A New Barcheek Darter Species from Buck Creek ... - BioOne

4 downloads 0 Views 4MB Size Report
1 Sportsman's Lane, Frankfort, KY 40601 USA. ABSTRACT. Etheostoma nebra, the Buck Darter, is described as a new species endemic to the Buck Creek sys-.
A New Barcheek Darter Species from Buck Creek (Cumberland River System), Kentucky (Percidae: Etheostomatinae: Catonotus: Oopareia) Thomas J. Near1 and Matthew R. Thomas2 1

Corresponding author: Department of Ecology and Evolutionary Biology, Osborn Memorial Labs, P.O. Box 208106, Yale University, New Haven, CT 06520-8106 USA; and Peabody Museum of Natural History, Yale University, P.O. Box 208118, New Haven, CT 06520-8118 USA —email: [email protected] 2

Fisheries Division, Kentucky Department of Fish and Wildlife Resources, 1 Sportsman’s Lane, Frankfort, KY 40601 USA

ABSTRACT Etheostoma nebra, the Buck Darter, is described as a new species endemic to the Buck Creek system of the Cumberland River drainage in Kentucky, USA. The earliest collection records of Etheostoma nebra date to 1955 and were considered a population of Etheostoma virgatum. Etheostoma nebra is delimited through morphological comparisons with Etheostoma virgatum and phylogenetic analyses using DNA sequences from a mitochondrial gene and five nuclear genes. Etheostoma nebra is distinguished from Etheostoma virgatum by a lower number of total lateral scales, fewer pored lateral scales and modally 13 compared with 12 pectoral fin rays. The two species also differ in patterns of pigmentation and nuptial male coloration. In the molecular phylogenies, Etheostoma nebra is not resolved as the sister species of Etheostoma virgatum. A review of all museum collection records of Etheostoma nebra and extensive field surveys in the Buck Creek system demonstrate a dramatic decline of the species over the past 30 years. Collections made from 1955 to 1981 show that Etheostoma nebra was widespread throughout the Buck Creek system, but the species is currently restricted to a small portion of Flat Lick Creek. Etheostoma nebra is critically imperiled based on its restricted geographic distribution and documented disappearance of populations within the Buck Creek system during the past three decades.

KEYWORDS Species delimitation, gene tree, species tree, new species, Teleostei, Actinopterygii, Etheostoma

Introduction Barcheek darters are a clade of seven described freshwater teleost fish species that exhibit strict allopatric distributions in the Cumberland, upper Green, upper Barren and Duck River systems of Kentucky and Tennessee (Page 1983; Hollingsworth and Near 2009). Morphological and molecular phylogenetic analyses have supported monophyly of barcheek darters, and a recent phylogeny-based classification of darters named this clade Oopareia (Braasch and Mayden 1985; Porterfield et al. 1999; Mendelson and Simons 2006; Hollingsworth and Near 2009; Near et al. 2011). Divergence time esti-

mates derived from fossil-calibrated molecular phylogenies indicate that the origin of Oopareia dates to the early to mid-Miocene with age estimates ranging between approximately 8.5 and 14.5 million years ago (Hollingsworth and Near 2009; Near et al. 2011). Five of the seven Oopareia species were discovered and described since 1971, and molecular phylogenies are a useful tool for species discovery in the clade (Kuehne and Small 1971; Page and Braasch 1976, 1977; Page et al. 2003). In addition to resolving as reciprocally monophyletic groups in molecular phylogenies, species of Oopareia are diagnosed by differences in meristic characters

Bulletin of the Peabody Museum of Natural History 56(2):127–146, October 2015. © 2015 Peabody Museum of Natural History, Yale University. All rights reserved. • http://peabody.yale.edu

128

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

(e.g., numbers of scale rows and fin elements), pigmentation patterns and male nuptial coloration (Kuehne and Small 1971; Page and Braasch 1976, 1977; Page et al. 2003). The strict allopatric distribution of species of Oopareia allows characterization of species in the clade, in part, by their specific geographic distributions. Historically, the presence of geographically disjunct barcheek darter populations—for example, Etheostoma striatulum in the upper portion of the Duck River system—was a preliminary indicator of undescribed species diversity in the clade (Page and Braasch 1977). Jordan (1880:236–237) described Etheostoma virgatum based on specimens sampled from the Rockcastle River at Livingston, Rockcastle County, Kentucky. When E. virgatum was redescribed by Page and Braasch (1977), the species was thought to exhibit a disjunct distribution of three populations in the Cumberland River system, which includes (1) the eastern part of the drainage below Cumberland Falls (Rockcastle River, Beaver Creek and Buck Creek); (2) the Caney Fork River system; and (3) the western portion of the middle Cumberland River system, which includes the Stones River, Harpeth River, Red River and several small direct tributaries to the Cumberland River. Phylogenetic analyses of mitochondrial and nuclear gene sequences revealed that the three disjunct sets of E. virgatum populations represented three distinct species, as each of the three “E. virgatum” populations were more closely related to other species of Oopareia (Page et al. 2003). Assessment of morphological differences among these three lineages resulted in the description of E. basilare (Caney Fork River system) and E. derivativum (Stones, Red and Harpeth Rivers). Characters that distinguish E. virgatum, E. basilare and E. derivativum include pigmentation on the margins of fins, coloration of pectoral fin in breeding males and several meristic characters that include modal number of anal fin rays, total lateral scales and pored lateral line scales (Page et al. 2003). As delimited by Page et al. (2003), Etheostoma virgatum is restricted to three adjacent tributaries of the Cumberland River in eastern Kentucky, the Rockcastle River, Beaver Creek and Buck Creek (Figure 1). Previous studies have noted appreciable differences in the mean number of lateral scales, pored lateral line scales and pectoral fin rays between Rockcastle River and Buck Creek

populations of E. virgatum (Page and Braasch 1977; Page et al. 2003); however, specimens of Oopareia from Buck Creek have never been included in a molecular phylogenetic analysis. In this study, we describe a new species of Oopareia endemic to Buck Creek in Kentucky, long recognized as Etheostoma virgatum, as a new species based on phylogenetic relationships inferred using molecular data and divergence in morphological characters traditionally used to diagnose and describe species of darters. Our investigation of this new species reveals a substantial reduction of its distribution in the Buck Creek system over the past 35 years. The restricted distribution of this species of Oopareia and its precipitous decline in the Buck Creek system indicates it is critically imperiled.

Materials and Methods Specimens for morphological analyses were obtained from field-based sampling and museum collections (Figure 1; Tables 1 and 2). Institutional abbreviations follow Sabaj Pérez (2014), except that YFTC refers to the Yale Fish Tissue Collection and UTFTC refers to the University of Tennessee Fish Tissue Collection. Meristic data on scale rows and fin elements were made as outlined in Hubbs and Lagler (1958) and Page (1981), except that the number of transverse scale rows was counted as described by Page (1983). Information on the number of infraorbital canal pores was taken from Page and Braasch (1977). Specimens of the new species and Etheostoma virgatum were examined from the following collections: Illinois Natural History Survey (INHS), Champaign, Illinois, USA; University of Michigan Museum of Zoology (UMMZ), Ann Arbor, Michigan, USA; Florida Museum of Natural History (UF), University of Florida, Gainesville, Florida, USA; and the Peabody Museum of Natural History (YPM ICH), Yale University, New Haven, Connecticut, USA (Tables 1 and 2). The phylogenetic relationships of Oopareia, including the new species, were investigated from analysis of DNA sequences of a single mitochondrial (NADH subunit 2 [ND2]) and five nuclear genes (plagl2, Ptr, rag1, S7 ribosomal protein intron 1 and SH3PX3). Multiple specimens were sampled from all species of Oopareia for DNA sequencing (Appendix), of which several were

A New Barcheek Darter Species from Buck Creek • Near and Thomas

129

FIGURE 1. Distribution of localities from which Etheostoma nebra (triangles) and E. virgatum (circles) have been collected. Solid symbols represent localities from which specimens were used in this study. Type locality for E. virgatum is labeled “T.” Tributaries of the Cumberland River: 1, Buck Creek; 2, Beaver Creek; 3, Rockcastle River; 4, Mill Creek.

