell, Mingo and Wayne counties, with stable populations occurring in Dry Fork, Mate, Panther, and Horse Creeks. All three states share the Tug Fork mainstem population. Cambarus hatfieldi appears to be replaced by Cambarus theepiensis in lower reaches of the Tug Fork River where the river loses gradient and gains sandy substrates.

All West Virginia collections are housed in the West Liberty University Astacology Collection, denoted with the prefix WLU and were collected in the summer of 2009 unless otherwise noted. West Liberty University field crew members for 2009 included ZJL, SAW, Nicole L. Garrison, David A. Foltz, Evan I. Hewitt, and Mathew I. M c Kinney. All Kentucky and Virginia specimens examined were collected by RFT and are housed in the Ohio State University Museum of Biological Diversity Crustacean Collection. Abbreviations are defined as follows: CR = county road; I = interstate; KY = Kentucky state highway; mi = miles; RD = road; US = U. S. route; VA = Virginia state highway; WV = West Virginia state highway; Rd = road; F = female; OF = ovigerous female; IM = Form I male; IIM = form II male; JV = juvenile.

A total of 256 specimens were examined from the following 24 localities. KENTUCKY: Martin Co: (1.) OSUM 7423, Knox Creek upstream of confluence with Tug Fork and intersection of Woodman Creek Road & unnamed road, 1.2 mi. NW of Woodman, 18 Sep 2009, 1 F. Pike Co: (2.) OSUM 7423, Knox Creek upstream of confluence with Tug Fork River and intersection of Woodman Creek Road & unnamed road 1.17 mi. NW of Woodman, 17 Sep 2009, 1 F. (3.) OSUM 7428, Peter Creek at Freeburn adjacent to KY 194 1.2 mi. W of Vulcan, 17 Sep 2009, 1 JV. (4.) OSUM 7431, Blackberry Creek adjacent KY Rt. 1056 2.26 mi. S of Matewan, 17 Sep 2009, 1 F, 2 IIM. (5.) OSUM 7423, Pond Creek at Sherondale adjacent US. 119 3.9 mi N of Pinsonfork, 17 Sep 2009, 6 F, 2 IIM, 9 JV. (6.) OSUM 7441, Big Creek adjacent to KY 468 at confluence of Lick Branch 1.29 mi. SW of Nolan, 18 Sep 2009, 3 JV. VIRGINIA: Tazewll Co.: (7.) OSUM 8737, Dry Fork of Tug Fork at VA Rt. 636 bridge; 1.42 Km NNW of Rourkes Gap, 10.53 Km NW of Tazewell, 28 Jul 2011, 2 F, 3 IIM, 23 JV. WEST VIRGINIA: McDowell Co: (8.) WLU 2, Crane Creek adjacent to CR 5/02, 2.0 mi SE of Avondale, 11 Jun 2009, 10 F, 5 IIM. (9.) WLU 3, Elk Horn Creek 0.1mi NW of intersection of Coe Street and E Main Street in Kimball, 13 Jun 2009, 2 F, 2 II M. (10.) WLU 7, Horse Creek adjacent to CR 1–4, 0.5 mi from Johnny Cake Road 3.4 mi SE of Panther, 10 Jun 2009, 2 OF. WLU 26, 10 Jul 2009, 22 F, 24 IIM, 16 JV. (11.) WLU 9, Dry Fork adjacent to SR 80, 2.7 mi SE of Iaeger, 1.0 mi SE of Apple Grove, 10 Jun 2009, 4 F, 2 IIM, 1 JV. (12.) WLU 10, Barrenshe Creek adjacent to CR 83–20/CR 83 intersection in Yukon, 10 Jun 2009, 1 OF, 2 F, 1 IM, 3 IIM, 2 JV. (13.) WLU 12, Tug Fork River parallel to SR 103, 0.6 mi NW of Gary, 10 Jun 2009, 3 F, 1 IIM. (14.) WLU 13, Clear Fork parallel to CR 2, 1.1 mi W of CR 2- 1 in Coalwood, 10 Jun 2009, 2 F, 8 IIM, 15 JV. (15.) WLU 23, Tug Fork at Clear Fork confluence in Roderfield, 13 Jun 2009, 9 F, 5 IIM. (16.) WLU 25, Spice Creek adjacent to US 52, 0.3 mi SE of Erin, 13 Jun 2009, 6 F, 10 IIM. (17.) Mingo Co: (17.) WLU 3, Left Fork of Gilbert Creek adjacent to CR 13 & CR 13-4 junction, 2.4 mi SE of Baisden, 11 Jun 2009, 1 F, 4 IIM. (18.) WLU 14, Pigeon Creek parallel to US 52 in Musick, 11 Jun 2009, 1 OF, 7 F, 4 IIM. (19.) WLU 16, Pigeon Creek at SR 65/ US 52 intersection 2.8 mi W of Varney, 11 Jun 2009, 1 F. (20.) TYPE SERIES (USNM 12861 holotype, USNM 122862 allotype, USNM 12863 morphotype; CMNH 3829.1,3829.2 paratypes, WLU 2000 paratypes, Mate Creek at CR 6 crossing in Red Jacket, 20 Apr 2013, 12 F, 9 IM, 3 IIM, 11 JV. (21.) WLU 18, Mate Creek parallel to CR 6, 0.1 mi NW of Mark's Branch Rd in Newtown, 11 Jun 2009. 2 F. (22.) Sycamore Creek parallel to US 52, 0.2 mi N of SR 49 in Williamson, 11 Jun 2009, 4 F, 3 IIM. (23.) WLU 20, Buffalo Creek adjacent to CR 14, at intersection with Mullberry Street in Chattaroy, 11 Jun 2009, 2 F, 1 IM, 5 IIM, 2 JV. Wayne Co: (24.) WLU 22, Mill Creek at intersection of CR 36-3/CR 34-3, 2.75 mi W of Radnor, 11 Jun 2009, 2 F, 2 IIM.

