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Zootaxa 4243 (3): 432 454 http://www.mapress.com/j/zt/ Copyright 2017 Magnolia Press Article https://doi.org/10.11646/zootaxa.4243.3.2 http://zoobank.org/urn:lsid:zoobank.org:pub:6771949e-c3b2-4a3f-abfd-fa3d90454e9a ISSN 1175-5326 (print edition) ZOOTAXA ISSN 1175-5334 (online edition) Cambarus (C.) appalachiensis, a new species of crayfish (Decapoda: Cambaridae) from the New River Basin of Virginia and West Virginia, USA ZACHARY J. LOUGHMAN 1, STUART A. WELSH 2 & ROGER F. THOMA 3 1 West Liberty University, Department of Natural Sciences and Mathematics, P.O. Box 295, West Liberty, West Virginia, 26074. E-mail: zloughman@westliberty.edu 2 U.S. Geological Survey, West Virginia Cooperative Fish and Wildlife Research Unit, 322 Percival Hall, Morgantown, WV 26506. E-mail: swelsh@mail.wvu.edu 3 Midwest Biodiversity Institute, 4673 Northwest Parkway, Hilliard, Ohio, 43026 USA. E-mail: cambarus1@mac.com Abstract Cambarus (Cambarus) appalachiensis is a stream-dwelling crayfish endemic to the greater New River basins of Virginia and West Virginia. The new species is morphologically most similar to Cambarus sciotensis. Cambarus appalachiensis can be differentiated from C. sciotensis by its more elongated chelae which possess a single mesial row of tubercles, reduced to no tuberculation on the dorsal-longitudinal ridge of the dactyl, and reduced lateral impression. Cambarus sciotensis has a more subrectangular chelae with two rows of mesial margin tubercles on the chelae, as well as both a pronounced dorsal-longitudinal ridge and pronounced lateral impression. Several chelae meristic ratios also differentiate C. appalachiensis from C. sciotensis. Within the New, Gauley, and lower portions of the Greenbrier basins C. appalachiensis is the dominant tertiary burrowing Cambarus species, and as such, is considered stable across its range. Key words: Crayfish; New River; Appalachian Mountains Introduction The disjunct distribution range of Cambarus sciotensis Rhoades, 1944 has been a point of discussion for many students of North American astacology (Hobbs and Bouchard 1994; Jezerinac et al. 1995; Loughman et al. 2013). Prior to 2013, the geographic distribution of C. sciotensis included the Scioto basin, the Little and Big Sandy basins, the Guyandotte and New river drainages, and several Ohio River direct drainage basins. Loughman et al (2013) investigated the morphology of type population (Scioto R.) C. sciotensis and compared that morphology to animals occurring throughout the previously mentioned basins. Results of that work discovered that animals from the Guyandotte and Big Sandy river drainages, and Ohio River direct drainages had unique morphologic characters compared to those of the Scioto and New river populations. This resulted in the description of Cambarus theepiensis. While performing the aforementioned analysis, animals were studied from the Tug Fork watershed of eastern Kentucky, southern Virginia, and southwestern West Virginia, and it was found that they had unique morphologic characters when compared with those from populations of both C. sciotensis and C. theepiensis. Subsequent analyses indicated that the population of C. sciotensis from the Tug Fork River drainage was unique both genetically and morphologically compared to those of the Scioto and New river drainages. Cambarus hatfieldi Loughman et al., 2014 was described based on those results, leaving C. sciotensis with two completely disjunct populations, one in the Scioto River of Ohio and another in the New River upstream of Kanawha Falls in Virginia and West Virginia. While performing the aforementioned analysis, specimens of C. sciotensis from the Tug Fork watershed of eastern Kentucky, southern Virginia, and southwestern West Virginia were observed to maintain unique morphologic characters compared to those of C. theepiensis and populations of C. sciotensis from other watersheds. Subsequent genetic and morphologic analyses indicated additional differentiation. Genetic analyses of 432 Accepted by J. Goy: 19 Jan. 2017; published: 16 Mar. 2017

