Zootaxa 4290 (1): 123–139 ISSN 1175-5326 (print edition) Article ZOOTAXA http://www.mapress.com/j/zt/ Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4290.1.7 http://zoobank.org/urn:lsid:zoobank.org:pub:8F0A8A4C-CD1D-4838-93F0-A821A4E81E11 A diamond in the rough desert shrublands of the Great Basin in the Western United States: A new cryptic toad species (Amphibia: Bufonidae: Bufo (Anaxyrus)) discovered in Northern Nevada MICHELLE R. GORDON1, ERIC T. SIMANDLE2 & C. RICHARD TRACY1 1 Department of Biology, University of Nevada, Reno, NV 89557. 2 Department of Natural Sciences, Paul Smith’s College, Paul Smiths, NY, 12970 * Correspondence: C. Richard Tracy (Email: [email protected]) Abstract We describe a new species of toad from the Great Basin region of northern Nevada belonging to the Bufo (Anaxyrus) boreas species complex. This cryptic species was detected through genetic analyses of toad populations sampled through- out the Great Basin and the morphological evidence was quantified through extensive sampling of live toads within the region. The new species has the smallest body size in the species complex, and can be further diagnosed from other species in the complex by its large tibial glands and unique coloration. The known distribution of the new species is restricted to an area less than 6 km2 in Dixie Valley, Churchill Co., Nevada. The Great Basin is an arid region where aquatic resources are both rare and widely scattered, making habitat suitable for anuran populations highly vulnerable to anthropogenic change. The habitat occupied by this newly described species is threatened by the incipient installation of geothermal and solar power development projects that require the water that defines its habitat. Keywords: Bufo (Anaxyrus) williamsi sp. nov., Dixie Valley Toad, Western Toad, Bufo(Anaxyrus) boreas species com- plex, cryptic species, morphology, new species, conservation, geothermal Introduction The Great Basin, which was covered by large marshes and giant inland lakes during the Pleistocene Epoch, is among the most arid regions in the United States (Sada & Vinyard 2002). Only one percent of the landscape contains an aquatic resource, often in the form of widely dispersed springs, seeps, and small streams. These rare habitats provide an enormous value to flora and fauna that are dependent on these aquatic resources (Shepard 1993; Bogan et al. 2014) and represent regional biodiversity hotspots (Shepard 1993). Aside from supporting widespread taxa, Great Basin springs and wetland habitats also harbor high levels of endemic species, including aquatic organisms such as desert fishes (Hubbs & Miller 1948; Hewitt 1996, 2000; Smith et al. 2002), springsnails (Hershler & Sada 2002; Sada & Vinyard 2002), and insects (Shepard 1992). Despite the relatively recent recognition of numerous new species of plants and animals associated with these rare habitats, undetected diversity is still suspected given the rarity and isolation of aquatic sites within the region (Sada & Vinyard 2002). The Bufo (Anaxyrus) boreas species complex occurs within the western United States (Blair 1972; Stebbins, 2003) and includes subspecies B. b. boreas (Baird & Girard 1852), B. b. halophilus (Baird & Girard 1853), and three narrow endemics known only to occur within the hydrological Great Basin: B. canorus (Camp 1916), B. exsul (Myers 1942) and B. nelsoni (Stejneger 1893). Studies of evolutionary divergence within this species complex have suggested that localized species are relics of more continuous toad populations that diversified allopatrically from B. boreas during the Pleistocene (Myers 1942; Karlstrom 1962; Feder 1973; Graybeal 1993; Goebel et al. 2009). Analyses of diversity across the large geographic range of B. boreas suggest that diversification and speciation within the complex has been underrepresented (Stephens 2001; Goebel 2005; Goebel et al. 2009). While prior studies have included samples of