used in previous molecular phylogenetic studies (Page et al. 2003; Hollingsworth and Near 2009; Near et al. 2011; Harrington and Near 2012, 2015; Near and Keck 2013). Two species from the Catonotus subclade Richiella, Etheostoma flabellare and E. percnurum, were included as outgroup taxa for the phylogenetic analyses. DNA was isolated from frozen or ethanol-preserved tissues using standard phenol-chloroform extraction followed by ethanol precipitation or using a protocol outlined in the Qiagen DNeasy tissue kit (http://www1.qiagen.com/Products). Previously published polymerase chain reaction primers and cycling conditions were used to amplify and sequence the targeted genes (Kocher et al. 1995; Chow and Hazama 1998; Li et al. 2007). Successful amplification products were prepared for sequencing using a polyethylene glycol

precipitation. Contiguous DNA sequences were assembled from individual sequencing reactions using the computer program Geneious, v. 7.2 (Kearse et al. 2012). The DNA sequences from each gene were aligned visually. Optimal nucleotide data partitioning and molecular evolutionary models for the phylogenetic analyses were determined using Partitionfinder, v. 1.1 (Lanfear et al. 2012). A mitochondrial gene tree was inferred from the ND2 sequences using the computer program MrBayes, v. 3.2 (Ronquist et al. 2012). The MrBayes analysis was run for 107 generations with two simultaneous runs each with four chains. Monitoring the average standard deviation of the split frequencies between the two runs allowed assessment of stationarity of the chains and convergence, which was less than 0.005 after 106 generations. The first

130

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

TABLE 1. Museum records of Etheostoma nebra. Collections containing specimens from which meristic data were recorded are indicated by an asterisk (*). All collections were made in Kentucky, USA. Catalog number

Date collected

UMMZ 171557* UMMZ 171553* UMMZ 171591* UF 14778* NLU 6053 NLU 10837 UAIC 2976.15 UF 235351* INHS 78872 INHS 78860 INHS 75518 INHS 76019* SIUC 9510 SIUC 13228 SIUC 9380 SIUC 13299 SIUC 13256

September 1955 September 1955 September 1955 March 1967 March 1967 August 1968 June 1968 October 1969 June 1970 March 1976 October 1976 March 1977 June 1978 April 1979 July 1979 July 1979 June 1979

SIUC 13503 SIUC 13560

June 1979 May 1979

SIUC 13533 SIUC 13266 SIUC 13271

March 1980 September 1980 April 1981

SIUC 3897 EKU 1414 MOSU 1791 EKU 1826 YPM ICH 023841* YPM ICH 023754* YPM ICH 027039*

September 1981 May 1996 August 2001 September 2001 July 2010 July 2010 April 2010

YPM ICH 027088* July 2011 YPM ICH 026966* May 2014 YPM ICH 027005* May 2014 YPM ICH 027331* April 2014 YPM ICH 028113* April 2014 YPM ICH 028203* May 2015 YPM ICH 028026* March 2015

Locality

Number of specimens

Brushy Creek, at Buck Creek confluence, Pulaski Co. Buck Creek, at KY-80 bridge, Pulaski Co. Buck Creek, at Brushy Creek confluence, Pulaski Co. Buck Creek, at Brushy Creek confluence, Pulaski Co. Buck Creek, at KY-461 bridge, Pulaski Co. Indian Creek, at KY-452 bridge, Pulaski Co. Buck Creek, at KY-39 bridge, Pulaski Co. Flat Lick Creek, at KY-461 bridge, Pulaski Co. Buck Creek, at Briary Creek confluence, Pulaski Co. Flat Lick Creek, at Dabney, Pulaski Co. Buck Creek, at KY-80 bridge, Pulaski Co. Buck Creek, at Kocher Ridge Rd. crossing, Lincoln Co. Buck Creek, at KY-1677 bridge, Pulaski Co. Buck Creek, at Kocher Ridge Rd. crossing, Lincoln Co. Brushy Creek, at KY-328 bridge, Rockcastle Co. Flat Lick Creek, at KY-461 bridge, Pulaski Co. Brushy Creek, at Smith Hollow Rd., crossing, Pulaski Co. Clifty Creek, 0.3 km upstream of mouth, Pulaski Co. Gilmore Creek at Ephesos School Rd. bridge, Lincoln Co. Buck Creek, at KY-39 bridge, Pulaski Co. Buck Creek, at Gilmore Creek confluence, Lincoln Co. Bee Lick Creek, at Friendship Church Rd. bridge, Pulaski Co. Buck Creek, at KY-461 bridge, Pulaski Co. Buck Creek, at Broughtentown Rd. bridge, Lincoln Co. Big Spring Branch, 4 km west of Shopville, Pulaski Co. Buck Creek, at Goochtown Rd. crossing, Pulaski Co. Flat Lick Creek, at Barnesburg Rd., Pulaski Co. Flat Lick Creek, at Barnesburg Rd., Pulaski Co. Flat Lick Creek, at Big Spring Branch confluence, Pulaski Co. Flat Lick Creek, at Herrin Lane, Shopville, Pulaski Co. Flat Lick Creek, at Big Spring Branch confluence, Pulaski Co. Flat Lick Creek, at Herrin Lane, Shopville, Pulaski Co. Flat Lick Creek, at Big Spring Branch confluence, Pulaski Co. Flat Lick Creek, at Big Spring Branch confluence, Pulaski Co. Stewart Branch, at Shopville Community Park, Pulaski Co. Stewart Branch, at Shopville, Pulaski Co.

3 3 1 5 12 10 1 13 1 1 1 3 2 7 28 2 5 1 3 3 2 2 2 1 7 1 4 7 12 6 4 2 1 1 4 6

131

A New Barcheek Darter Species from Buck Creek • Near and Thomas

TABLE 2. Specimens of Etheostoma virgatum from which meristic data were recorded. All collections were made in Kentucky, USA. Catalog number

Date collected

UF 15489 UMMZ 139054 UMMZ 154515 UMMZ 154528 UMMZ 96942 YPM ICH 022055 YPM ICH 022980 YPM ICH 023710 YPM ICH 023772 YPM ICH 027040 YPM ICH 027041 YPM ICH 027042 YPM ICH 027086 YPM ICH 028201

September 1967 August 1936 April 1947 April 1947 July 1928 April 2009 November 2009 July 2010 July 2010 June 2010 November 2009 June 2009 May 2010 May 2015

Locality Roundstone Creek, Rockcastle Co. Rockcastle River, Rockcastle/Laurel Co. line Clear Creek, Rockcastle Co. Roundstone Creek, Rockcastle Co. Parker Branch, Laurel Co. Roundstone Creek tributary, Rockcastle Co. Middle Fork Rockcastle River, Jackson Co. Indian Creek, Jackson Co. Beaver Creek, McCreary Co. Mill Creek, McCreary Co. Indian Creek, Jackson Co. Birch Lick Creek, Jackson Co. Beaver Creek, McCreary Co. Sinking Creek, Rockcastle Co.

50% of the sampled generations was discarded as burn-in, and the posterior phylogeny was summarized as a 50% majority-rule consensus tree. A mitochondrial DNA (mtDNA) haplotype network was constructed from the ND2 sequences sampled from the new species using TCS 1.21 (Clement et al. 2000). The computer program BEAST, v. 1.8 (Heled and Drummond 2010; Drummond et al. 2012) was used to perform two sets of multispecies coalescent analyses to estimate a species tree for Oopareia, one restricted to the five sampled nuclear genes and another that included the mtDNA ND2 gene. The number of specimens sampled from each species is given in Appendix. A birth–death speciation branching prior was used for the species tree inferences. The uncorrelated log normal model of molecular evolutionary rate heterogeneity was used for all loci, and the molecular evolutionary rate for each locus was scaled to the gene with the highest rate (Drummond et al. 2006). The chain lengths were 507 generations with parameters sampled every 103 generations. Convergence of parameter values in the Markov chain Monte Carlo were assessed by the effective sample sizes that were calculated using Tracer, v. 1.5 and visualizing the cumulative split frequencies in the set of posterior trees using AWTY (Nylander et al. 2008). Generations sampled before convergence were discarded as burn-in.

Number of specimens 7 7 4 2 12 10 3 2 10 22 1 5 17 4

All meristic data, specimen information, DNA sequence alignments and BEAST formatted xml files are available from the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad. 13nj6; Near and Thomas 2015). All new DNA sequences generated in this study were submitted to GenBank (Benson et al. 2013; Appendix).