Loughman et al. (2014) and Fetzner and Thoma (2014) retrieved clades indicating that the Scioto River and New River populations were separate monophyletic groups. Loughman et al. (2014) concluded that further work was needed to determine the true nature of this relationship; Fetzner and Thoma (2014) found that COI divergence values did not indicate a significant taxonomic relationship worth further study, and New River populations were C. sciotensis. Fetzner and Thoma (2014) also retrieved results indicating that C. theepiensis was not monophyletic, and needed further taxonomic investigation. We believe that this conclusion was in error, given both morphological and biogeographic data were not considered by Fetzner and Thoma (2014), which relied solely on COI divergence. Both Evolutionary and Unified species concepts rely on the ultimate evolutionary legacy of populations as well as their trajectories (De Queiroz 2007). Given that populations in the Scioto and New rivers are completely disjunct from each other both geographically and genetically (Loughman et al. 2014; Fetzner and Thoma 2014) and currently lack the possibility of interbreeding, and thus have their own evolutionary trajectories, we completed a morphological analysis comparing Scioto and New River populations of C. sciotensis. Results of this work indicated that the populations differ significantly morphologically and meristically, are morphologically unique from each other, and thus warrant the description of the New River form as Cambarus appalachiensis, species nova herein. Materials and methods Morphometric measurements were taken from 98 adult individuals of C. sciotensis from the New River drainage (32 form I males, 31 form II males, and 35 females) and 40 adult individuals of C. sciotensis from the Scioto River drainage (18 form I males, 6 form II males, and 16 females). The following morphometric distances were measured with hand-held digital calipers (nearest 0.1 mm); abdominal length and width, acumen length, antennal scale length, areola length and width, total carapace length (TCL), cephalon width, carapace depth, postorbital carapace length (PCL), post orbital carapace width, cephalic width, palm depth, dactyl length and width, palm length and width, propodus length and width, and rostral length and width. Additionally, three gonopod measurements were taken for form I males using an ocular micrometer; gonopod width, length of central projection, and length of mesial process. Sixteen morphometric ratios were calculated as follows: acumen length/rostrum length, areola length/tcl, areola length/carapace width, areola width/areola length, carapace width/tcl, carapace width/ carapace width, dactyl length/propodus length, dactyl width/dactyl length, palm length/propodus length, palm length/palm width, palm depth/palm length, palm width/propodus length, propodus width/dactyl length, propodus width/propodus length, rostrum length/tcl, and rostrum width/rostrum length. Meristic data, presence/absence data, as well as orientation of tubercles and spines were also recorded from each specimen. Meristic data included the number of tubercle rows on the mesial margin of the chela palm, and the counts of tubercles from the first and second rows on the mesial margin of the chela palm. Presence/absence data were recorded for subpalmer tubercles. Additionally, orientation was recorded for the terminal tubercle or spine of the post orbital ridge; straight vs. dorsally deflected. Morphometric distances of New River C. appalachiensis and Scioto River C. sciotensis were examined separately for form I males, form II males, and females using sheared principal component analysis (SPCA), a multivariate method that quantifies morphologic variation along the second and third principal component axes, while restricting size variation to the first principal component. Morphometric variation within and between taxa were depicted by plotting the second sheared principal component versus the third sheared principal component. Statistical differences (p < 0.05) between ratios of C. appalachiensis and C. sciotensis were examined separately for form I males, form II males, and females using t-tests for normally distributed data or Mann-Whitney tests for non-normal data. The SPCAs were conducted with the Statistical Analysis System Software, and Shapiro-Wilks normality tests, t-tests, and Mann-Whitney tests were conducted with SAS-JMP (SAS Institute, Inc., Cary, NC; http://www.sas.com). Plates and figures were created following Schuster et al. (2015) and Schuster and Taylor (2016). CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 433

FIGURE 1. Sheared PCA plot for (A) form 1 males, (B) form II males, and (C) females of Cambarus appalachiensis and C. sciotensis. Cambarus appalachiensis = solid dots; Cambarus sciotensis = open dots. 434 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

Results Plots of the second sheared principal component vs. the third sheared principal component depicted morphologic distinctness between C. appalachiensis and C. sciotensis for form I males, form II males, and females (Fig. 1). Totals of 35.9, 26.2, and 30.0 % of morphologic variation were explained along the SPC2 axes for form I males, form II males, and females, respectively. The SPC3 axes explained 11.6, 18.6, and 18.0 % of morphologic variation of form I males, form II males, and females, respectively. For the form I male SPC2 axis, highest loadings were propodus width (0.56), palm length (-0.44), dactyl length (-0.43), rostrum length (0.36), and dactyl width (0.27). Cambarus appalachiensis Form I male had a narrower propodus and dactyl, a shorter rostrum, and a longer palm and dactyl than C. sciotensis. For the form II male SPC2 axis, highest loadings were propodus width (0.67), rostrum length (-0.42), dactyl length (-0.30), and dactyl width (0.30). Form II males of C. appalachiensis had a narrower propodus and dactyl, and a longer rostrum and dactyl than that of C. sciotensis. For the female SPC2 axis, highest loadings were dactyl length (-0.44), propodus width (0.44), propodus length (-0.40), palm length (-0.40), dactyl width (0.34), and rostrum length (0.28). Female C. appalachiensis had a longer dactyl, propodus, and palm, a narrower propodus and dactyl, and a shorter rostrum than that of C. sciotensis. Morphometric ratios differed significantly between C. appalachiensis and C. sciotensis for form I males (13 of 16 ratios), form II males (8 of 16 ratios), and females (13 of 16 ratios). For form I males, ratios of C. appalachiensis (n=32) were significantly larger than that of C. sciotensis (n=18) for palm length/propodus length, rostrum width/rostrum length, palm length/palm width, acumen length/rostrum length, areola width/areola length, and dactyl length/propodus length. Ratios of C. appalachiensis form I males were significantly smaller than that of C. sciotensis for carapace width/tcl, palm depth/width, rostrum length/tcl, areola length/tcl, dactyl width/ length, propodus width/length, and propodus width/dactyl length. For form II males, ratios of C. appalachiensis (n=31) significantly exceeded those of C. sciotensis (n=6) for palm length/width, areola width/length, rostrum length/tcl. Ratios of C. appalachiensis form II males were significantly smaller than those of C. sciotensis for rostrum width/length, palm depth/width, cephalon width/carapace width, propodus width/length, and propodus width/dactyl length. For females, ratios of C. appalachiensis (n=35) were significantly larger than those of C. sciotensis (n=16) for rostrum width/length, palm length/width, areola width/length, cephalon width/carapace width, and areola length/carapace width. Ratios of C. appalachiensis females were significantly smaller than those of C. sciotensis for carapace width/tcl, palm depth/width, rostrum length/tcl, areola length/tcl, dactyl width/ length, propodus width/length, and propodus width/dactyl length. Systematics and description of species Cambarus (Cambarus) appalachiensis new species Diagnosis. Body and eyes pigmented. Rostrum broad, moderately excavated, and deflected anteriorly, margins thickened, parallel though rarely sub-parallel; converging to acumen abruptly at terminance of rostral margins. Acumen distinctly triangular with prominent dorsally deflected spiniform tubercle at terminus. Areola 2.2 5.1 ( = 3.4, n = 98, σ = 0.6) times as long as wide with 5 8 (usually 7) punctations across narrowest point. No cervical spines; 4 7 ( = 6.1, SE = 0.8) tubercles present. Mandibular, branchiostegal, and orbital regions of carapace with well-developed tubercles. Postorbital ridges short; pronounced, spiniform, dorsally deflected cephalic tubercle present in juveniles and subadults; adult postorbital ridge terminating in reduced to prominent dorsally deflected cephalic tubercle. Suborbital angle acute. Total carapace length (TCL) 1.8 2.1 ( = 1.9, n = 98, SE = 0.1) times longer than width. Form I and II males possessing hook on ischium of third pereopod 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 chela nearly always possesses a single mesial row of tubercles; but rarely (3% of individuals; n = 98) an incomplete second row of tubercles also present; mesial most row with 6 10 ( = 7.9, n = 98, SE = 0.5) tubercles, second incomplete row when present with 2 4 ( = 2.2, n = 3, SE = 0.6) tubercles. Ventral surface of chelae with 1 3 ( = 1.9, n = 98, SE = 0.7) subpalmar tubercles near the dactyl/ propodus junction. Dorsomedian ridge of fixed finger of propodus pronounced. Lateral impression at base of fixed finger shallow (61% of individuals) or absent (39% of individuals). Dactyl and fixed finger each with sharp CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 435