Conservation status. It is recommended Cambarus hatfieldi be listed as vulnerable (V) according to the American Fisheries Society criteria (Taylor et al. 2007), and assigned a G3 ranking according to the global conservation criteria (Masters 1991) for conservation listing as a consequence of its limited range. Cambarus hatfieldi should be listed as near threatened (NT) using the International Union for the Conservation of Nature (IUCN 2001) criteria due to its narrow distribution. All of the range of C. hatfieldi occurs in portions of West Virginia, Virginia, and Kentucky that currently undergo extensive amounts of surface mining. The majority of streams harboring C. hatfieldi experience elevated siltation rates and high conductivity due to mine drainage issues (Pond et al. 2008). At present, the impact of environmental change on C. hatfieldi populations remains unknown.

Habitat and life history notes. Cambarus hatfieldi occurs in small to large, moderate to high gradient streams with substrates composed of cobbles, boulders and slabs. Slab boulders are the preferred refuge of C. hatfieldi , though when absent course woody debris snags, leaf packs, and rootwads are readily used as shelter. As stream gradient decreases and sandy substrates pervade, C. hatfieldi numbers decline (ZJL, personal observation). In situations with these habitat parameters in lower reaches of the Tug Fork River, C. hatfieldi is replaced by C. theepiensis .

Cambarus hatfieldi male reproductive form state is seasonal. Nine percent of males collected in June and July were first form, compared to 89% collected in March and April. These results suggest the majority of males molt from form II to form I in late summer/early fall, with an additional molting event occurring in May/Jun back to form II. Females collected from Mate Creek, Mingo Co. West Virginia in March and April 2013 exhibited active glair glands. Three ovigerous females were collected from Horse Creek, McDowell Co. West Virginia and a single female with stage 1 instars and unhatched eggs was collected on 11 Jul 2009 from Barenske Creek, McDowell Co. West Virginia ( Table 4 ). Hatched eggs were still attached to the Barenske Creek females pleopods, indicating hatching had occurred very close to the time of capture (ZJL, personal observation). All ovigerous females were taken from embedded boulders in slack water environments with sandy substrates (ZJL, personal observation). Young of the year were collected in September 2011 from the same environs ovigerous females were collected from in Horse Creek during the preceding years.