Taxonomy Etheostoma nebra Near and Thomas, new species Buck Darter Figures 2, 3A and B, 4A–C and 5; Tables 1 and 3–12 Etheostoma virgatum. Page and Braasch, 1977:17, fig. 4, tbls. 1–7 (“Striped Darter,” geographic distribution, presence in Buck Creek and morphology); Page and Schemske, 1978:407–408 (morphology and distribution in Buck Creek); Harker et al., 1979:397 (presence in Buck Creek); Harker et al., 1980:525 (presence in Brushy Creek, Buck Creek system); Page, 1980:707 (“Striped Darter,” in part, presence in Buck Creek); Burr, 1980:78 (“Striped Darter,” in part, presence in Buck Creek); Kuehne and Barbour, 1983:155 (“Striped Darter,” in part, presence in Buck Creek); Page, 1983:149–150 (“Striped Darter,” in part, presence in Buck Creek, distribution map and morphological divergence); Cicerello and Butler, 1985:146, tbl. 4 (“Striped Darter,” distribution in Buck Creek and distribution map); Burr and Warren, 1986: 326 (“Striped Darter,” distribution in Buck Creek and distribution map); Moeykens, 1997:75 (presence in Buck Creek); Page et al., 2003:521, fig. 1, tbls. 4–10 (“Striped Darter,” in part, presence in Buck Creek, distribution map and morphology); Thomas and Brandt, 2012:10–11 (presence in Flat Lick Creek, Buck Creek system).

132

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

FIGURE 2. Live holotype and allotype specimens of Etheostoma nebra. A, Etheostoma nebra holotype, YPM ICH 027331, 58.8 mm standard length (SL) male. B, E. nebra allotype, YPM ICH 028113, 52.3 mm SL female. Both freshly preserved specimens, Flat Lick Creek, Pulaski Co., Kentucky, 8 April 2010. Holotype. YPM ICH 027331 (Figure 2A), adult male, 58.8 mm standard length (SL). Flat Lick Creek, immediately below confluence with Big Spring Branch, latitude 37.15689, longitude 84.4789, access via Mark Welborn Rd., Pulaski Co., Kentucky, USA, 8 April 2010, M.R. Thomas and S.L. Brandt. Allotype. YPM ICH 028113 (Figure 2B), adult female, 52.3 mm SL. Collected with the holotype. Paratypes. UMMZ 171553 (three males, 27.4–40.6 mm SL), 14 September 1955, Buck Creek at KY 80, east of Somerset, Pulaski Co., Kentucky, USA; UMMZ 171577 (three males, 39.4–40.8 mm SL), 14 September 1955, UMMZ 171591 (one male, 38 mm SL), 14 September 1955 and UF 14788 (four males, 29.9–44.9 mm SL; one female, 42.5 mm SL), 25 March 1967, Buck Creek at confluence with Brushy Creek, latitude 37.212255, longitude -84.467324, Pulaski Co., Kentucky, USA; UF 235351 (six males, 36.3–51.8 mm SL; six females, 30.1–36.3 mm SL; meristic data not collected for one specimen less than 29 mm SL), Flat Lick Creek at KY 461, latitude 37.212255, longitude -84.467324, Pulaski Co., Kentucky, USA; INHS 76019 (three males, 45.7–51.2 mm SL; meristic data not collected for 10 juveniles, 19.0–25.8 mm SL), 14 March 1977, Buck Creek at Kocher Ridge Rd., latitude 37.403875, longitude -84.614385, Lincoln Co., Kentucky, USA; YPM ICH 027039 (five males, 32.4–54.6 mm SL;

seven females, 32.3–52.3 mm SL), 8 April 2010, YPM ICH 023754 (seven males, 35.8–51.4 mm SL), 29 July 2010, YPM ICH 027088 (four males, 36.9–50.8 mm SL; one female, 41.8 mm SL), 19 July 2011, YPM ICH 026966 (one male, 57.4 mm SL; three females, 37.6–44.1 mm SL), 13 May 2014, same locality as holotype; YPM ICH 028203 (four males, 58–61 mm SL), 14 May 2015, Stewart Branch at Shopville Community Park, latitude 37.15699, longitude -84.48392, Pulaski Co., Kentucky, USA; YPM ICH 028206 (four males, 51.8–60.1 mm SL; two females, 37.6–49.2 mm SL), 24 March 2015, Stewart Branch at Shopville, Pulaski Co., Kentucky, USA. Material examined not designated as types. YPM ICH 023841 (four males, 38.3–46.0 mm SL), 27 July 2010, same locality as holotype; YPM ICH 027005 (two females, 37.4–44.2 mm SL), 13 May 2014, Flat Creek at Herrin Lane (CR-1009), at Shopville, Pulaski Co., Kentucky, USA. Diagnosis and description. A moderate-sized species of Oopareia, as diagnosed in previous studies (Page 1975; Braasch and Mayden 1985; Page et al. 2003; Near et al. 2011). Characters present in Etheostoma nebra that are synapomorphies for Oopareia include the presence of an iridescent bar on the cheek and red or orange coloration in the fins of nuptial males. Maximum body size is 60.1 mm SL, 71.7 mm total length (YPM ICH

A New Barcheek Darter Species from Buck Creek • Near and Thomas

133

FIGURE 3. Live adult male and female specimens of Etheostoma nebra and E. virgatum. A, Etheostoma nebra paratype, YPM ICH 27039, 55 mm standard length (SL) male, Flat Lick Creek, Pulaski Co., Kentucky, 8 April 2010. B, E. nebra paratype, YPM ICH 026966, YFTC 24305, 44 mm SL female, Flat Lick Creek, Pulaski Co., Kentucky, 13 May 2014. C, E. virgatum, YPM ICH 027042, 53 mm SL male; D, E. virgatum, YPM ICH 027042, 42 mm SL female; both Birch Lick Creek, Jackson Co., Kentucky, 29 June 2009.

134

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

FIGURE 4. Live adult male specimens of Etheostoma nebra and E. virgatum, all collected and photographed on 14 May 2015. Etheostoma nebra, YPM ICH 028203: A, 58 mm standard length (SL). B, 58 mm SL. C, 61 mm SL. All Stewart Branch, Pulaski Co., Kentucky. E. virgatum, YPM ICH 028201: D, 59 mm SL. E, 62 mm SL. F, 60 mm SL. All Sinking Creek, Laurel Co., Kentucky.

028203). Faint brown stripes on side of body are overlain with irregular shaped black-brown blotches that extend from the dorsum to the midline of the body and from the midline to the last three or four scale rows toward the ventral portion of the body (Figures 2, 3 and 4). Modal scale counts are 43 lateral scales and 19 pored lateral scales (Tables 3 and 4), 6 scale rows above the lateral line (Table 5), 7 scale rows below the lateral line (Table 6), 14 transverse scale rows (Table 7) and 18 scales around the caudal peduncle (Table 8). Modal counts of fin elements are 13 pectoral rays (Table 9), 9 anal rays (Table 10), 9 dorsal fin spines (Table 11) and 13 dorsal fin rays (Table 12). The infraorbital canal is interrupted, with four anterior and two posterior pores (Page and Braasch 1977, tbl. 1; Page et al. 2003, tbl. 10). Nuptial condition males have an intense red-orange coloration in the pectoral, first and second dorsal, anal and caudal fin membranes (Figures 2A, 3 and 4). The red-orange coloration of the pectoral fin is interrupted with irregular-shaped bright white patches that are restricted to the distal half of the fin (Figures 2A, 3 and 4). The first four to six membranes of the first dorsal fin are colored black with the darkest pigment in the second and third membranes. The distal margin of the anal fin is pigmented with a continuous black stripe. Infrequently, there is black pigment in the caudal fin that is restricted to a small area of the distal edge of the ventral fin margin (see Figure 4A and C). Comparisons. The only other species of Oopareia that have brown stripes on the side of the body are Etheostoma virgatum, E. derivativum and E. basilare (Page et al. 2003). E. nebra differs morphologically from E. virgatum in pigmentation/coloration and meristic traits (Tables 3, 4 and 9). Compared with E. virgatum, the brown stripes on the side of the body in E. nebra are

much less pronounced (Figures 2, 3 and 4). The patches of pigment along the midline of the side of the body in E. virgatum consist of intense black melanophores along the brown stripe that are one or two scales wide and no more than three scale rows deep, with diffuse black dusky pigmentation between the black melanophores (Figures 3 and 4). Pigmentation along the midline of the side of the body in E. nebra consists of a much less intense black pigmentation, extends across four or more scale rows and is more irregular in shape and usually taller than wide (Figures 2, 3 and 4). The coloration of the first dorsal fin in E. virgatum is a yellow orange and is typically more diffuse than the intense red-orange coloration in E. nebra (Figures 2, 3 and 4). Pigment on head and predorsal area in E. virgatum is darker overall, with more prominent vermiculated patterns than in most E. nebra examined (Figures 3 and 4). Nuptial condition males of E. virgatum have a slight to intense wash of black pigmentation distally on the ventral portion of the caudal fin that can extend around the margin of most of the fin (see Figure 4E and F). When present, black pigmentation in the caudal fin of E. nebra is fainter and occurs on a smaller area on the distal edge of the ventral margin than typically observed in E. virgatum (Figure 4A and C). Compared with E. virgatum, E. nebra has modally 43 compared with 48 total lateral scales (Table 3), an average of 18.5 compared with 14.9 pored lateral scales (Table 4) and modally 13 compared with 12 pectoral fin rays (Table 9). Etymology. The name nebra is from the Greek word ␯␧␤␳óc¸, meaning “young deer or fawn,” in reference to the endemic distribution in Buck Creek and the fact that the authors are both presently parents of young children.