corneous tip. Form I male palm length 64.3 77.1% ( = 69.9%, n = 32, SE = 2.8%) of palm width, form I male palm length 27.1 31.3% ( = 29.6%, n = 32, SE = 1.2%) of total propodus length; female dactyl length 55.2 64.1% ( = 61.5%, n = 35, SE = 2.2%) of total propodus length. First pleopod of form I male with short terminal elements. Central projection not tapering distally; recurved >90º to main shaft, with distinct subapical notch. Mesial process directed 90 to shaft, bent cephalolaterally; inflated proximally, tapering to a point distinctly caudal to slighty cephalic to terminance of central projection and not projecting beyond the edge of the margin of gonopod shaft. Annulus ventralis immovable; distinctly asymmetrical caudally; cephalic portion with median trough leading to strongly sculptured central fossa; exaggerated S bend in sinus terminating at caudal edge. Description of holotypic male, form I. (Fig. 2 A C, G J; Table 1). Body somewhat compressed dorsoventrally (Fig. 2A); thoracic section of carapace slightly wider than abdomen. Carapace depth less than carapace width at caudodorsal margin of cervical groove. Total carapace length 38.1 mm; post orbital carapace length 32.1 mm. Areola 2.8 times longer than wide, with 7 punctations across narrowest part (Fig. 2G); length of areola 34.9% of TCL (41.4% of postorbital carapace length (PCL). Rostrum weakly excavated, more so anteriorly than posteriorly; margins thickened and parallel; acumen margins noticeably thinner than rostral margin forming distinct 90 0 break from rostrum margins, continuous to base of acumen; floor of rostrum with numerous punctations. Rostrum 1.7 times longer than wide. Acumen distinctly triangular, ending in dorsally deflected corneous tip (Fig. 2A). Postorbital ridges well developed, terminating in dorsally deflected spiniform tubercles. Suborbital angle acute, with tubercle (Fig. 2A). Cervical spine absent; several weakly to moderately developed tubercles present. Mandibular, branchiostegal, and orbital regions of carapace punctated with well-developed tubercles; greatest tubercle density in hepatic region. Abdomen slightly longer than carapace, pleura rounded cephaloventrally, angled caudoventrally. 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 14 immovable, small spines and 1 lateral, large, movable spine. Cephalomedian lobe of epistome subtriangular, zygoma moderately arched (Fig. 2J); cephalolateral margins thickened, forming sharp angle at junction with endostyle; main body possessing prominent cephalomedian fovea. Antennal scale broadest anteriorly; lateral margin thickened, terminating in large corneous spine; (Fig. 2H). Right antennal scale 5.4 mm long, 2.5 mm wide. Tip of right antenna reaching middle of telson when appressed. Mesial surface of right chela with one well-formed row of 8 tubercles (Fig. 3K). Palm length 68.1 % of palm width; depth of palm 9.6 mm. Ventral surface of palm lacking subpalmar tubercles. Dorsal longitudinal ridge of dactyl developed and possessing moderate sized tubercles (Fig. 2I); dactyl terminating in large corneous spine. Dorsomedian ridge of fixed finger of propodus weakly developed. Weakly developed lateral impression at the junction of fixed finger and palm; numerous setiferous punctations present. Dactyl and fixed finger with sharp, corneous tip. All measurements and counts from right chela. Carpus with prominent dorsal furrow (Fig. 2I) and 2 weak dorsomesial tubercles; rest of surface with setiferous punctations; mesial margin with large, procurved spine at about midlength, and one reduced proximal tubercle. Distodorsal surface of merus with 9 spiniform tubercles; ventrolateral ridge with 2 small spines and one large, corneous distal spine; ventromesial ridge with two welldeveloped spines. Carapace depth less than width. Hook on ischium of third pereopod only; hook gently curved at apex, overarching basioischial joint, not opposed by tubercle on basis. Form I gonopod as described in diagnosis (Fig. 2B C); tip reaching anterior margin of fourth caudomesial boss when abdomen flexed. Description of allotypic female. (Fig. 2F, Table 1). Differing from holotype in following respects; carapace height less than carapace width (16.9 and 21.1 mm, respectively); TCL 39.6 mm, PCL 33.5 mm. Areola length 35.1% of TCL (41.4% of PCL), 3.0 times as long as wide. Posterior portion of rostrum more excavated than anterior portion; rostrum 1.7 times longer than wide. Abdomen length 41.1 mm. Mesial surface of chelae with single row of 8 tubercles. Palm length (10.0 mm) 62.5% of palm width (16.0 mm); depth of palm 9.2 mm. All measurements and counts from right chela. Antennal scale 5.7 mm long, 2.3 mm wide. Annulus ventralis as described in diagnosis (Fig. 2F); width of postannular sclerite half total width of annulus ventralis; first pleopods uniramous, reaching central region of annulus ventralis when abdomen flexed. 436 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