populations across much of the geographic distribution of the B. boreas species complex, samples were limited or absent from the interior of the Great Basin. Our recent morphological and Accepted by D. Baldo: 9 Jun. 2017; published: 6 Jul. 2017 123 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 genetic analyses of B. cf. boreas populations within the Great Basin (Tracy et al. unpubl. data) has uncovered significant divergences among toads in the isolated basin of Dixie Valley, Nevada, indicating that this population should be recognized as a new species. Here, we use morphological and genetic evidence to describe and diagnose this new endemic member of the B. boreas species complex. Materials and Methods Morphological data collection. Measurements were collected from live adult toads (n = 380) from 17 distinct populations throughout the hydrological Great Basin (Fig. 1a) including B. boreas (n = 289), B. nelsoni (n = 31), B. exsul (n = 30), and B. sp. nov (n = 76). Fourteen morphological characters were recorded: snout–vent length (SVL; tip of snout to posterior end of urostyle), head length (HL; tip of snout to occiput), head width (HW; at widest part of the head), snout length (SL; tip of snout to anterior corner of eye), internarial distance (IND; distance between nares), eye diameter (ED; at widest part of eye), interorbital space (IOD; shortest distance between medial margin of upper eyelids), tympanum diameter (TYM; at maximum width of tympanum), parotoid length (PTL; horizontal length of parotoid gland) and width (PTW; maximum width of parotoid), interparotoid distance (IPD; shortest distance between medial margin of parotoid glands), femur length (FML; distance between vent and knee), tibia length (TBL; distance between knee and heel), and hind foot length (FTL; distance from anterior margin of heel to distal end of the third toe). All morphological characters were measured using Mitutoyo digital calipers to a precision of 0.01mm. ETS measured all individuals with the exception of 46 individuals collected from Dixie Valley, Nevada, which were measured by MRG, including the holotype and paratypic series. Sex was determined in the field near breeding sites where adults congregate, noting behavior and secondary sex characteristics such as the presence of nuptial pads on males as identifiers. FIGURE 1. Sampling localities of populations included for morphological (a) and DNA (b) collections within the hydrological Great Basin and surrounding states. a) Colors indicate species-specific populations measured for morphological analysis. b) Colors correspond with localized species and B. boreas colors correspond with major mtDNA haplotype clades (ONV- Oregon- NW Nevada (yellow), HL-Humboldt-Lahontan (blue), M-Mojave (aqua)) identified in Tracy et al. (in progress) molecular study of B. boreas diversity. Maps created using ArcGIS software by ESRI (2011: Release 10). 124 · Zootaxa 4290 (1) © 2017 Magnolia Press GORDON ET AL. Individuals selected to represent the type series were euthanized and preserved following the guidelines under the Institutional Animal Care and Use Committee (IACUC) from University of Nevada (UNR IACUC #00066). Tissue samples were extracted and preserved in 70% ethanol and specimens were fixed in 10% buffered formalin and transferred to 70% ethanol. Morphological analyses. We used multivariate analysis of covariance (MANCOVA) to quantify morphological differences among species and populations. We used SVL as a covariate to account for the effect of body-size variability in regressions against each morphological variable (Dahl & Peckarsky 2002; McCoy et al. 2006). This analysis results in least squares means from each regression for each size corrected morphological character which identify subtle, but significant differences in the fine features of these toads. Additionally, we log transformed the morphological measurements as an additional way to account for allometric