A New Barcheek Darter Species from Buck Creek • Near and Thomas

135

FIGURE 5. Changes in the distribution of Etheostoma nebra in Buck Creek based on collections made during the past 60 years. Tributaries of Buck Creek: 1, Flat Lick Creek; 2, Brushy Creek; 3, Indian Creek; 4, Caney Creek; 5, Crab Orchard Creek; 6, Gilmore Creek. Distribution and habitat. Etheostoma nebra is restricted to the Buck Creek drainage, a fifth-order tributary of the impounded Cumberland River (Lake Cumberland) downstream of Cumberland Falls (Figures 1 and 5). Buck Creek is part of the middle Cumberland River subsystem (Harker et al. 1980) and has a watershed area of 767 km2 draining portions of Lincoln, Rockcastle and Pulaski Counties in southeastern Kentucky. Surface geology within the Buck Creek drainage consists primarily of Mississippian Age limestone with limited exposures of shale bedrock (Quarterman and Powell 1978). Most of the drainage, from Flat Lick Creek and state route (KY) 80 upstream, lies within the Eastern Highland Rim Subsection of the Interior Plateau Ecoregion. Topography is characterized by gently rolling hills with an expansive floodplain. Here streams are nutrient rich and have moderate gradients with riffles composed of cobble, gravel and bedrock; pools are frequent and underlain with sand, detritus, cobble and areas of exposed bedrock (Woods et al. 2002). Approximately 80% of the watershed is used for agriculture with the remainder consisting of isolated parcels of forest and rural dwellings (Harker et al. 1979, 1980). Karst topography and sinking creeks are common in the watershed, especially south of latitude 37°17⬘00⬙N (Cicerello and Butler 1985). The lower section of the drainage, downstream of Flat Lick Creek confluence, lies within the Plateau Escarpment Subsection of the Southwestern Appalachians Ecoregion. This section of Buck Creek is deeply entrenched and surrounded mostly by forest, some of which is included within the proclamation boundary of the Daniel Boone National Forest (Woods et al. 2002). The substrate in lower Buck Creek gradually changes from coarse sand, gravel and small

cobble to limestone rubble, slab boulders and extended areas of cracked bedrock (Schuster et al. 1989). Although 36 vouchered collections exist for the main stem of Buck Creek and seven of its tributaries (Table 1), Etheostoma nebra is currently known to occur only in the Flat Lick Creek drainage in Pulaski County (Figure 5). Flat Lick Creek, a second-order tributary with a 56.5 km2 watershed, submerges underground before entering lower Buck Creek. During 2010 to 2015, E. nebra was collected only in Big Spring Branch and Stewart Branch, two small tributaries of Flat Lick Creek, and the main stem of Flat Lick Creek near the confluences of these two tributaries. Individuals were most frequently captured in clear flowing pools and runs, at depths of less than 0.5 m, and substrate consisting of a loose mixture of coarse sand, gravel, cobble and scattered large slab rocks. In Big Spring Branch and Stewart Branch, E. nebra was the only darter species encountered. Other species often captured with E. nebra were typical inhabitants of cool, clear headwater streams, including Semotilus atromaculatus (Creek Chub), Rhinichthys obtusus (Western Blacknose Dace) and Chrosomus erythrogaster (Southern Redbelly Dace). Despite survey attempts at other locations in Flat Lick Creek, the species was not found elsewhere in the drainage. Molecular phylogeny. Details on the size, in base pairs, and optimal molecular evolutionary models for each gene are given in Table 13. Uncorrected genetic distances observed in the ND2 gene between Etheostoma nebra and E. virgatum ranged between 9.7% and 10.2%, and distances between E. nebra and E. obeyense ranged between 9.2% and 10.3%. There were eight

136

2.46 2 5

1

13 2 3 8 15 3 4 8 11 1 3 7 23 10 2 11 6 2 2 2

5 1 2 2

6 1 1 4

14 6 3 5

1 1 2

3

7

13

12

12

11

7

4

1

1 4

5 1 1 3

2

1

27 22 57

106 48.75 47.96 2.03 48.45 2.58 49.21 2.52

2.24 44.60 74

SD Mean N 55 56 54 53 52 51 50 49 48 47 46 45 44 43 42 41 40

Buck Creek

Total Beaver Creek Mill Creek Rockcastle River

Etheostoma nebra

Etheostoma virgatum

39 Drainage Species

Number of lateral scales

TABLE 3. Counts of lateral scales in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation.

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015 observed haplotypes among the 17 sampled specimens of E. nebra, and a maximum number of four nucleotide changes between any two haplotypes (Figure 6). The Bayesian-inferred mtDNA ND2 gene tree did not resolve the relationships among Etheostoma barbouri, the E. basilare complex and a strongly supported clade containing all other species of Oopareia (Figure 6). E. nebra was resolved as the sister lineage of a clade containing E. obeyense, E. derivativum, E. striatulum and E. smithi; however, the nodes relating both E. nebra and E. obeyense are not supported with strong (greater than 0.95) Bayesian posteriors (Figure 6). E. nebra and E. virgatum were not resolved as sister species in the mtDNA gene tree. The coalescent species trees inferred using a combination of the mtDNA ND2 gene and the five nuclear loci and a species tree inferred using only the nuclear genes both resolve a clade containing Etheostoma nebra, E. virgatum, E. obeyense, E. derivativum, E. striatulum and E. smithi (Figure 7). The node support for this clade is strong in the mtDNA nuclear gene species tree, but only moderate in the nuclear gene only phylogeny. In both species tree analyses, E. nebra and E. obeyense are resolved as sister species, and neither species is sister to E. virgatum (Figure 7).

Discussion Phenotypic differences and molecular phylogenetic analyses support the recognition of Etheostoma nebra as a distinct species. Specimens of E. nebra were first collected in the mid-1950s (Table 1), about 75 years after E. virgatum was described from the Rockcastle River (Jordan 1880). The first use of molecular phylogenies for species discovery in darters resulted in the description of E. basilare and E. derivativum (Page et al. 2003), both of which were long considered geographically disjunct populations of E. virgatum (Page and Braasch 1977; Burr and Warren 1986; Etnier and Starnes 1993). Several morphological studies (Page and Braasch 1977; Page et al. 2003), including our new data, which also consider pigmentation and male nuptial coloration (Figures 2, 3 and 4), show that E. nebra is quite divergent from E. virgatum (Tables 3, 4 and 9). Similar to results presented in Harrington and Near (2015), molecular phylogenetic analyses either resolve E. nebra as the sister species of E. obeyense (Figure 7) or unresolved in a lineage containing E. obeyense and a clade composed of E. derivativum, E. striatulum and E. smithi (Figure 6). No molecular phylogeny resolves E. nebra and E. virgatum as sister species (Figures 6 and 7). The three species previously masquerading as Etheostoma virgatum are not cryptic species in the classic sense of being morphologically indistin-

3.15 2.60 2.81 3.50 14.88 13.41 16.82 14.66 106 27 22 57

3.28 18.49 74 2

1 1 2 2 2 2 2 1 4 8

1

3

2 1 2

Total Beaver Creek Mill Creek Rockcastle River Etheostoma virgatum

2 1 Buck Creek Etheostoma nebra

2

1 1

1

4 3

4 2

6 1 1 4

11 5 1 5

17 4 3 10

20 5 4 11

13 5 1 7

12

1

4

2

3

1

1 6 2 10 9 11 4 6 10 5 2 3 2 1

SD Mean N 25 26 24 23 22 21 19 20 18 17 16 15 14 13 12 11 10 9 8 7 Drainage Species

Number of pored lateral scales

TABLE 4. Counts of pored lateral scales in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation.