Crayfish associates. Cambarus (Cambarus.) hatfieldi has been collected with Cambarus (C.) bartonii cavatus Hay, 1902 , Cambarus (Jugicambarus.) dubius Faxon, 1884, Cambarus (Puncticambarus.) veteranus Faxon, 1914 , and Orconectes (Procericambarus) cristavarius Taylor, 2000 .

Variation. Morphological variation was homeoplasic across Cambarus hatfieldi’s range, with little variation observed between similar age cohorts occurring in different streams. Ontogenic morphologic variation does occur in the following respects. Juvenile rostrums and chelae are noticeably elongate compared to adults. With age, both structures become both broader and deeper in form. Mesial margin tubercles on chelae are reduced in juveniles and become steadily more pronounced as adulthood is reached. Tubercles on the post-orbital ridge and acumen are always spinose in juveniles, compared to adults where they can either be spinose or truncated. Coloration also differs between juveniles and adults, with juvenile coloration dominated by various shades of brown. With maturity, blues, greens, and grays begin to dominate the walking legs, cephalothorax, and abdomen as described in the color section of the manuscript.

Relationships and comparisons. Cambarus hatfieldi is placed in the subgenus Cambarus based on the presence of a subapical notch in the form I gonopod and the lack of a well-developed mesial second tubercle row on the palm (Hobbs 1969). Among described members of the subgenus, C. hatfieldi is most similar to C. sciotensis and C. angularis in overall body size and shape and thickening of the rostral margins.

Meristic percentages/ratios that distinguish C. hatfieldi from C. sciotensis include areola length/carapace length, propodus length/areola length, two chelae ratios and size of the central projection and mesial process in comparison to gonopod shaft in form I-males. Areola length on average represents 30.4 % (n = 52; SE ± 3.9%) of the carapace length compared to 36.5% (n = 30; SE ± 0.1%) in C. sciotensis . Cambarus hatfieldi areola length is 2.3 (n = 52; SE ± 0.2) times the propodus length compared to 2.8 (n = 30; SE ± 0.2) times in C. sciotensis . Cambarus hatfieldi’s palm length/dactyl length ratio is smaller (x = 1.9; n = 52; SE ± 0.3) than C. sciotensis’s (x = 2.3; n = 48; SE = 2.1–2.6); palm depth to palm length ratio is also smaller in C. hatfieldi (x = 1.4; n = 52; SE ± 0.1) compared to C. sciotensis (x = 1.8; n = 30; SE ± 0.3).

The length of C. hatfieldi central projection and mesial process both extend to the margin of the shaft of the gonopod and not beyond compared to C. sciotensis with the central projection extending beyond the margin of the gonopod shaft ( Fig. 4 ). Cambarus hatfieldi can be distinguished from Scioto River C. sciotensis easily by the absence of a second row of mesial tubercles on the chelae palm, both New and Scioto River C. sciotensis also have noticeably elongate chelae compared to C. hatfieldi’s squamous, truncated chelae. Finally, C. hatfieldi’s average total body length is smaller (x = 67.6 mm; n = 52; SE ± 7.3 mm) as an adult compared to C. sciotensis (x = 80.5 mm; n = 30; SE ± 6.5 mm).

Cambarus angularis and C. hatfieldi share in common short, subrectriangular chelae with swollen palms and a single row of mesial tubercles on the chelae. Cambarus hatfieldi can be differentiated from C. angularis by the rostrum width/length ratio, areola length/carapace length ratio, shape of the cephalothorax, and length of the central projection and mesial process in comparison to gonopod shaft in Form I-males ( Fig. 4 ). Cambarus hatfieldi rostrum width constitutes a smaller percentage of the rostrum length (x = 59.4%; n = 52; SE ± 5.2%) compared to C. angularis (x = 67.2%; n = 22; SE ± 8.3%), and a smaller areola length/carapace length ratio (x = 30.4%; n = 52; SE ± 2.1%) compared to C. angularis (x = 36.7%; n = 22; SE ± 1.7%).

Cambarus hatfieldi’s abdominal pleura are rounded ventrally compared to C. angularis’s subtruncate pleura. Dorsally C. angularis cephalon is anteriorly swollen compared to C. hatfieldi cephalon, which begins to taper anteriorly at the junction of the cephalon with the cervical groove. The length of C. hatfieldi central projection and mesial process both extend to the caudal margin of the shaft of the gonopod but not beyond when compared to C. angularis where both extending beyond the caudal margin of the gonopod shaft ( Fig. 4 ). Finally, like C. sciotensis , C. angularis’ total body length on average is larger (x = 84.1 mm; n = 22; 9.8 mm) than C. hatfieldi (x = 67.6 mm; n = 52; SE ± 7.3 mm).