differences among measured toads. Likewise, we analyzed these scaled data using MANCOVA to quantify morphological differences by population and by species (Lleonart et al. 2000). Tukey HSD post-hoc tests were used to identify significant differences among the morphological characters in pairwise comparisons by species resulting from the MANCOVA analyses for both size-corrected data sets. We used a cross-validated discriminant function analysis (DFA) to evaluate the variation in multivariate space to identify variables that best discriminated among the species. Hayek et al. (2001) cautioned that multiple measurers, despite care, result in interobserver error, particularly on fine features of amphibian anatomy and that these biases could result in different biological interpretations of morphometric analyses. To avoid interobserver biases, only measurements by ETS were used in the multivariate analyses, whereas the means table (Table 1), including the holotype are raw, unadjusted measures from both ETS and MRG. Our Table 2 provides the least squares means generated from the regression analyses, plus results from post hoc tests where significant differences were detected. All statistics were conducted using JMP Pro v. 10 (SAS Institute Inc. Cary, N.C.). TABLE 1. Morphological variation of four species of the Bufo (Anaxyrus) boreas species complex within the Great Basin. Fourteen morphological measurements (in mm) are as follows: snout–vent length (SVL), head length (HL), head width (HW), snout length (SL), internarial distance (IND), eye diameter (ED), interorbital distance (IOD), tympanum diameter (TYM), parotoid width (PTW), parotoid length (PTL), interparotoid distance (IPD), femur length (FML), tibial length (TBL), and foot length (FTL). Data include sample size (n), character mean ± standard deviation, and range. The values reflect unadjusted raw data, which includes additional individuals of B. williamsi from sampling efforts in 2014 and 2015 (n = 46), plus the holotype. B. williamsi B. exsul B. nelsoni B. boreas Holotype (n=76) (n=30) (n=31) (n=289) 54.6±4.73 64.0±8.43 80.8±13.0 82.3±12.2 SVL 52.92 44.01–69.97 53.00–79.00 57–122 53–113 16.02±1.62 18.64±2.42 23.99±3.34 23.96±2.98 HL 17.45 11.63–19.98 15.04–22.90 17.2–31.8 16.9–30.9 18.23±1.50 20.6±3.00 28.1 ±4.35 27.6±3.89 HW 18.35 14.81–24.32 16.55–25.02 19.0–37.7 18.5–36.21 5.43±0.90 4.42±0.65 5.41±0.79 5.57±0.77 SL 6.61 3.92–7.41 3.30–6.21 4.46–7.56 3.67–8.44 3.19±0.86 4.31±0.51 5.23±0.72 4.97±0.62 IND 2.13 1.50–4.61 3.08–5.56 3.99–7.16 3.1–6.70 5.76±0.92 6.04±0.70 7.59±1.09 7.47±0.96 ED 4.94 3.17–7.70 4.68–7.10 5.39–10.91 4.65–10.31 5.37±3.22 9.73±1.04 12.2±2.03 12.9±1.68 IOD 3.83 1.69–10.55 7.79–11.85 9.3–18.66 9.09–17.79 2.81±0.50 3.23±0.34 4.17±0.70 4.28±0.83 TYM 2.89 1.84–3.88 2.74–4.16 3.19–5.54 2.43–6.62 ......continued on the next page A NEW NEARCTIC TOAD SPECIES Zootaxa 4290 (1) © 2017 Magnolia Press · 125 TABLE 1. (Continued) B. williamsi B. exsul B. nelsoni B. boreas Holotype (n=76) (n=30) (n=31) (n=289) 5.18±0.79 5.38±0.82 6.89±1.01 7.1±1.14 PTW 3.90 3.36–7.40 3.76–6.52 5.12–9.47 4.49–10.59 6.50±0.95 6.82±0.92 8.83±1.33 9.99±1.58 PTL 5.56 4.20–9.49 5.53–8.75 5.69–11.52 6.61–14.58 10.35±1.11 11.9±1.53 15.6±2.66 15.8±2.39 IPD 9.18 8.23–14.00 10.00–15.00 11.50–23.0 11.00–23.0 19.60±2.65 24.5±3.30 31.13±4.59 32.8±4.38 FML 19.54 14.22–27.00 19.0–30.0 20.0–41.0 22.0–44.0 18.20±2.68 24.2±3.36 30.3±4.57 32.2±4.43 TBL 18.75 13.21–24.0 18.0–29.0 19.0–38.0 22.0–43.0 26.52±4.18 26.2±3.84 32.6±5.02 33.8±4.44 FTL 33.10 19.0–38.68 19.0–31.0 23.0–45.0 21.0–44.0 Genetic data. Tissue samples were collected from measured individuals of B. boreas populations (Fig.1b: n = 308), B. nelsoni (n = 32), B. exsul (n = 30), and B. canorus (n = 32) for a broader study of B. boreas phylogeography and species diversity within the hydrological Great Basin (Tracy et al. unpubl. data). Whole genomic DNA was extracted from dried toes or liver tissues stored in 