A New Barcheek Darter Species from Buck Creek • Near and Thomas

137

guishable (e.g., Egge and Simons 2006), but rather remained undiscovered because there was no phylogenetic context to interpret patterns of phenotypic divergence among these distinct evolutionary lineages. The geographically disjunct populations previously considered as E. virgatum were assumed to share common ancestry, so the observed divergence in morphology was considered a curious pattern of intraspecific variation (Page and Braasch 1977). Our approach to species discovery in Oopareia roughly follows the approach advocated by Berendzen et al. (2009), in which divergence in traditional meristic and morphometric traits is assessed by partitioning among resolved clades in molecular phylogenies. This approach does not rely on the assumption that deep divergences within species, or nonmonophyly of species, are characterized by morphologically cryptic lineages. Instead, the molecular phylogenies provide a tool for species discovery that provides a sound evolutionary perspective to interpret divergence in morphological traits traditionally used to describe and delimit species of North American freshwater fishes. The continued integration of classic meristic traits and patterns of male nuptial coloration that have long been used to delimit species of darters will result in the discovery and description of additional species of Oopareia. There is a noticeable and precipitous decline of Etheostoma nebra throughout the Buck Creek system over the past 35 years (Figure 5). During a survey of the fishes of Buck Creek, Cicerello and Butler (1985) reported the species to be generally distributed throughout the drainage; specimens were collected at 22 of 39 sites sampled, including the main stem of Buck Creek and seven tributaries. Despite extensive sampling of these and other historical collection localities over the past 10 years and a survey of fishes at 47 sites throughout the Buck Creek drainage during 2010 to 2012, E. nebra was found at less than 10% of sites having historical presence and now seems to be restricted globally to the Flat Lick Creek drainage (Figure 5; Thomas and Brandt 2012). Repeated sampling at 10 selected historic sites outside of Flat Lick Creek during 2014 to 2015 also failed to detect E. nebra. Within the Flat Lick Creek drainage, backpack electrofishing surveys conducted during March 2015 in Big Spring Branch and Stewart Branch detected Etheostoma nebra in the lower and upper reaches of each stream. At these sample locations,

138

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

TABLE 5. Counts of scales above the lateral line in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of scales above lateral line Species

Drainage

5

6

7

N

Mean

SD

Etheostoma nebra

Buck Creek

9

62

3

74

5.92

0.41

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

37 9 17 11

66 18 5 43

3

106 27 22 57

5.68 5.67 5.23 5.58

0.53 0.48 0.43 0.87

3

TABLE 6. Counts of scales below the lateral line in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of scales below lateral line Species

Drainage

6

7

8

9

N

Mean

SD

Etheostoma nebra

Buck Creek

12

41

20

1

74

7.14

0.69

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

11 3 2 6

80 24 17 39

15

106 27 22 57

7.04 6.89 7.05 6.92

0.50 0.32 0.49 1.01

3 12

TABLE 7. Counts of transverse scale rows in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of transverse scale rows Species

Drainage

12

13

14

15

16

N

Mean

SD

Etheostoma nebra

Buck Creek

6

14

38

14

2

74

13.89

0.90

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

1 1

12 3 3 6

69 23 16 30

20

106 27 22 57

14.09 13.81 14.00 13.84

0.61 0.48 0.53 1.92

E. nebra was the only species of darter encountered, and individuals (n ⫽ 9–25) were captured in short periods of time (217–795 shocking seconds) despite obvious habitat disturbance throughout each watershed. In Flat Lick Creek, the species was present only at locations sampled within 400 m downstream of the confluences of Big Spring Branch and Stewart Branch. These survey data suggest that the two tributary streams are essential to the persistence of the species, and its

3 21

presence in Flat Lick Creek is sparse and limited to the portion that is downstream of Big Spring Branch. Factors responsible for the apparent decline of Etheostoma nebra in the Buck Creek drainage are uncertain, and it is puzzling why the species has persisted only in Flat Lick Creek and two of its small tributaries. As with much of the upper Buck Creek watershed, land use within the Flat Lick Creek drainage is predominantly agriculture

139

A New Barcheek Darter Species from Buck Creek • Near and Thomas

TABLE 8. Counts of scale rows around the caudal peduncle in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of caudal scale rows Species

Drainage

16

17

18

19

Etheostoma nebra

Buck Creek

1

24

30

19

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

7

33 9

7

24

40 14 10 16

25 4 11 10

20

1 1

N

Mean

SD

74

17.89

0.80

106 27 22 57

17.81 17.81 18.59 17.51

17.81 0.68 0.59 2.47

TABLE 9. Counts of left pectoral fin rays in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of left pectoral fin rays Species

Drainage

Etheostoma nebra

Buck Creek

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

11

6 2 4

12

13

14

N

Mean

SD

11

62

1

74

12.87

0.38

95 27 18 50

5

106 27 22 57

11.99 12.00 12.00 11.78

0.33 0.00 0.44 1.61

2 3

TABLE 10. Counts of anal fin rays in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of anal fin rays Species

Drainage

8

9

10

N

Mean

SD

Etheostoma nebra

Buck Creek

13

55

6

74

8.91

0.50

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

12 2 9 1

68 24 13 31

26 1

106 27 22 57

9.14 8.96 8.59 8.98

0.61 0.34 0.50 1.31

(mostly pasture). Narrow forested areas occur intermittently along stream channels, with intervening areas of open pasture that allow cattle access to the stream at multiple locations. Rural residential settlement occurs throughout the Flat Lick Creek drainage, including bridge crossings at three state routes (KY 80, KY 461 and KY 692) and several local access roads. Flat Lick Creek is situated on the boundary between the Plateau Escarpment (Knobs physiographic region) and the Eastern Highland Rim (Mississippi Plateau physiographic region). Big Spring Branch and

25

Stewart Branch, which drain from the south (Knobs), receive cool, clear groundwater discharge from multiple karst springs. Although other portions of Flat Lick Creek are spring fed, our sampling failed to detect E. nebra outside of Big Spring Branch, Stewart Branch and Flat Lick Creek within close proximity to the mouths of these two tributaries. In general, Buck Creek is one of the least affected tributaries of the upper Cumberland River drainage below Cumberland Falls and supports a rich aquatic fauna, with nearly 56% of the

140

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

TABLE 11. Counts of first dorsal fin spines in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of dorsal fin spines Species

Drainage

8

9

10

N

Mean

SD

Etheostoma nebra

Buck Creek

11

61

2

74

8.88

0.40

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

10 2

92 24 20 48

4 1 2 1

106 27 22 57

8.94 8.96 9.09 8.79

0.36 0.34 0.29 1.21

8

TABLE 12. Counts of second dorsal fin rays in Etheostoma nebra and E. virgatum. Numbers in bold are total counts for each species. Abbreviations: N, number of specimens; SD, standard deviation. Number of dorsal fin rays Species

Drainage

Etheostoma nebra

Buck Creek

Etheostoma virgatum

Total Beaver Creek Mill Creek Rockcastle River

12

13

14

N

Mean

SD

3

57

14

74

13.15

0.46

16 4 10 2

68 22 11 35

22 1 1 20

106 27 22 57

13.06 12.89 12.59 12.82

0.60 0.42 0.59 1.78

TABLE 13. Size of sampled genes in base pairs and optimal molecular evolutionary models Gene

Number of base pairs

Optimal molecular evolutionary model

ND2 (mtDNA)

1,047

plagl2 Ptr rag1 S7 intron 1 SH3PX3

814 766 1,305 527 687

HKY+G (first codon) HKY+I (second codon) GTR+G (third codon) HKY+I HKY+I HKY+G GTR+G HKY+I

144 fish species known from the combined middle and upper Cumberland River basin in Kentucky (Thomas and Brandt 2012). Six freshwater mussel species that are federally listed as endangered are known from Buck Creek (US Fish and Wildlife Service 2013; R. Cicerello, pers. comm.). Etheostoma nebra is one of three species in the Buck Creek drainage known to be a host to Villosa trabalis (Cumberland Bean), a federally listed freshwater mussel with a small population in the main stem of Buck Creek (Layzer and Anderson 1991). How the decline or localized extirpation of

E. nebra has affected the V. trabalis population in Buck Creek is uncertain; however, limited host fish availability has been suggested as a factor limiting recruitment for this rare mussel (US Fish and Wildlife Service 2010). Sixty-one river kilometers of the main stem of Buck Creek has been designated as a critical habitat unit for the six freshwater mussels protected under the Endangered Species Act (US Fish and Wildlife Service 2013). Critical habitat designated for these mussels is the only form of protection for Etheostoma nebra in Buck Creek. Apart from

A New Barcheek Darter Species from Buck Creek • Near and Thomas

141

FIGURE 6. Bayesian-inferred phylogeny of Oopareia based on the mitochondrial NADH subunit 2 (ND2) gene. Filled black circles identify clades supported with a Bayesian posterior of 1.00, and filled gray circles identify clades supported with a posterior ranging between 0.99 and 0.95. The value of all nodes with posterior support less than 0.95 is given at nodes. Inset shows an unrooted mitochondrial DNA (mtDNA) haplotype network of 17 sampled Etheostoma nebra specimens.