Trees resulting from ML, MP and NJ analyses were identical in their placement of taxa within clades. The phylogenetic tree based on ML analysis is shown in Fig. 5 . Cambarus sciotensis sequences form two groups, New River Basin, WV (Glade Creek, Meadow River, Cherry River, Bluestone River, Gauley River, and Whitewater Branch) and Scioto River, OH, clustered separately and formed a large, well-supported clade. Both groups (i.e., New River Basin and Scioto River) are phylogenetically related ( Fig. 5 ). Cambarus hatfieldi sequences (from Browns Creek and Mate Creek, WV) are more distantly related to C. sciotensis and are placed in a well-supported cluster outside of the above mentioned clade consisting of C. sciotensis taxa.

Pairwise nucleotide sequence divergence rates within the (i) C. sciotensis clade consisting of sequences from New River Basin, WV, (ii) C. sciotensis cluster from Sciotto River, OH, (iii) C. hatfieldi clade (from Browns Creek, Mate Creek, WV), and (iv) C. angularis from Sycamore Creek, TN, were 0.002–0.008, 0.002, 0.002–0.005 and 0.022–0.037, respectively. In contrast, C. sciotensis from New River Basin, WV, and C. sciotensis from Scioto River, OH, diverged on average, 0.026 (approx. 3%) and 0.029 (approx. 3%), respectively, from C. hatfieldi (from Browns Creek, Mate Creek), WV ( Table 2 ). It is our hypothesis that divergence results indicate that C. hatfieldi is an incipient species, and likely diverged from Teays River C. sciotensis populations sometime in the Pleistocene epoch. GenBank accession numbers, specimen locations are provided in Table 3 .

C. sciotensis New River C. sciotensis Sciotto C. hatfieldii Tug Fork C. angularis

Basin, WV River, OH River, WV Sycamore Cr, TN.

Three additional Cambarus species occur in the Tug Fork River system that can easily be differentiated from C. hatfieldi . Cambarus theepiensis replaces C. hatfieldi in the Big Sandy River system, but is syntopic with C. hatfieldi in lower reaches of the Tug Fork River (ZJL personal observation). Cambarus bartonii cavatus occurs throughout the headwaters of the Tug Fork River system, and is the dominant species in ephemeral streams throughout the watershed. Both C. theepiensis and C. b. cavatus possess two rows of tubercles on the mesial surface of the chelae’s palm, and 1–2 subpalmer tubercles on the ventral surface of the chelae. Cambarus hatfieldi possesses a single row of adpressed tubercles on the mesial surface of the palm and lacks subpalmer tubercles. Cambarus veteranus is syntopic with C. hatfieldi in mid to headwaters reaches of Tug Fork, though noticeably rarer than the latter (ZJL and RFT, personal observation). Cambarus veteranus rostrum is lanceolate compared to C. hatfieldi’s broad rostrum. In addition to rostrum morphology, C. veteranus possess strong cervical spines; C. hatfieldi lacks both cervical spines and cervical tubercles.

Distribution of C. hatfieldi , C. theepiensis , and C. sciotensis in Kentucky and West Virginia. In light of C. hatfieldi’s description herein, and the recent description of C. theepiensis , the distribution of C. sciotensis in both Kentucky and West Virginia comes into question. Cambarus sciotensis previously was recorded in the Big and Little Sandy River drainages of Kentucky, and hadn’t formerly been documented in the Tug Fork drainage in the state (Taylor and Schuster 2004). Currently, populations previously recognized as C. sciotensis in the Big and Little Sandy River watersheds of Kentucky are synonymized under C. theepiensis (Loughman et al. 2013) . Records for C. sciotensis do exist for Kentucky in Tygart Creek, a direct tributary to the Ohio River in the vicinity of the Scioto River confluence with the Ohio River. At present Tygart Creek and streams in its associated watershed possess the only populations of C. sciotensis known to occur in Kentucky. Thoma (2010) was the first to document C. hatfieldi in Kentucky, and reported the species as C. angularis .