95% ethanol and extracted using DNeasy Blood and Tissue Kit (Qiagen). Primers were designed based on the sequenced whole mitochondrial genome of B. boreas (not published). We used the mitochondrial control region (CR) as our genetic marker. This is a rapidly evolving region of mtDNA that is ideal for evaluating intraspecific polymorphism (Avise et al. 1987) and has been used in previous phylogenetic studies of B. boreas (Stephens 2001; Goebel et al. 2009). A 1.6 kb fragment of the CR was amplified via PCR with primers Bmt14844F and Bmt14200R. The PCR reagent included 1-5ng/uL template DNA, 1X PCR buffer, 4 mM dNTP mix, 0.5 uM of each primer, and TaqPlus Long PCR enzyme (Stratagene) in water to achieve the desired reaction volume. PCR conditions for the target genetic marker consisted of 30 cycles for 30 s at 95˚C, 45 s at 55˚C, and 1 min at 68˚C, followed by 5 min for final elongation at 68˚C. Fragments were purified by either gel purification (fragment sizes over 1 kb) or by column filtration. DNA concentration was determined by fluorescence and then sequenced with the same primers used for PCR and with primers internal from the PCR primers: Bmt14844F, Bmt14999R, Bmt 14223F, Bmt 15273F, Bmt 15400R, Bmt 15612R, Bmt 15777F, Bmt 15930R, Bmt 16207F, Bmt 16237R, Bmt 14200R. In previous studies of the B. boreas complex (Stephens 2001; Goebel et al. 2009), distantly related bufonids within the genus were adequate outgroups to evaluate genetic variation of the CR, and Bufo punctatus was selected for our study. For B. punctatus, primers were designed first based on the B. boreas sequence, then later based on D-loop sequences for B. punctatus that are within the CR. We amplified 300 to 500 bp fragments of D-loop and then sequenced these fragments using the same PCR primers. DNA was sequenced by an ABI 3730 Sequencer and data were analyzed with Sequencher software (Gene Codes, Ann Arbor, Michigan). The final alignment of the B. boreas group (CR 1622bp) was completed in ClustalW (Larkin et al. 2007) within Mega 7.0 (Kumar et al. 2015) resulting in 72 unique haplotypes, which were included in subsequent analyses. To examine pairwise genetic distances among sequences relative to the haplotypes identified, a Jukes- Cantor model (Jukes & Cantor 1969) was applied in Mega 7.0 (Kumar et al. 2015). Genetic analyses. Previous molecular studies have shown that taxa within the B. boreas species complex are close relatives that have diverged recently (Graybeal 1993; Shaffer et al. 2000; Stephens 2001; Pauly et al. 2004; Goebel et al. 2009). To examine population level genealogy, a TCS haplotype network was constructed in PopART (Clement et al. 2002; Leigh & Bryant 2015). Phylogenetic hypotheses were tested using both Bayesian inference (BI) and maximum likelihood (ML) methods to compare tree reconstructions highlighting relationships between taxa of this species complex. 126 · Zootaxa 4290 (1) © 2017 Magnolia Press GORDON ET AL. Bayesian inference analyses were conducted using MrBayes v.3.1.2 (Ronquist & Huelsenbeck 2003). The BI analysis included 2 x 107 generation of Markov chains sampled every 1000 generations. A standard 25 % burn-in was performed. In Mega 7.0 (Kumar et al. 2015), a ML phylogeny was constructed with the model GTR + G+ I. The program Tracer v. 1.6 (Rambaut et al. 2014) confirmed that stationarity was obtained and trees were constructed using FigTree v. 1.4.2 (Rambaut 2014). A condensed tree was constructed in Mega 7.0 (Kumar et al. 2015) for simplicity as the broader phylogeographic analyses for the B. boreas group is in progress. FIGURE 2. Bufo (Anaxyrus) boreas species complex distribution. a) Bufo (Anaxyrus) boreas distribution (shown in brown) across the Western United States with hydrological Great Basin shown with black outline and hash mark interior; b) Bufo (Anaxyrus) boreas species complex and ranges for toads including new species, illustrating the narrow distribution of localized endemics. Spatial