142

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

FIGURE 7. Maximum clade credibility species trees of Oopareia. A, Estimated from the mitochondrial NADH subunit 2 (ND2) gene and five nuclear genes. B, Estimated from five nuclear genes. Bars represent the 95% credible interval of the relative divergence times, and the Bayesian posterior clade support is given at each node.

143

A New Barcheek Darter Species from Buck Creek • Near and Thomas

small parcels of land managed by the Nature Conservancy (Pumphrey Tract, 109 hectares) and Kentucky Department of Fish and Wildlife Resources (Buck Creek Wildlife Management Area, 344 hectares; Ping-Sinking Valley Wildlife Management Area, 325 hectares), land within the Buck Creek watershed is under private ownership. In 2005, the Kentucky Department of Fish and Wildlife Resources listed E. virgatum, which now includes E. nebra, as a Species of Greatest Conservation Need under the State Wildlife Grant Program (Kentucky CWCS 2013). This program provides funding for research, monitoring and other conservation actions for rare or at-risk species not protected under the Endangered Species Act; however, it provides no legal protection from activities that could result in habitat loss or degradation. Etheostoma nebra should be assigned the rank of G1 (critically imperiled globally) using global conservation criteria for conservation listing (Master 1991) and conservation status of endangered (E) using American Fisheries Society criteria (Jelks et al. 2008) based on (1) its narrowly restricted range and (2) documented decline within its range during the past 35 years. Recommended conservation measures include (1) cooperative agreements with landowners and local managing entities that promote habitat protection or enhancement within Flat Lick Creek and other Buck Creek tributaries with known occurrences of E. nebra; (2) additional surveys throughout the

Buck Creek drainage using detection probability and occupancy estimation; (3) life history research that identifies potential factors responsible for the decline of E. nebra, specifically habitat and water quality variables that could be present in Flat Lick Creek but have changed or diminished elsewhere in the Buck Creek drainage; and (4) protocols for captive propagation to determine thermal tolerances and survivorship, and potentially for reintroducing or augmenting populations. Any plans for propagation, translocation, reintroduction or augmentation should follow guidelines established by George et al. (2009) and must be done in conjunction with genetic analyses given the restricted range and potentially small population size of this species.

Acknowledgments We thank the following individuals and institutions for loans of material: C.A. Taylor and D. Wylie (INHS), D. Nelson (UMMZ) and R. Robbins and L. Page (UF). G. Watkins-Colwell (YPM) provided collections assistance. We were assisted in the field by S. Brandt, D. Murray and B. Keck. Molecular work was coordinated by M. Correa and J. Glass. This research was supported by the Peabody Museum of Natural History, Yale University. Received 4 June 2015; revised and accepted 10 July 2015.

Appendix Specimens Sampled for DNA Sequencing and GenBank Accession Numbers Abbreviations: INHS, Illinois Natural History Survey; UT, University of Tennessee Etnier Ichthyology Collection; YFTC, Yale Fish Tissue Collection; YPM ICH, Peabody Museum of Natural History, Yale University, New Haven, Connecticut, USA; NA, not sampled. Species

Catalog number

Etheostoma nebra YPM ICH 023754 YPM ICH 023754 YPM ICH 027005 YPM ICH 027005 YPM ICH 023754 YPM ICH 023754

Tissue number

YFTC 19190 YFTC 19191 YFTC 20067 YFTC 20068 YFTC 19192 YFTC 19193

Specimen code

EbckA EbckB EbckJ EbckK EbckP EbckQ

ND2

KT258080 KT258081 KT258082 KT258083 KT258084 KT258085

Plagl2

KT258133 KT258134 KM393469 KT258135 NA NA

Ptr

KT258173 KT258174 KM393545 KT258175 NA NA

Rag1

KT258212 KT258212 JF742867 JF742868 NA NA

S7

SH3PX3

KT258276 KT258277 JF742843 JF742844 NA NA

KT258251 KT258252 KM393695 KT258253 NA NA

Continued

144

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

APPENDIX continued. Species

Catalog number

Tissue number

Specimen code

ND2

Plagl2

Ptr

Rag1

S7

SH3PX3

YPM ICH 023754 YPM ICH 023754 YPM ICH 023754 YPM ICH 023841 YPM ICH 023841 YPM ICH 023841 YPM ICH 023841 YPM ICH 027088 YPM ICH 027088 YPM ICH 027088 YPM ICH 027088

YFTC 19188 YFTC 19189 YFTC 19187 YFTC 19183 YFTC 19184 YFTC 19185 YFTC 19186 YFTC 20106 YFTC 20107 YFTC 20108 YFTC 20109

EbckR EbckS EbckT EbckU EbckV EbckW EbckX EbckY EbckZ EbckAA EbckAB

KT258086 KT258087 KT258088 KT258089 KT258090 KT258091 KT258092 KT258093 KT258094 KT258095 KT258096

NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA

E. barbouri

INHS 27864 INHS 27864 None UT 91.7332 YPM ICH 019951

YFTC 1083 YFTC 1084 DNA extraction only YFTC 6398 YFTC 11746

EbarA EbarB EbarD EbarE EbarF

AF412542 AF412543 FJ012509 KT258053 KT258054

KC135241 KT258106 KT258107 KT258108 KT258109

KC134428 KT258145 KT258146 KT258147 KT258148

HQ127760 KT258185 KT258186 KT258187 KT258188

AF412559 AF412543 FJ012657 KT258257 KT258258

KC134698 KT258224 KT258225 KT258226 KT258227

E. basilare

UT 91.6589 UT 91.6704 UT 91.6592 UT 91.6624 YPM ICH 028243 UT 91.6939 UT 91.6944 YPM ICH 017620 UT 91.7022 YPM ICH 016575 UT 91.7880 UT 91.7880 UT 91.7889 YPM ICH 022186

YFTC 2407 YFTC 2426 YFTC 3156 YFTC 3138 YFTC 2605 YFTC 4122 YFTC 5341 YFTC 9479 YFTC 5404 YFTC 9577 UTFTC 1305 UTFTC 1306 UTFTC 1324 YFTC 15910

col_EbasAJ col_EbasAM cne_EbasBB cne_EbasBS rky_EbasAZ rky_EbasCK bfk_EbasDR bfk_EbasFS cfk_EbasEM cfk_EbasM mtn_EbasFA mtn_EbasFB hky_EbasFD hky_EbasFV

FJ012518 FJ012520 FJ012534 FJ012539 FJ012532 FJ012544 FJ012576 KT258047 FJ012595 KT258048 KT258049 KT258050 KT258051 KT258052

KT258097 KT258098 KT258099 KT258100 KT258101 KM393455 KM393459 KT258102 KM393460 KM393466 KT258103 KT258104 KM393461 KT258105

KT258136 KT258137 KT258138 KT258139 KT258140 KM393531 KM393535 KT258141 KM393536 KM393542 KT258142 KT258143 KM393537 KT258144

KT258176 KT258177 KT258178 KT258179 KT258180 KM393609 KM393613 KT258181 KM393614 KM393619 JF742865 KT258183 JF742866 KT258184

FJ012666 FJ012668 FJ012682 FJ012687 FJ012680 FJ012692 FJ012724 KT258254 FJ012743 KM393654 JF742841 KT258255 JF742842 KT258256

KT258215 KT258216 KT258217 KT258218 KT258219 KM393681 KM393685 KT258220 KM393686 KM393692 KT258221 KT258222 KM393687 KT258223

E. derivativum

YPM ICH 028244 INHS 88930 UT 91.7001 UT 91.7001 YPM ICH 28110

YFTC 1298 YFTC 1851 YFTC 5517 YFTC 5518 YFTC 3314

EderB EderH EderZ EderAA EderAF

KT258055 FJ012608 FJ012620 FJ012621 KT258056

KM393478 KM393479 KM393480 KT258110 KT258111

KM393555 KM393556 KM393557 KT258149 KT258150

HQ127812 KM393624 KM393625 KT258189 KT258190

KM393658 FJ012756 FJ012768 FJ012769 KT258259

KM393704 KM393705 KM393706 KT258228 KT258229

E. obeyense

INHS 41894 None YPM ICH 26996 YPM ICH 26998 UT 91.7523 UT 91.7523 YPM ICH 018510 YPM ICH 016192 YPM ICH 018220 YPM ICH 027038 YPM ICH 027038 YPM ICH 27001 YPM ICH 20091