TABLE 1. Morphological measurements (mm) of Cambarus appalachiensis, new species. Holotype Allotype Morphotype Carapace Total carapace length 38.1 39.6 44.0 Postorbital length 32.1 33.5 37.8 Length cephalic section 24.8 25.7 27.8 Width 20.2 21.1 4.3 Depth 16.2 16.9 19.0 Length rostrum 8.7 9.1 9.1 Length acumen 2.6 2.9 2.8 Length areola 13.3 13.9 16.2 Width areola 4.4 4.4 4.5 Abdomen Width 16.4 20.0 19.7 Length 38.8 41.1 43.0 Cheliped Length mesial margin palm 11.8 10.0 13.3 Width palm 17.3 16.0 20.3 Depth palm 16.2 9.2 11.2 Length dactyl 24.5 20.0 19.7 Length carpus 11.7 12.2 15.6 Width carpus 9.6 8.7 11.5 Length dorsal margin merus 10.5 9.5 9.9 Depth merus 9.9 9.8 12.2 Gonopod length 8.1 9.9 Description of morphotypic male, form II. (Fig. 2D E, Table 1). Differing from holotype in the following respects: Carapace height less than carapace width (19.0 and 24.3 mm respectively); TCL 44.0 mm and PCL 37.8 mm. Areola length 36.8% of TCL (42.8% of PCL), 3.6 times longer than wide. Rostrum margins parallel to base of acumen and thickened; rostrum ventrally deflected and excavated; rostrum 1.6 times as long as wide. Abdomen 43.0 mm long. Single mesial row of tubercles on palm of chela with 7 tubercles. Palm length (13.3 mm) 65.5% of palm width (20.3 mm). All measurements and counts from right chela. Antennal scale 6.6 mm long, 2.9 mm wide. Gonopods reaching anterior margin of 4 th pereopod caudomesial boss. Central projection curved 90 to shaft, with complete apex; rounded (Fig. 2D E). Mesial process tapered, bulbous, directed caudolaterally. Hook on ischium of third pereopod small, not reaching basioischial joint. Size. Form I male (n = 32) TCL ranges in size from 30.1 48.6 mm (PCL 25.4 41.0 mm) with a mean TCL of 37.4 mm. Form II male (n = 31) mean TCL is 36.6 mm and ranges in size from 24.9 46.9 mm (PCL 20.7 40.0 mm). Female (n = 35) TCL mean is 37.0 mm and ranges from 11.1 45.0 mm (PCL 8.5 38.1 mm). The largest specimen examined was a form I male with TCL of 48.6 mm (PCL 41.0 mm). CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 437

FIGURE 2. Cambarus appalachiensis, new species: (A.) Lateral view of carapace; (B.) Lateral and (C.) mesial view of form I gonopod; (D.) Lateral and (E.) mesial view of form II gonopod; (F.) Annulus ventralis; (G.) Dorsal view of carapace; (H.) Right antennal scale; (I.) Dorsal view of right carpus and chelae; (J.) epistome. A-C and G-I from holotype; F from allotype; DE from morphotypes. 438 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

FIGURE 3. Cambarus appalachiensis morphotype in life. Color. Cambarus appalachiensis (Fig. 3) carapace ground color is chestnut brown to pink to orange-brown; posterior margin of carapace dark brown to black. Hepatic and antennal region of carapace punctuated with cream, olive, or tan tubercles. Postorbital ridge orange, red-brown, to light. Rostrum margins and acumen cream to orange, orange-brown, red-brown or tan. Cephalic section of carapace immediately anterior to and including cervical groove is black, forming weak saddle; mandibular abductor scars mottled, ranging from light-brown, brown, to dark-brown. Lateral margin of antennal scale olive to light brown; body of antennal scale brown to cream. Antennal flagellum and antennules green-brown, with olivaceous hue; dorsal surface of lamellae tan to brown; ventral surface light-green to olivaceous. Dorsal surface of chelae variable ranging from green, green-brown, olivaceous, orange, light-brown, or brown; occasionally with mesial row of tubercles cream, tan or orange. Denticles on opposable surfaces of fingers yellow, white, or tan. Ventral surface of chelae cream or tan. Dorsal surface of carpus light green, green-brown, or olivaceous; region adjacent to and including furrow green-brown to green; carpus spine orange, orange brown or cream. Merus green-brown, grey, or olivaceous brown. Podomeres of pereopods light blue, cream, or grey-blue; joints of pereopod podomeres pink. Dorsal and dorsolateral surface of abdomen same colors as carapace; tergal margins brown to olivaceous brown; abdomen lacking dorsal stripe. Uropods same colors as abdomen. Ventral surface of abdomen and carapace cream. 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. Pipestem Creek at intersection of Tom-Honaker Road (CR 20-3) and State Route 20, 3.3 km (2.04 mi) north-east of Pipestem, Summers County, West Virginia. The type series was collected on 11 October, 2016 by Z. J. Loughman. At this site (Fig. 4), Pipestem Creek is 4 10m wide, and consists of a series of high gradient riffles and glides, followed by moderate to low gradient runs with occasional pools. Substrates utilized by C. appalachiensis included large slab-rock, boulders, and large cobbles, as well as leaf packs. Riparian conditions ranged from residential yards to intact mesophytic forest bordered by SR 20. Water depth ranged from 0.2 1.0 m deep. Here, C. appalachiensis was both abundant and the only crayfish species observed. The type series was collected (n=3), along with several additional specimens (n = 15). Disposition of types. The holotype, allotype, and morphotype are deposited in the North Carolina Museum of Science (NCSM), Raleigh, NC. (catalogue numbers NCSM 1251657, 1251658, 1251659, respectively). Paratypes consisting of 1 M1, 1 MII, and 1 F are deposited in the West Liberty University Astacology Collection (WLU 3003), the United States National Museum (USNM 1422178) and the Ohio State Museum of Zoology (OSUMC 9916). CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 439