data for all toads except B. williamsi provided by IUCN (2015). Images taken by M.R.Gordon except B. canorus with photo credit to G. Nafis. Bufo (Anaxyrus) williamsi. sp. nov. Dixie Valley Toad (Fig. 2b, Fig.4) Holotype. CAS 259271 (California Academy of Science Herpetology Collection), adult male (Fig. 4, Table 1), Dixie Valley, Churchill County, Nevada, United States (39°47'39.02"N, 118° 3'32.08"W), on 3 June 2015 by M. R. Gordon, K. Nicholson, C. Mo and C. Gibson. Paratypes. UNR 7918, adult male; UNR 7919, adult female; UNR 7920, subadult; UNR 7921, adult male; UNR 7922, adult female; UNR 7923, subadult; UNR 7924, adult male. Same locality, collection date, and collectors as holotype. Diagnosis. Bufo (Anaxyrus) williamsi is distinguishable from B. boreas by a combination of diagnostic morphological characters (Fig. 4; Table 1, Table 2), genetic evidence (Fig.3, Fig. 6), and localized distribution (Fig. 2b). Bufo (Anaxyrus) williamsi is distinct from B. boreas by: a small adult body size (SVL is more than 2.5 cm smaller than B. boreas; Table 1); significantly, but modestly, larger, closely-set eyes, and smaller head (Table 2); statistically and perceptibly larger tympanum, and shorter hind limbs; conspicuously large and elevated tibial glands; and distinctive color pattern (Fig. 4a, Fig. 4b). A NEW NEARCTIC TOAD SPECIES Zootaxa 4290 (1) © 2017 Magnolia Press · 127 FIGURE 3. Molecular examination of Bufo (Anaxyrus) boreas species complex.The TCS haplotype network was constructed using 246 sequences (1622 aligned sites) obtained from toad sampling (Fig.1b) resulting in 72 unique haplotypes, with circle sizes corresponding with the number of individuals of a particular haplotype. Haplotype colors correspond geographically (Fig. 1b) and to localized species (B. canorus (purple), B. exsul (green) and B. nelsoni (orange)) and highlight the genetic divergence of B. williamsi (red). The condensed phylogeny identifying Great Basin Bufo (Anaxyrus) boreas species complex major haplotype clades: maximum likelihood of 10 samples (1436 aligned sites) using GTR +G+I evolutionary model. The terminals are identified by taxon name and followed by locality of collection for B. boreas. Bufo williamsi, noted with a red circle, is sister to boreas of the HL clade. Heavy bars correspond with major haplotype clades. TABLE 2. Least squares means, confidence intervals and Tukey HSD post-hoc test results for four species of the Bufo (Anaxyrus) boreas species complex within the Great Basin. The least square values were generated from the MANCOVA analysis of 14 size-corrected morphometric characters described in Table 1 with corresponding lower and upper 95% confidence intervals (CI) and sample size for each species (n). Tukey HSD post-hoc tests results identify significant smaller (↓) or larger (↑) states exhibited by congeneric species when compared to B. williamsi. B. williamsi B. exsul B. nelsoni B. boreas (n=30) (n=30) (n=31) (n=289) SVL 56.3 64.0 ↑ 80.8 ↑ 82.3 ↑ CI 52.1, 60.4 59.8, 68.1 76.8, 84.9 81.0, 83.7 HL 22.2 22.0 23.4 ↑ 23.1 ↑ CI 21.7, 22.6 21.5, 22.4 23.0, 23.8 23.0, 23.3 HW 25.2 25.0 27.4 ↑ 26.5 ↑ ......continued on the next page 128 · Zootaxa 4290 (1) © 2017 Magnolia Press GORDON ET AL. TABLE 2. (Continued) B. williamsi B. exsul B. nelsoni B. boreas (n=30) (n=30) (n=31) (n=289) CI 24.6, 25.8 24.5, 25.6 27.0, 27.9 26.4, 26.7 SL 5.62 5.10↓ 5.31 5.40 CI 5.43, 5.83 4.91, 5.30 5.14, 5.50 5.34, 5.46 IND 4.87 4.81 5.15 4.84 CI 4.68, 5.05 4.64, 4.99 4.99, 5.31 4.79, 4.90 ED 7.83 6.98 ↓ 7.45 7.24 ↓ CI 7.58, 8.01 6.77, 7.19 7.26, 7.65 7.18, 7.31 IOD 11.8 11.4 11.9 12.5 ↑ CI 11.5, 12.2 11.07, 11.77 11.58, 12.22 12.40, 12.62 TYM 4.46 4.06 ↓ 4.06 ↓ 4.10 ↓ CI 4.28, 4.65 3.88, 4.23 3.89, 4.22 4.04, 4.15 PTW 6.65 6.32 6.75 6.87 CI 6.33, 6.97 6.02, 6.62 6.47, 7.02 6.78, 6.96 PTL 8.57 8.04 8.65 9.69 ↑ CI 8.12, 9.02 7.62, 8.46 8.26, 9.05 9.55, 9.82 IPD 14.9 14.4 15.2 15.2 CI 14.5, 15.4 14.0, 14.9 14.8, 15.6 15.1, 