YFTC 923 DNA extraction only YFTC 3146 YFTC 3193 YFTC 7106 YFTC 7107 YFTC 7214 YFTC 9450 YFTC 11758 YFTC 20060 YFTC 20061 YFTC 20090 YFTC 20091

EobyA EobyB EobyG EobyJ EobyY EobyZ EobyAB EobyAC EobyAD EobyAE EobyAF EobyAG EobyAH

AF412544 FJ012625 FJ012628 FJ012629 FJ012635 FJ012636 KT258062 KT258063 KT258064 KT258065 KT258066 KT258067 KT258068

KM393490 KT258112 KT258113 KT258114 KT258115 KT258116 KT258117 KT258118 KT258119 KT258120 KT258121 KT258122 KT258123

KM393569 KT258151 KT258152 KT258153 KT258154 KT258155 KT258156 KT258157 KT258158 KT258159 KT258160 KT258161 KT258162

HQ127888 HQ127889 KT258192 KT258193 KT258194 KT258195 KT258196 KT258197 KT258198 KT258199 KT258200 KT258201 KT258202

HQ127888 FJ012773 FJ012776 FJ012777 FJ012783 FJ012784 KT258260 KT258261 KT258262 KT258263 KT258264 KT258265 KT258266

KM393718 KT258230 KT258231 KT258232 KT258233 KT258234 KT258235 KT258236 KT258237 KT258238 KT258239 KT258240 KT258241

E. smithi

INHS 51266 UT 91.7100 UT 91.7290 YPM ICH 018215 YPM ICH 021927

YFTC 1297 YFTC 5696 YFTC 6266 YFTC 10993 YFTC 16099

EsmiA EsmiW EsmiAC EsmiBB EsmiBJ

AF412545 FJ012649 FJ012651 KT258069 KT258070

KC135285 KM393512 KM393511 KT258124 KT258125

KC134472 KM393592 KM393591 KT258163 KT258164

HQ127946 KM393637 KM393636 KT258203 KT258204

AF412562 FJ012797 FJ012799 KT258267 KT258268

KC134742 KM393741 KM393740 KT258242 KT258243

E. striatulum

YPM ICH 028245 YPM ICH 018280 YPM ICH 028246 YPM ICH 021083

YFTC 2269 YFTC 7271 YFTC 7549 YFTC 12342

EstrC EstrD EstrF EstrN

FJ012653 KT258071 KT258072 KT258073

KM393514 KM393515 KM393516 KT258126

KM393594 KT258165 KM393595 KT258166

KM393639 KM393640 KM393641 KT258205

FJ012801 KM393672 KM393673 KT258269

KM393743 KM393744 KM393745 KT258244

Continued

145

A New Barcheek Darter Species from Buck Creek • Near and Thomas APPENDIX continued. Tissue number

Specimen code

Species

Catalog number

E. virgatum

INHS 27832 INHS 37939 UT 91.7238 UT 91.7238 UT 91.7238 YPM ICH 028249 YPM ICH 028249 YPM ICH 027086 YPM ICH 027086

YFTC 778 YFTC 781 YFTC 6533 YFTC 6534 YFTC 6536 YFTC 20065 YFTC 20066 YFTC 20072 YFTC 20073

EvirE EvirF EvirG EvirH EvirI EvirL EvirM EvirN EvirO

AF412552 AF412553 FJ012654 KT258074 KT258075 KT258076 KT258077 KT258078 KT258079

KM393522 KM393523 KM393524 KT258127 KT258128 KT258129 KT258130 KT258131 KT258132

KM393601 KM393602 KM393603 KT258167 KT258168 KT258169 KT258170 KT258171 KT258172

HQ127977 KM393645 KM393646 KT258206 KT258207 KT258208 KT258209 KT258210 KT258211

AF412569 AF412570 FJ012802 KT258270 KT258271 KT258272 KT258273 KT258274 KT258275

KM393751 KM393752 KM393753 KT258245 KT258246 KT258247 KT258248 KT258249 KT258250

E. flabellare

YPM ICH 028247

YFTC 1438

EflaB

AF412540

NA

NA

NA

NA

NA

E. percnurum

YPM ICH 028248

YFTC 921

EpcnA

AF412539

NA

NA

NA

NA

NA

Literature Cited BENSON, D.A., M. CAVANAUGH, K. CLARK, I. KARSCH-MIZRACHI, D.J. LIPMAN, J. OSTELL AND E.W. SAYERS. 2013. Jan. GenBank. Nucleic Acids Research 41(Database Iss.):D36–D42. Published online 2012 Nov 27; doi:10.1093/nar/gks1195 BERENDZEN, P.B., W.M. OLSON AND S.M. BARRON. 2009. The utility of molecular hypotheses for uncovering morphological diversity in the Notropis rubellus species complex (Cypriniformes: Cyprinidae). Copeia 2009(4):661–673. BRAASCH, M.E. AND R.L. MAYDEN. 1985. Review of the subgenus Catonotus (Percidae) with descriptions of two new darters of the Etheostoma squamiceps species group. Occasional Papers of the Museum of Natural History, The University of Kansas 119:1–83. BURR, B.M. 1980. A distributional checklist of the fishes of Kentucky. Brimleyana 3:53–84. BURR, B.M. AND M.L. WARREN JR. 1986. A Distributional Atlas of Kentucky Fishes. Frankfort, KY: Kentucky Nature Preserves Commission. 398 pp. (Scientific and Technical Series No. 4.) CHOW, S. AND K. HAZAMA. 1998. Universal PCR primers for S7 ribosomal protein gene introns in fish. Molecular Ecology 7:1255–1256. CICERELLO, R.R. AND R.S. BUTLER. 1985. Fishes of Buck Creek, Cumberland River drainage, Kentucky. Brimleyana 11: 133–159. CLEMENT, M., D. POSADA AND K.A. CRANDALL. 2000. TCS: A computer program to estimate gene genealogies. Molecular Ecology 9(10):1657–1659. DRUMMOND, A.J., S.Y.W. HO, M.J. PHILLIPS AND A. RAMBAUT. 2006. Relaxed phylogenetics and dating with confidence. PLoS Biology 4(5):699–710. DRUMMOND, A.J., M.A. SUCHARD, D. XIE AND A. RAMBAUT. 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29(8):1969–1973. EGGE, J.J.D. AND A.M. SIMONS. 2006. The challenge of truly cryptic diversity: Diagnosis and description of a new madtom catfish (Ictaluridae: Noturus). Zoologica Scripta 35(6):581–595. ETNIER, D.A. AND W.C. STARNES. 1993. The Fishes of Tennessee. Knoxville: University of Tennessee Press. 681 pp. GEORGE, A.L., B.R. KUHAJDA, J.D. WILLIAMS, M.A. CANTRELL, P.L. RAKES AND J.R. SHUTE. 2009. Guidelines for propagation