FIGURE 4. Cambarus appalachiensis type locality. Pipestem Creek at intersection of Tom-Honaker Road (CR 20-3) and State Route 20, 3.3 km (2.04 mi) north-east of Pipestem, Summers County, West Virginia 11 October, 2016. (A.) Run downstream of Tom-Honaker Road bridge where holotype and allotype were collected; (B.) pool immediately upstream of Tom-Honaker Road bridge. 440 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

FIGURE 5. Cambarus appalachiensis distribution. Range and specimens examined. Cambarus appalachiensis (Fig. 5) occurs in the Ridge and Valley, Appalachian Plateau, and Allegheny Mountain physiographic provinces in the upper New River watershed in the middle New River basin in Virginia and West Virginia, and the lower New, upper Kanawha, Gauley and Greenbrier river basins in West Virginia (Fig. 5). The southern/upstream portion of the range of C. appalachiensis occurs in the Middle New River mainstem. Robust populations currently persist in the New River, as well as Peak Creek and Little River and their associated tributaries. Cambarus appalachiensis is the dominant tertiary burrower in the middle New River basin, with large populations occurring in the New River mainstem and the Bluestone River, as well as all major New River tributaries. Greenbrier watershed populations are limited to the lower reaches of the basin, with the farthest upstream populations reported in Wolf Creek, Monroe County. Within the lower New River, C. appalachiensis occurs within the mainstem as well as all large tributaries. Gauley River drainage populations are stable and robust in the mainstem of the Gauley River, as well as the Williams, Cherry, Cranberry and Meadow Rivers and all associated tributaries, where C. appalachiensis is the dominant Cambarus species. Streams immediately downstream of Kanawha Falls in the upper Kanawha River are the only waterbodies within the basin that maintain C. appalachiensis. Downstream of Kanawha falls C. appalachiensis is replaced by its ecological equivalent, Cambarus robustus Girard, 1852. Material examined included 1,089 specimens from 189 collections housed at either The Ohio State University Museum of Biological Diversity Collection or the West Liberty University Astacology Collection and are presented in Appendix 1. Habitat and life history notes. The within-basin abundance and distribution of Cambarus appalachiensis differs among drainage basins. In both the upper and middle New River, C. appalachiensis is abundant in larger ordered streams, as well as the New River mainstem. As the New River increases in order, C. appalachiensis CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 441

occurrence decreases in the mainstem, and is limited primarily to larger ordered tributaries. Throughout the Gauley River system C. appalachiensis is the dominant tertiary burrowing crayfish and occurs in all stream orders from small headwater systems through the Gauley River mainstem. In all situations, C. appalachiensis occurs in streams with substrates of mixed cobble and gravels with isolated large boulders and slabs, which are the species preferred habitat (RFT and ZJL personal observation). Cambarus appalachiensis also occurs in a myriad of gradients, from slack water eddies and slow runs, to rapids and high gradient step-pool streams. Based on collections examined to complete the description, C. appalachiensis appears to have a life history similar to that of other larger tertiary burrowing Cambarus species in Appalachia (Jezerinac et al. 1995; Foltz et al. 2016). Form I males dominate over Form II males during autumn, winter and early spring months. Population wide molts occur in West Virginia beginning in mid-may, and are complete by mid-june (Z.J.L. personal observation). The majority of adult males are form II from the latter part of June through mid-september, when another population wide molt occurs, at which time the majority of males are Form I. This was evident during the collection of the type series in Pipestem Creek, Summer County West Virginia. Not all animals captured were retained, though basic biological data was ascertained. Fifty-one males were collected on 11 October 2016; only three were Form II. Mating likely occurs in the fall, winter and spring months. During the collection of the type series on three separate instances Form I males were collected alongside two to three females under large slabrock boulders. While mating was not observed, it was possible cohabitation in this scenario was associated with possible mating efforts. Active glare glands were not observed on any females collected in the type series (n = 89). Females collected in early and mid-summer in both June and July in West Virginia frequently display active glare glands (ZJL personal observation). Ovigerous females as well as females carrying instars have been collected on several occasions in both Virginia and West Virginia (Table 2). The earliest recorded date for females carrying eggs in this study was 24 July, 1989 with the latest occurrence of eggs on 19 August (Table 2). The earliest recording of juveniles was 2 August when a female was collected with stage I juveniles. Females likely maintain juveniles throughout the fall, and possibly into the spring. During the collection of the type series two females were collected maintaining 56 and 78 stage IV juveniles capable of living freely from their mothers (11 October, 2016). Mean egg counts for nine females was 105.9 eggs; mean number of juveniles for 6 females was 43.9 juveniles. The highest fecundity observed was 337 juveniles from a female collected in the Gauley River, Webster County, West Virginia (Table 2). TABLE 2. Cambarus appalachiensis pleopodal egg and juvenile counts. OSUMC # # Eggs # Instars Date County Stream County State 7647 159 0 7/24/1989 Monroe Turkey Cr. Monroe WV 7647 117 0 7/24/1989 Monroe Turkey Cr. Monroe WV 7647 98 0 7/24/1989 Monroe Turkey Cr. Monroe WV 6869 82 0 8/2/2007 Tazewell Mud Fork Tazewell VA 6874 0 58 8/2/2007 Tazewell Bluestone R. Tazewell VA 7123 161 0 8/18/1988 Monroe Wolf Cr. Monroe WV 7189 118 0 8/18/1988 Monroe Indian Cr. Monroe WV 7189 88 0 8/18/1988 Monroe Indian Cr. Monroe WV 7189 15 0 8/18/1988 Monroe Indian Cr. Monroe WV 7124 115 0 8/19/1988 Summers Hungard Cr. Summers WV 7276 0 10 8/20/1988 Greenbrier Little Clear Cr. Greenbrier WV 7191 0 337 8/22/1988 Webster Gauley R. Webster WV 7118 0 18 9/1/1989 McDowell Elkhorn Cr. McDowell WV 7266 0 54 9/10/1988 Nicholas Cranberry R. Nicholas WV 7543 0 62 9/10/1988 Nicholas Cherry R. Nicholas WV X egg/instar count = 105.9 150.8 SD egg/instar count = 43.9 123.0 442 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