15.3 FML 29.5 29.3 30.4 31.6 ↑ CI 28.8, 30.2 28.6, 30.0 29.8, 31.0 31.4, 31.8 TBL 28.3 29.1 29.6 ↑ 31.0 ↑ CI 27.6, 29.0 28.5, 29.8 29.0, 30.2 30.8, 31.2 FTL 29.9 30.8 31.9 ↑ 32.7 ↑ CI 28.9, 30.8 30.0, 31.7 31.1, 32.7 32.4, 32.9 Bufo (Anaxyrus) williamsi is the smallest bufonid within the B. boreas species complex (Table 1, Table 2). This new species has a statistically, but modestly short, narrow head similar to the small sized B. exsul, but B. williamsi can be distinguished from B. exsul by a significantly, but modestly, longer relative snout length comparable to that B. boreas and B. nelsoni (Table 2). Bufo (Anaxyrus) williamsi has relatively large, closely set eyes and perceptively large tympanum, which distinguishes this toad from all taxa within the B. boreas species complex. The parotoid glands are slightly longer than wide, but are comparatively shorter overall than parotoids of B. boreas (Table 2). Bufo (Anaxyrus) williamsi has hind legs that are similar in relative size to B. exsul, but significantly and perceptibly shorter than those of B. boreas and B. nelsoni. The tibial glands exhibited in B. williamsi are conspicuous and approximately the width of the parotoid glands, regular in shape and rust colored with little variation among individuals of this species. In addition to morphological shape differences, B. williamsi exhibits unique coloration different from taxa of the B. boreas species complex. The dorsal ground color consists of olive shades that contain minute black flecks, rust colored warts are bordered by fine, black halos, and prominent parotoid glands are pale tan and black specked. The venter of B. williamsi is similar to B. exsul, exhibiting sharply contrasted black marbling against a white background color on the anterior sides of the limbs and belly. The presence of a dorsal stripe is variable among individuals of B. williamsi, as is similar to the other members of the B. boreas complex, with the exception of B. exsul. Distinct nuptial pads develop on the dorsal side of the thumb in males of B. williamsi, a typical secondary sexual characteristic exhibited among most bufonids. This species lacks an advertisement call, but retains a release call that sounds like the weeping of a chick (Stebbins 2003). The call is emitted when males come into contact with one another, similar to congeners of the B. boreas complex. A NEW NEARCTIC TOAD SPECIES Zootaxa 4290 (1) © 2017 Magnolia Press · 129 FIGURE 4. Photographs of Bufo (Anaxyrus) williamsi sp. nov. holotype (CAS 259271). Adult male toad presented live: (a) dorsal view and (b) ventral view; and preserved: (c) dorsal view and (d) ventral view. Photographs taken by M.R.Gordon. Description of holotype. Body small (SVL = 52.92 mm), robust; head nearly long (17.45 mm) as wide (18.35 mm; 95 % head length to head width). Dorsal outline of snout is moderately truncate; snout long in lateral view (6.61 mm; 1.3 times longer than eye diameter). Canthus rostralis distinct, slightly concave and abrupt at nares, sloping up to anterior margin of orbits. Loreal region moderately concave. Nostrils protuberant, directed dorsolaterally and closer to anterior corner of eye than end of snout. Internarial distance 75% of eye-to-naris distance. Eyes large (4.94 mm), close spaced (3.83 mm); interorbital distance 75% of eye diameter. Eyes prominent, breaching snout profile in dorsal view. Tympanum distinct, ovoid, relatively large (2.89 mm; 58% of eye diameter). Supratympanic fold present. Parotoid glands sub-elliptical, tapered at posterior margin of eye, longer (5.56 mm) than wide (3.90 mm; 77%). Parotoids elevated dorsally, slightly divergent and separated (9.18 mm); gland width smaller than eye diameter (75%). Forearms robust. Fingers unwebbed; relative lengths III > VI > I > II; nuptial pads present, raised on dorsal side of digit I; tips rounded, subarticular tubercles moderate, round; accessory palmar tubercles small and round. Thenar tubercle