ND2

Plagl2

Ptr

Rag1

S7

SH3PX3

and translocation for freshwater fish conservation. Fisheries 34(11):529–545. HARKER, D.F., JR., S.M. CALL, J.M.L. WARREN, K.E. CAMBURN AND P. WIGLEY. 1979. Aquatic Biota and Water Quality Survey of the Appalachian Province, Eastern Kentucky. Frankfort, KY: Kentucky Nature Preserves Commission. HARKER, D.F., JR., J.M.L. WARREN, K.E. CAMBURN, S.M. CALL, G.J. FALLO AND P. WIGLEY. 1980. Aquatic Biota and Water Quality Survey of the Upper Cumberland River Basin. Frankfort, KY: Kentucky Nature Preserves Commission. HARRINGTON, R.C. AND T.J. NEAR. 2012. Phylogenetic and coalescent strategies of species delimitation in snubnose darters (Percidae: Etheostoma). Systematic Biology 61(1):63–79. —2015. Phylogenetic relationships of Goneaperca and the evolution of parental care in darters (Teleostei: Percidae). Molecular Phylogenetics and Evolution 84:158–165. HELED, J. AND A.J. DRUMMOND. 2010. Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution 27(3):570–580. HOLLINGSWORTH, P.R., JR. AND T.J. NEAR. 2009. Temporal patterns of diversification and microendemism in Eastern Highland endemic barcheek darters (Percidae: Etheostomatinae). Evolution 63:228–243. HUBBS, C.L. AND K.F. LAGLER. 1958. Fishes of the Great Lakes Region. Ann Arbor: University of Michigan. 213 pp. JELKS, H.L., S.J. WALSH, N.M. BURKHEAD, S. CONTRERASBALDERAS, E. DIÁZ-PARDO, D.A. HENDRICKSON, J. LYONS, N.E. MANDRAK, F. MCCORMICK, J.S. NELSON, S.P. PLATANIA, B.A. PORTER, C.B. RENAUD, J.J. SCHMITTER-SOTO, E.B. TAYLOR AND M.L. WARREN JR. 2008. Conservation status of imperiled North American freshwater and diadromous fishes. Fisheries 33(8):372–407. JORDAN, D.S. 1880. Description of new species of North American fishes. Proceedings of the United States National Museum 2(84):235–241. KEARSE, M., R. MOIR, A. WILSON, S. STONES-HAVAS, M. CHEUNG, S. STURROCK, S. BUXTON, A. COOPER, S. MARKOWITZ, C. DURAN, T. THIERER, B. ASHTON, P. MEINTJES AND A. DRUMMOND. 2012. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28(12):1647–1649. [KENTUCKY CWCS] KENTUCKY’S COMPREHENSIVE WILDLIFE CONSERVATION STRATEGY. 2013. Kentucky Department of Fish and Wildlife Resources, #1 Sportsman’s Lane, Frankfort, Ken-

146

Bulletin of the Peabody Museum of Natural History 56(2) • October 2015

tucky 40601. [updated 2/5/2013]. http://fw.ky.gov/WAP/ Pages/Default.aspx KOCHER, T.D., J.A. CONROY, K.R. MCKAYE, J.R. STAUFFER AND S.F. LOCKWOOD. 1995. Evolution of NADH dehydrogenase subunit 2 in East African cichlid fish. Molecular Phylogenetics and Evolution 4(4):420–432. KUEHNE, R.A. AND R.W. BARBOUR. 1983. The American Darters. Lexington: University Press of Kentucky. 177 pp. KUEHNE, R.A. AND J.W. SMALL JR. 1971. Etheostoma barbouri, a new darter (Percidae, Etheostomatini) from the Green River with notes on the subgenus Catonotus. Copeia 1971(1):18–26. LANFEAR, R., B. CALCOTT, S.Y.W. HO AND S. GUINDON. 2012. PartitionFinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29(6):1695–1701. LAYZER, J.B. AND R.M. ANDERSON. 1991. Fish hosts of the endangered Cumberland bean pearly mussel (Villosa trabalis). North American Benthological Society Bulletin 8:110. LI, C., G. ORTÍ, G. ZHANG AND G. LU. 2007. A practical approach to phylogenomics: The phylogeny of ray-finned fish (Actinopterygii) as a case study. BMC Evolutionary Biology 7:44. MASTER, L.L. 1991. Assessing threats and setting priorities for conservation. Conservation Biology 5(4):559–563. MENDELSON, T.C. AND J.N. SIMONS. 2006. AFLPs resolve cytonuclear discordance and increase resolution among barcheek darters (Percidae: Etheostoma: Catonotus). Molecular Phylogenetics and Evolution 41(2):445–453. MOEYKENS, M.D. 1997. Bioassessment of the fish and macroinvertebrates of the upper section of Buck Creek, Lincoln and Pulaski Counties, Kentucky [master’s thesis]. Richmond: Eastern Kentucky University. 76 pp. NEAR, T.J., C.M. BOSSU, G.S. BRADBURD, R.L. CARLSON, R.C. HARRINGTON, P.R. HOLLINGSWORTH JR., B.P. KECK AND D.A. ETNIER. 2011. Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae). Systematic Biology 60(5):565–595. NEAR, T.J. AND B.P. KECK. 2013. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae). Molecular Phylogenetics and Evolution 66(3):868–876. NEAR, T.J. AND M.R. THOMAS. 2015. A New Barcheek Darter Species from Buck Creek (Cumberland River System), Kentucky (Percidae: Etheostomatinae: Catonotus: Oopareia). Dryad Digital Repository. http://dx.doi.org/10.5061/dryad. 13nj6 NYLANDER, J.A.A., J.C. WILGENBUSCH, D.L. WARREN AND D.L. SWOFFORD. 2008. AWTY (are we there yet?): A system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. Bioinformatics 24(4):581–583. PAGE, L.M. 1975. Relations among the darters of the subgenus Catonotus of Etheostoma. Copeia 1975(4):782–784. —1980. Etheostoma virgatum (Jordan) striped darter. In: D.S. Lee, C.R. Gilbert, C.H. Hocutt, R.E. Jenkins, D.E. McAllister and J.J.R. Stauffer, eds. Atlas of North American Freshwater Fishes. Raleigh, NC: North Carolina State Museum of Natural History. p. 707. —1981. The genera and subgenera of darters (Percidae, Etheostomatini). Occasional Papers of the Museum of Natural History, The University of Kansas 90:1–69.

—1983. Handbook of Darters. Neptune City, NJ: T.F.H. Publications. 271 pp. PAGE, L.M. AND M.E. BRAASCH. 1976. Systematic studies of darters of the subgenus Catonotus (Percidae), with the description of a new species from the lower Cumberland and Tennessee River systems. Occasional Papers of the Museum of Natural History, The University of Kansas 60:1–18. —1977. Systematic studies of darters of the subgenus Catonotus (Percidae), with the description of a new species from the Duck River system. Occasional Papers of the Museum of Natural History, The University of Kansas 63:1–18. PAGE, L.M., M. HARDMAN AND T.J. NEAR. 2003. Phylogenetic relationships of barcheek darters (Percidae: Etheostoma, Subgenus Catonotus) with descriptions of two new species. Copeia 2003(3):512–530. PAGE, L.M. AND D.W. SCHEMSKE. 1978. The effect of interspecific competition on the distribution and size of darters of the subgenus Catonotus (Percidae: Etheostoma). Copeia 1978(3):406–412. PORTERFIELD, J.C., L.M. PAGE AND T.J. NEAR. 1999. Phylogenetic relationships among fantail darters (Percidae: Etheostoma: Catonotus): Total evidence analysis of morphological and molecular data. Copeia 1999(3):551–564. QUARTERMAN, E. AND R.L. POWELL. 1978. Potential Ecological/Geological Natural Landmarks on the Interior Low Plateaus. Washington, DC: US Department of the Interior. RONQUIST, F., M. TESLENKO, P. VAN DER MARK, D.L. AYRES, A. DARLING, S. HOHNA, B. LARGET, L. LIU, M.A. SUCHARD AND J.P. HUELSENBECK. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3):539–542. SABAJ PÉREZ, M.H., ED. 2014. Standard Symbolic Codes for Institutional Resource Collections in Herpetology and Ichthyology: An Online Reference. Version 5.0 (22 September 2014). Washington, DC: American Society of Ichthyologists and Herpetologists. Available from: http://www.asih. org/sites/default/files/documents/resources/symbolic_codes _for_collections_v5.0_sabajperez_2014.pdf SCHUSTER, G.A., R.S. BUTLER AND D.H. STANSBERY. 1989. A survey of the unionids (Bivalvia: Unionidae) of Buck Creek, Pulaski County, Kentucky. Transactions of the Kentucky Academy of Science 50(1–2):79–85. THOMAS, M.R. AND S.L. BRANDT. 2012. Ichthyofaunal Assessment of Buck Creek, Upper Cumberland River Drainage, Kentucky. Section I of the Interim Annual Performance Report for State and Tribal Wildlife Grant: T-60, Fish Surveys in Kentucky. Frankfort, KY: Kentucky Department of Fish and Wildlife Resources. US FISH AND WILDLIFE SERVICE. 2010. Cumberland Bean (Villosa trabilis) 5-Year Review: Summary and Evaluation. Frankfort, KY: US Fish and Wildlife Service, Kentucky Ecological Services Field Office. —2013. Endangered and threatened wildlife and plants: Designation of critical habitat for the fluted kidneyshell and slabside pearlymussel. Federal Register 78(187):59556– 59620. WOODS, A.J., J.M. OMERNIK, W.H. MARTIN, G.J. POND, W.M. ANDREWS, S.M. CALL, J. A. COMSTOCK AND D.D. TAYLOR. 2002. Ecoregions of Kentucky (color poster with map, descriptive text, summary tables, and photographs). Reston, VA: US Geological Survey.