In West Virginia, C. sciotensis is distributed in the greater New River system upstream of Kanawha Falls, Kanawha County as well as the Kanawha River mainstem and tributaries immediately downstream of Kanawha Falls. Cambarus robustus replaces C. sciotensis downstream of Kanawha Falls, and is the dominant tertiary burrowing Cambarus throughout the Coal, Upper, and Lower Kanawha drainages in West Virginia. What previously was documented as C. sciotensis in the Guyandotte and Twelvepole systems of West Virginia (Jezerinac et al. 1993; Loughman et al. 2009) is synonymized with C. theepiensis (Loughman et al. 2013).

Resultant of the description of C. hatfieldi and C. theepiensis , the distribution of C. sciotensis , as it is currently is understood, is disjunct with populations in the Scioto River basin of Central Ohio as well as direct Ohio River tributaries in Kentucky near the Ohio /Scioto River confluence and in the New River system of West Virginia and Virginia. Both populations were likely connected via the ancient Teays River, which is evident given the low COI divergence values exhibited between both populations ( Table 2 ). That being said, Scioto and New river populations of C. sciotensis differ morphologically and are readily identifiable from each other using morphological characters (Z. J Loughman and R. F. Thoma personal observation). Given morphologic and zoogeographic differences, further taxonomic investigation is warranted between the Scioto and New River populations of C. sciotensis .

Etymology. Latinized form of Hatfield in honor of the Hatfield and M c Coy feud which occurred in the Tug Fork River Valley of Kentucky and West Virginia in the 1860s–1870s.

Common name. Tug Valley Crayfish.

Figures

FIGURE 1. Cambarus hatfieldi n. sp.: A. lateral view of carapace; B. lateral and C. mesial view of first gonopod of form I male; D. lateral and E. mesial view of form II male gonopod; F. antennal scale; G. dorsal view of carapace; H. dorsal view of distal podomere of right cheliped of form I male; I. caudal view of in situ form I male gonopods; J. annulus ventralis; K. epistome; L. ischial hook; A – C, F – I, K – L from holotype; J from allotype; D – E from morphotype.

FIGURE 2. Cambarus hatfieldi n. sp., holotype (USNM 1227861) in life.

FIGURE 3. Known range of Cambarus hatfieldi n. sp. in Kentucky, Virginia, and West Virginia. Tug Fork River basin is highlighted in red; yellow circles represent C. hatfieldi collection locales.

FIGURE 4. Form I gonopod’s of (A.) Cambarus hatfieldi, (B.) Cambarus sciotensis, and (C.) Cambarus angularis.

FIGURE 5. Maximum Likelihood tree of partial COI sequences from analysis of 17 unique sequences (599 bp) retrieved in this study. The species and locations of Cambarus sp. are listed in Table 3. The COI gene of C. bartonii was used as the outgroup. Accession numbers of sequences are indicated in parentheses. Bootstrap values from 1,000 replicates are indicated at the nodes of branches (if> 50). The scale bar represents the number of nucleotide changes.

Tables

TABLE 1. Measurements (mm) of Cambarus hatfieldi, new species.

	Holotype	Allotype	Morphotype
Carapace
Total carapace length	38.9	37.7	26.0
Postorbital length	33.0	32.0	21.8
Length cephalic section	24.7	18.0	16.8
Width	21.6	20.2	13.6
Depth	14.9	15.8	10.6
Length rostrum	8.6	8.2	5.0
Length acumen	2.6	2.8	1.6
Length areola	14.2	14.0	9.2
Width areola	3.7	3.8	1.8
Antennal scale
Width	2.5	2.7	1.1
Abdomen
Width	16.6	18.2	10.6
Cheliped (Right)
Length mesial margin palm	10.7	9.4	6.2
Width palm	15.5	9.3	8.9
Depth palm	8.6	8.0	5.4
Length dactyl	20.4	18.1	10.3
Length carpus	11.0	10.4	7.9
Width carpus	7.3	9.2	6.0
Length dorsal margin merus	13.0	11.3	10.8
Depth merus	7.7	7.4	5.9
Gonopod length	7.1	N.A.	5.1

TABLE 4. Pleopodal egg diameters and instar condition for Cambarus hatfieldi, n. sp. ED denotes average egg diameter in mm; asterisk denotes incomplete complement of eggs or instars.