raised, prominent, and round. Palmar tubercle is distinct, large, subovoid, separated from medial margin of lesser thenar tubercle. Hind limbs short (FML =19.54 mm; TBL =18.75 mm; FTL =33.10 mm), robust; femur slightly longer than tibia. Tarsal fold present. Hind feet webbed proximally. Relative toe lengths IV > III > V > II > I; tips rounded. Subarticular tubercles moderate, small, 130 · Zootaxa 4290 (1) © 2017 Magnolia Press GORDON ET AL. round; plantar tubercles small, numerous. Inner metatarsal tubercle pronounced, elevated, and elliptical. Outer metatarsal tubercle smaller than inner metatarsal tubercle, conspicuous, ovoid. Longitudinally along dorsum, dorsal stripe broken, weakly present, originating posterior to interorbital space and terminating at sacral hump; irregular, elevated, scattered tubercles present, increasing in size from interorbital space to posterior margin of urostyle. Skin between tubercles nearly smooth; forearms smooth; hind leg tubercles vary in size. Tibial glands present on dorsal surface of legs, prominent, equivalent to the width of parotoid gland. Small densely concentrated tubercles present, originating posterior to labial commissure, inferior to tympanum, terminating in axillary region. Small, densely concentrated tubercles present longitudinally along mid axillary line, terminating at articulation of femur. Venter granular; seat patch dark, conspicuous. Color in life. Dorsal ground color of the holotype is complex, with chromatic hues of olive with small, diverse and irregular black flecks, (Fig. 4a). Face heavily specked. Upper eyelids flecked black against olive background color. Pupil black, horizontal, with gold-streaked iris. Parotoid glands tan; minor black spotting on crown of gland with black streaks along margins. Small, dense tubercles occur between labial commissure and axillary region and are rust colored. Rust colored tubercles irregularly distributed across dorsum, small but variable in size, with black margins. Tubercles between mid-axillary line and articulation with femur rust colored, bordered by fine black halos. Dorsal stripe cream, originating at interorbital space, broken just posterior to terminal margin of parotoid glands, resumes along vertebral region, and terminates at sacral hump. Forearms with black flecks dorsally and medium to dark brown overlying olive background. Hind legs with rusty tubercles arranged atop dark brown banding overlying ground olive color with black flecking. Inferior to midaxillary line, tubercles diminish in size until absent. Inferior mid-axillary line with heavy black mottling against white. Small black spots along inferior lower labial margin. Anterior forearms and hind legs heavily marbled black against the white background color. Throat white, immaculate. White venter heavily mottled in black; seat patch conspicuous and dark brown, with round, white spotting (Fig. 4b). Undersides of hands and feet dark gray. Tubercles of hands and feet, fingers, and toes bright orange. Color in preservative. Color is notably different and muted (Fig. 4c, Fig. 4d) relative to life (Fig. 4a, Fig. 4b). Distinctive differences include nearly monotone ground color which is dark greenish gray, warts to dark brown, dorsal stripe faint. Parotoid glands pale brown and conspicuous, streaked, and spotted a muted black color. In preservative, the bright coloration of the spinose tubercles inferior to tympanum and tubercles of hands and feet fade to white. Black mottle on the venter and limbs appears duller than in life. Tubercles on feet and hands are white with brown tips. Morphological results. Results of statistical analyses were consistent for both log-transformed data, and for using regression of SVL against the morphological variables. Both analyses detected significant differences for all 14 morphological characters evaluated at the species level among B. boreas, B. nelsoni, B. exsul, and B. williamsi (Table 1, Table 2). Bufo (Anaxyrus) williamsi is the smallest of this group (F = 77.9, p < 0.0001; F = 63.4, p 3, 376 3, 376 < 0.0001) with a relatively short (F = 903.8, p < 0.0001; F = 830.8, p < 0.0001; Table 2) and narrow head (F 4, 379 4, 379 4, =1219.0, p < 0.0001;F = 1080.1, p < 0.0001; Table 2). There were significant differences in snout length 379 4, 379 among species (F = 164.9, p < 0.0001; F =160.9, p < 0.0001), and in pairwise comparisons of Tukey HSD 4, 379 4, 379 post-hoc tests, B. williamsi differed significantly from B. exsul by having a relatively longer snout more like larger species B. boreas and B. nelsoni, a similarity detected in the least squares means generated from the MANCOVA analyses and corresponding linear regression that normalize SVL against this character (Table 2). Bufo (Anaxyrus) williamsi has relatively large eyes (F = 259.9, p < 0.0001; F = 240.0, p < 0.001) that are close together (F 4, 379 4, 379 4, 379 = 422.5, p < 0.0001; F = 371.1, p < 0.0001), which is distinct from B. boreas, and a larger tympanum compared 4, 379 to all three other species examined (F = 231.4, p < 0.0001). While the width of the parotoid gland in B. williamsi 4, 379 is similar to B. boreas, the comparative length of the parotoid is shorter (Table 2). Additionally, the characters that define the length of the leg of B. williamsi are short (FL: F = 910.4, p < 0.0001; F = 801.0, p < 0.0001; TL: 4, 379 4, 379 F = 1063.8, p < 0.0001; F = 909.0, p < 0.0001; FTL: F = 571.8, p < 0.0001; F = 470.7, p < 0.0001), 4, 379 4, 379 4, 379 4, 379 and differ significantly from B. boreas (Tukey HSD post-hoc pairwise comparisons, p < 0.0001). MANCOVA results evaluating log-transformed data by population yielded similar results with significant differences detected among localities sampled (Fig. 1a), and this analysis confirmed that B. williamsi is the smallest toad among all populations examined (F = 34.7, p < 0.0001), with a comparatively short, narrow head (HL: F = 285.1, p < 16, 379 17,379 0.0001; HW: F = 353.3, p < 0.0001), long snout (F = 55.1, p < 0.0001) and relatively the largest eyes among 17,379 17,379 A NEW NEARCTIC TOAD SPECIES Zootaxa 4290 (1) © 2017 Magnolia Press · 131 regional B. boreas and congeneric taxa examined (F = 86.07, p < 0.0001). This additional analysis confirmed that 17,379 the parotoid glands of B. williamsi are relatively shorter in length (F = 81.1, p < 0.0001) compared to B. boreas. 17, 379 However, the size of the parotoids and internarial distance (IND) were among those traits that were similar in relative sizes to B. boreas, B exsul, and B. nelsoni. On the other hand, the tympanic diameter is relatively large (F = 82.4, 17, 379 p < 0.0001) in B. williamsi, and its legs are the shortest among all populations sampled (FL: F 248.9, p < 0.0001; 17, 379 = TB: F = 312.6, p < 0.0001; FTL: F = 139.1, p < 0.0001), similar to leg sizes among B. exsul. 17, 379 17, 379 Discriminant function analysis (DFA) illustrates significant morphological differences among species (F = 42, 173 2.80, p < 0.0001; Fig. 5). The DFA correctly classified 77.3 % of predicted species, with some morphological overlap detected among B. boreas, B. nelsoni and B. exsul (Fig. 5). The morphological characters were accurate predictors of B. williamsi in all thirty predictions. The first canonical axis explained 60% of the variation in the DFA with tibial length loading most heavily, while the second canonical axis accounted for 24 % of the variation with head width loading more heavily than other characters. FIGURE 5. Discriminant function analysis (DFA). Cross validated DFA using 14 size corrected morphological characters measured from 380 live adult toads (Fig. 1a) examined within the hydrological Great Basin Bufo (Anaxyrus) boreas species complex. Species identified as B. boreas (red circle), B. nelsoni (blue diamond), B. exsul (green circle), and B. williamsi (yellow square). 132 · Zootaxa 4290 (1) © 2017 Magnolia Press GORDON ET AL.