FIGURE 6. Cambarus appalachiensis chelae morphotypes: (A.) Elongate; (B.) Truncated. Over 90% of C. appalachiensis examined maintained the elongated morphotypes. Conservation status. Cambarus appalachiensis should be listed as currently stable (CS) using the American Fisheries Society criteria (Taylor et al. 2007), and assigned a G4/G5 ranking using the Master (1991) global conservation criteria for conservation listing because of its broad range and stable status. Cambarus appalachiensis should be listed as least concern (LC) using the International Union for the Conservation of Nature (IUCN 2001) criteria because of its broad distribution (ZJL and RFT personal observation). Crayfish associates. Cambarus appalachiensis has been collected with Cambarus (Cambarus) carinirostris Hay, 1914, Cambarus (Hiaticambarus) chasmodactylus James 1966, Orconectes (Crockerinus) sanbornii (Faxon,1884), Orconectes (Gremicambarus) virilis (Hagen, 1840), and Orconectes (Procericambarus) cristavarius Taylor, 2000. CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 443

FIGURE 7. Differences in chelae structure between Cambarus appalachiensis (Top) and Cambarus sciotensis (Bottom). Variation. As in many Cambarus species, C. appalachiensis undergoes an ontogenic change in morphology, with juveniles and sub-adults being noticeably more spinose than adults. Specifically, juvenile postorbital ridges as well as acumen terminate in well pronounced spines, compared to adults whose postorbital ridges and acumens often terminate in either tubercles or spinose tubercles, rarely in defined spines. Juvenile/sub-adult rostra also are less broad and more lanceolate than adult rostra, which normally consist of parallel margins that are thickened, ending in strongly angled acumen, which are often absent in juveniles/sub-adults. Finally, chelae architecture is reduced in juvenile/subadult C. appalachiensis and defined in adult animals as explained below. Two distinct chelae types exist in C. appalachiensis, with both forms present in Form I and Form II individuals (Fig. 6A B). Elongated chelae (Fig. 6A) are far more frequently encountered than truncated chelae (Fig. 6B). Among adults, there is variation between animals in the Greenbrier, the lower, middle, and upper New River Basins, and the Gauley River basin. Chelae of adult C. appalachiensis from the Greenbrier and New river basins lack well defined lateral impressions, lack extensive tuberculation on the dorsal surface of the dactyl, and often possess an extensive gape between the dactyl and fixed finger of the propodus when the dactyl is closed. Gauley 444 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