Stream	Date	Female TCL	Condition of eggs/instars
Horse Creek, McDowell Co. WV	10 July 2009	26.1	13 attached eggs*; ED = 1.9
Horse Creek, McDowell Co. WV	10 July 2009	33.3	33 attached eggs; ED = 1.6
Horse Creek, McDowell Co. WV	10 July 2009	39.5	36 attached eggs; ED = 1.8
Barenske Creek, McDowell Co. WV	11July 2009	32.1	64 attached instars; 3 attached eggs* ED = 1.5

TABLE 2. Proportion of uncorrected base substitutions (from sequence comparisons of 599 bp) between all taxa (as average values) from the identified clusters (above diagonal) and associated standard errors (below diagonal). Analyses were performed using ClustalW 2. New River sites include Glade Creek, Meadow River, Cherry River, Bluestone River, Gauley River, Whitewater Branch; Tug Fork sites include Browns Creek and Mate Creek. Both the Scioto River and Sycamore Creek represent type locations for their representative species.

C. sciotensis New River Basin, WV	0.019	0.026	0.057
C. sciotensis Sciotto River , 0.001 OH		0.029	0.061
C. hatfieldii Tug Fork 0.000 River, WV	0.000		0.055
C.angularis Sycamore Cr , 0.001 TN.	0.002	0.001

TABLE 3. Genbank accession numbers and locations for Cambarus sciotensis, Cambarus hatfieldi, and Cambarus angularis sequences used in this study.

Specimen# GenBank Accession # Species	Location	Coordinates
CS1 KF437297 C. sciotensis	Meadow River, WV	37.446123 N 81.120730 W
CS2 KF437298 C. sciotensis	Meadow River, WV	37.446123 N 81.120730 W
CS3 KF437299 C. sciotensis	Meadow River, WV	37.446123 N 81.120730 W
CS4 KF437300 C. sciotensis	Meadow River, WV	37.446123 N 81.120730 W
CS5 KF437301 C. sciotensis	Meadow River, WV	37.446123 N 81.120730 W
CS6 KF437302 C. sciotensis	Gauley River, WV	38.290968 N 80.640797 W
CS7 KF437303 C. sciotensis	Whitewater Branch, WV	38.274921 N 80.9324328 W
CS8 KF437304 C. sciotensis	Whitewater Branch, WV	38.274921 N 80.9324328 W
CS9 KF437305 C. sciotensis	Cherry River, WV	38.254229 N 80.510992 W
CS10 KF437306 C. sciotensis	Glade Creek, WV	37.704200 N81.052423 W
CS11 KF437307 C. sciotensis	Glade Creek, WV	37.704200 N81.052423 W
CS12 KF437308 C. sciotensis	Glade Creek, WV	37.704200 N81.052423 W
CS13 KF437309 C. sciotensis	Bluestone River, WV	37.446120 N81.120730 W
CS14 KF437310 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CS15 KF437311 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CS16 KF437312 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CS17 KF437313 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CS18 KF437314 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CS19 KF437315 C. sciotensis	Scioto River, OH	40.141670 N 83.11968 W
CA1 KF437316 C.angularis	Little Sycamore Creek, TN	36.442270 N 83.51006 W
CA2 KF437317 C.angularis	Little Sycamore Creek, TN	36.442270 N 83.51006 W
CA3 KF437318 C.angularis	Little Sycamore Creek, TN	36.442270 N 83.51006 W
CA4 KF437319 C.angularis	Little Sycamore Creek, TN	36.442270 N 83.51006 W
CH1 KF437320 C.hatfieldii	Browns Creek, WV	37.443050 N 81.56886 W
CH2 KF437321 C.hatfieldii	Browns Creek, WV	37.443050 N 81.56886 W
CH3 KF437322 C.hatfieldii	Mate Creek, WV	37.648070 N 82.13524 W
CH4 KF437323 C.hatfieldii	Mate Creek, WV	37.648070 N 82.13524 W