River C. appalachiensis have a more defined lateral impression, moderate tuberculation on the dorsal surface of the dactyl, and moderate to weak gape between the dactyl and propodus when the dactyl is closed. In addition to differences in chelae architecture, Greenbrier and New river animal s rostra are normally broad, with steeply angled acumen. Gauley River C. appalachiensis rostra can be less broad, occasionally approaching a sublanceolate condition, and can have less steeply angled acumen, though most animals observed possess rostra similar to those from other basins. The latter is an exception in the Gauley, but does occur with enough frequency to be mentioned (ZJL personal observation). Relationships and comparisons. Cambarus appalachiensis is placed in the subgenus Cambarus based on (1) the presence of a subapical notch in the form I gonopod and (2) the lack of a well-developed mesial second tubercle row on the palm (Hobbs 1969). Among described members of the subgenus, C. appalachiensis is most similar to C. sciotensis, C. hatfieldi, and C. angularis in overall body size and shape and thickening of the rostral margins. Cambarus appalachiensis and C. sciotensis were distinguished by morphometric ratios of palm length/width, and palm depth/length, as well as chelae architecture. Mean ± SE of palm length/width ratios of form I males, form II males, and females were 70.7% ± 0.49, 70.0% ± 0.40, and 70.6% ± 0.54 for C. appalachiensis and 62.6% ± 0.65, 64.2% ± 0.90, and 63.8% ± 0.80 for C. sciotensis, respectively. Also, mean ± SE of palm depth/length ratios of form I males, form II males, and females were 83.7% ± 0.82, 85.6% ± 0.52, and 85.1% ± 0.61 for C. appalachiensis and 93.1% ± 1.1, 90.8% ± 1.2, and 92.7% ± 1.1 for C. sciotensis, respectively. The result of the previously mentioned ratios is C. appalachiensis has a much more elongated chelae in profile than C. sciotensis, which possess a subrectangular chela. In addition to the previous ratios, C. appalachiensis is readily differentiated from C. sciotensis by its lack of a second mesial row of tubercles, lack of a pronounced lateral impression, and lack of pronounced tubercles on the longitudinal ridge of the dactyl (Fig. 7). Cambarus sciotensis has two mesial rows of tubercles, moderate to strong lateral impression, and pronounced tubercles on the dorsal longitudinal ridge of the dactyl (Fig. 7). Finally, C. sciotensis postorbital ridge terminate in a straight tubercle or spine; C. appalachiensis Post orbital ridges terminate in dorsally deflected spines or tubercles. Cambarus appalachiensis can be differentiated from both Cambarus angularis Hobbs and Bouchard, 1994 and Cambarus hatfieldi Loughman et al. 2014 by its more elongate chelae, compared to both the latter species subrectangular chelae. In addition to chelae shape, C. appalachiensis has less thickened rostral margins than C. angularis, and a more elongate rostrum than C. hatfieldi. Cambarus appalachiensis central projection does not extend past the gonopod shaft; both C. angularis and C. hatfieldi central projections typically extend past the gonopod shaft, and normally past the distal margin of the mesial process. Cambarus appalachiensis is also on average longer in total body length ( = 74.4 mm; n = 98; SE ± 9.8%) than C. hatfieldi ( = 67.6 mm; n = 52; SE ± 7.3 mm; Loughman et al. 2014). Four additional Cambarus species occur in the greater New River system that can easily be differentiated from C. appalachiensis. Both Cambarus smilax Loughman et al. 2011 and Cambarus cf. robustus Girard, 1852 have two mesial rows of tubercles as well as lanceolate rostra, compared to C. appalachiensis single row of mesial tubercles and broad rostra. Cambarus smilax does not appear to be sympatric with C. appalachiensis, and is endemic to mid and headwater reaches of the Greenbrier River (Loughman et al. 2011). Cambarus cf. robustus, like C. smilax, also is a headwater species and endemic to the upper New River system of Virginia and North Carolina, and does not occur in larger ordered streams typically utilized by C. appalachiensis. Only two sites have been found to harbor both species. Cambarus chasmodactylus is the third Cambarus species in the New River system, and does on occasion occur in sympatry with C. appalachiensis. Cambarus appalachiensis is readily differentiated from C. chasmodactylus by its elongate chelae, broad rostrum, and brown coloration compared to C. chasmodactylus broad chelae, lanceolate rostrum, and blue, green, turquoise coloration. Cambarus chasmodactylus also has an extremely large gape in the chelae, which is substantially larger than the chelae gape observed in C. appalachiensis. Cambarus carinirostris occurs in the northern New River system and functions as the basins secondary burrowing species. As such, it occurs primarily in headwater streams, and normally does not occur in sympatry with C. appalachiensis. Cambarus carinirostris has a broad rostrum like C. appalachiensis, but can be differentiated from C. appalachiensis by its subrectangular chelae which maintain a single row of appressed tubercles, compared to C. appalachiensis elongate chelae which possess a defined single mesial row of tubercles. The upper Guyandotte River system is geographically proximate to the upper and middle New River system and is occupied by C. theepiensis. Cambarus appalachiensis and C. theepiensis have similar broad rostra, but can CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 445

be differentiated from each other by the number of tubercles rows on the mesial margin of the palm as well as subpalmar tubercles. Cambarus theepiensis has two rows of tubercles on the mesial margin of the chelae, as well as 2 4 subpalmar tubercles (Loughman et al. 2013). Cambarus appalachiensis has a single row of tubercles, and normally lacks or possesses only a single subpalmar tubercle. Etymology. Cambarus appalachiensis is named after the Appalachian Mountains where C. appalachiensis entire range occurs. The common name Conhaway Crayfish is in reference to Conhaway River, which was the name of both New and Bluestone Rivers in the 1700 s, and was the name Christopher Gist referred to the New River by in his field journal. It is theorized that Conhaway was the Shawnee name for the New River. Gist s expeditions throughout the New River basin in 1750 and 1751 were among the first expeditions throughout the New River valley by Europeans. Common name. Conhaway Crayfish. Acknowledgements We would like to acknowledge both the West Virginia Division of Natural Resources and the Virginia Department of Game and Inland Fisheries for providing funding that enabled the collection of specimens needed to complete this description. We would also like to thank our field assistants who generously gave their time. Finally, we appreciate the time and effort undertaken by two anonymous reviewers whose comments improved the quality of our manuscript. References De Queiroz, K. & Weins, J. (2007) Species concepts and species delimitation. Systematic Biology, 56, 879 886. https://doi.org/10.1080/10635150701701083 Faxon, W.A. (1884) Descriptions of new species of Cambarus; to which is added a synonymical list of the known species of Cambarus and Astacus. Proceedings of the American Academy of Arts and Sciences, 20, 107 158. https://doi.org/10.2307/25138768 Fetzner, Jr., J.W. & Thoma, R.F. (2014) Resolving taxonomic issues with Cambarus sciotensis and Cambarus robustus (Decapoda: Cambaridae) in Virginia using population genetic data. Final Project Report submitted to the Virginia Department of Game and Inland Fisheries, Forrest, Virginia, v + 21 pp. Foltz, D.A., Nolan IV., E.J., McGill, K.T. & Loughman, Z.J. (2016) Life history of the coal fields crayfish Cambarus theepiensis Loughman, Foltz, Garrison and Welsh 2013 (Decapoda: Astacoidea: Cambaridae) in southwestern West Virginia, USA. Journal of Crustacean Biology, 36, 628 636. https://doi.org/10.1163/1937240x-00002470 Girard, C. (1852) A revision of the North American Astaci, with observations on their habits and geographic distribution. Proceedings of the Academy of Natural Sciences of Philadelphia, 6, 87 91. Hagen, H.A. (1840) Monograph of the North American Astacidae. Illustrated Catalogue of the Museum of Comparative Zoology. Welch, Bigelow & co., University Press, Cambridge, 109 pp. Hay, W.P. (1914) Cambarus bartonii carinirostris In: Faxon, W. (1914) Notes on the crayfishes in the United States National Museum and the Museum of Comparative Zoology with descriptions of new species and subspecies to which is appended a catalogue of the known species and subspecies. Memoirs of the Museum of Comparative Zoology, Harvard College, 40, 351 427. Hobbs, H.H. Jr. (1969) On the distribution and phylogeny of the crayfish genus Cambarus. In: Holt, P.C. & Hoffman, R.L, (Eds.), The Distributional History of the Biota of the Southern Appalachians. Part I: Invertebrates. Research Division Monograph 1. Virginia Polytechnic Institution, Blacksburg, Virginia. pp. 93 178. Hobbs, H.H. Jr. & Bouchard, R.W. (1994) Cambarus (Cambarus) angularis, a new crayfish (Decapoda:Cambaridae) from the Tennessee River basin of northeastern Tennessee and Virginia. Jeffersoniana, 5, 1 13. IUCN (2001) Categories & Criteria (version 3.1). Available from: http://www.iucnredlist.org/static/categories_criteria_3_1 (accessed 15 February 2017) James, H.A. (1966) Range and variations of the subspecies of Cambarus longulus (Decapoda, Astacidae). Proceedings of the United States National Museum, 119, 1 24. https://doi.org/10.5479/si.00963801.119-3544.1 Jezerinac, R.F., Stocker, G.W. & Tarter, D.C. (1995) The crayfishes (Decapoda: Cambaridae) of West Virginia. Bulletin of the Ohio Biological Survey, 10, 1 193. Loughman, Z.J., Simon, T.P. & Welsh, S.A. (2011) Cambarus (Puncticambarus) smilax, a new species of crayfish 446 Zootaxa 4243 (3) 2017 Magnolia Press LOUGHMAN ET AL.

(Crustacea:Decapoda:Cambaridae) from the Greenbrier River basin of West Virginia. Proceedings of the Biological Society of Washington, 124, 99 111. https://doi.org/10.2988/10-09.1 Loughman, Z.J., Foltz, D.A., Garrison, N.L. & Welsh, S.A. (2013) Cambarus (P.) theepiensis, a new species of crayfish (Decapoda:Cambaridae) from the coalfields region of eastern Kentucky and southwestern West Virginia, USA. Zootaxa, 3641 (1), 63 73. https://doi.org/10.11646/zootaxa.3641.1.7 Loughman, Z.J., Fagundo, R.A., Lau, E., Welsh, S.A. & Thoma, R.F. (2014) 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), 223 236. https://doi.org/10.11646/zootaxa.3750.3.3 Master, L. (1991) Assessing threats and setting priorities for conservation. Conservation Biology, 5, 559 563. https://doi.org/10.1111/j.1523-1739.1991.tb00370.x Rhoades, R. (1944) Further studies on distribution and taxonomy of Ohio crayfishes, and the description of a new subspecies. Ohio Journal of Science, 44, 96 99. Schuster, G.A., Taylor, C.A. & Adams, S.B. (2015) Procambarus (Girardiella) holifieldi, a new species of crayfish (Decapoda: Cambaridae) from Alabama with a revision of the Hagenianus Group in the subgenus Girardiella. Zootaxa, 4021 (1), 1 32. https://doi.org/10.11646/zootaxa.4021.1.1 Schuster, G.A. & Taylor, C.A. (2016) Cambarus (Depressicambarus) claritae, a new species of crayfish (Decapoda: Cambaridae) from Alabama with a review of the halli Group in the subgenus Depressicambarus. Zootaxa, 4193 (2), 332 346. https://doi.org/10.11646/zootaxa.4193.2.8 Taylor, C.A. (2000) Systematic studies of the Orconectes juvenilis complex (Decapoda: Cambaridae), with descriptions of two new species. Journal of Crustacean Biology, 20, 132 152. https://doi.org/10.1163/20021975-99990023 Taylor, C.A., Schuster, G.A., Cooper, J.E., Distefano, R.J., Eversole, A.G., Hamr, P., Hobbs H.H.III, Robison, H.W., Skelton, C.E. & Thoma, R.F. (2007) Reassessment of the conservation status of crayfishes of the United States and Canada after 10+ years of increased awareness. Fisheries, 32, 372 389. https://doi.org/10.1577/1548-8446(2007)32[372:arotcs]2.0.co;2 CAMBARUS APPALACHIENSIS Zootaxa 4243 (3) 2017 Magnolia Press 447

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