Mitochondrial DNA Variation in the Ozark Highland Members of the ...

2 downloads 124 Views 197KB Size Report
sequence data is concordant with morphological divergence in the five Ozark Highlands members of the complex of banded sculpin Cottus carolinae: eyelash ...
Transactions of the American Fisheries Society 136:1742–1749, 2007 Ó Copyright by the American Fisheries Society 2007 DOI: 10.1577/T06-025.1

[Article]

Mitochondrial DNA Variation in the Ozark Highland Members of the Banded Sculpin Cottus carolinae Complex ANDREW P. KINZIGER,* DAMON H. GOODMAN,1

AND

REBECCA S. STUDEBAKER2

Department of Fisheries Biology, Humboldt State University, 1 Harpst Street, Arcata, California 95521, USA Abstract.—The goal of this study was to determine whether variation in mitochondrial DNA (mtDNA) sequence data is concordant with morphological divergence in the five Ozark Highlands members of the complex of banded sculpin Cottus carolinae: eyelash sculpin, fringehead sculpin, Black River race, grotto sculpin, and midlands race. We generated a phylogenetic hypothesis for the banded sculpin complex based on a data set composed of 841 base pairs of mtDNA (ATPase-8 and ATPase-6) for 58 individuals representing all banded sculpin complex members that occur in the Ozark Highlands and comparative material collected outside of the highlands. Divergence in mtDNA was found to be generally concordant with morphological distinctiveness in the fringehead sculpin, eyelash sculpin, Black River race, and grotto sculpin; however, genetic divergence of these taxa was generally low (sequence divergence maximum ¼ 0.2–1.4%) relative to the marked morphological distinctness exhibited by these taxa. In contrast, the midlands race was recovered as nonmonophyletic and contained a relatively large amount of within-race sequence divergence (uncorrected p-distance ¼ 0–5%), illustrating that this race is poorly understood.

The genus Cottus contains approximately 66 described species (Eschmeyer 1998); however, many species in the genus are poorly understood, and the actual number of species is probably much higher. One of the most poorly understood and taxonomically complex members of the genus is the banded sculpin C. carolinae. The banded sculpin encompasses a complex of 14 taxa that have been confusingly assigned to different taxonomic ranks, including six forms, five races, two subspecies, and one species (Table 1). The forms and races lack standard scientific names. The different taxonomic ranks assigned to them are probably a result of differing opinions in the delineation of taxa. Each taxon is morphologically distinct (Robins 1954; Kinziger 2003) and will be considered as a separate taxonomic unit herein. The goal of this study was to determine whether variation in mitochondrial DNA (mtDNA) sequence data is concordant with morphological variation in the five Ozark Highlands members of the banded sculpin complex. Four members of the complex are endemic to the Ozark Highlands: eyelash sculpin, fringehead sculpin, Black River race, and grotto sculpin. One taxon, the midlands race, occurs in the Ozark Highlands, Shawnee Hills, and Appalachian Highlands (Table 1; Figure 1). Morphological divergence in each * Corresponding author: [email protected] 1 Present address: U.S. Fish and Wildlife Service, 1655 Heindon Road, Arcata, California 95521, USA. 2 Present address: California Department of Fish and Game, 619 2nd Street, Eureka, California 95501, USA. Received February 1, 2006; accepted March 8, 2007 Published online December 13, 2007

taxon is remarkable, as most are completely distinguishable from all other members of the banded sculpin complex. The eyelash and fringehead sculpins are distinguished from other members of the banded sculpin complex by possessing cirri, the eyelash sculpin is discernible by few cirri positioned above the eyes, and the fringehead sculpin is distinguished by its many cirri positioned on the head and nape. The presence of cirri in these taxa is a unique characteristic, as cirri have never been reported in any other Cottus species (Kinziger 2003). The Black River race is identifiable by its 18 pectoral fin rays (Kinziger 2003). The high pectoral fin ray count in the Black River race is distinctive among Cottus; only four other taxa are known to have pectoral fin ray counts that are this high (fourspine sculpin C. kazika, prickly sculpin C. asper, and eyelash and fringehead sculpins; Watanabe 1960; Berg 1965; Page and Burr 1991; Kinziger 2003). The grotto sculpin possesses characters associated with cave habitation (Burr et al. 2001). Although members of the banded sculpin complex often occur in caves, the grotto sculpin and the albino cave sculpin are the only members of the complex known to exhibit cave habitation characteristics (Williams and Howell 1979; Burr et al. 2001). Unlike other Ozark members of the banded sculpin complex, the grotto sculpin does not represent a single lineage but instead is composed of three different phenotypes (Burr et al. 2001). The midlands race is diagnosed by a lack of characteristics defining the other members of the complex: it has no cirri, has fewer than 18 pectoral fin rays, and exhibits no cave habitation characteristics (Robins 1954; Burr et al. 2001; Kinziger 2003). This taxon is probably the least

1742

1743

GENETIC VARIATION IN THE BANDED SCULPIN COMPLEX

TABLE 1.—Fourteen members of the banded sculpin Cottus carolinae complex and their geographic distribution. Taxon

Geographic distribution

Eyelash sculpin Fringehead sculpin Black River race Grotto sculpin Midlands race Alexander County race Sequatchie River race Pigeon River race Dusky banded sculpin C. carolinae zopherus Kanawha sculpin Alabama banded sculpin C. carolinae infernatus Fall-line sculpins Albino cave sculpin Bluestone Sculpin

Reference

Ozark Highlands Ozark Highlands Ozark Highlands Ozark Highlands Ozark Highlands, Shawnee Hills, and Appalachian Highlands Shawnee Hills Appalachian Highlands Appalachian Highlands Mobile Basin

Robins Robins Robins Robins

Appalachian Highlands Mobile Basin

Robins 1954, 2005 Williams and Robins 1970

Mobile Basin

Williams and Robins 1970, Robins 1954, Warren et al. 2000 Williams and Howell 1979 Jenkins and Burkhead 1994

Appalachian Highlands Appalachian Highlands

understood member of the complex; for this reason, it probably contains unrecognized taxonomic diversity. Methods We generated complete mtDNA sequence data for two genes, ATPase-8 and ATPase-6, in 58 individuals. Our data matrix included representatives of all banded sculpin complex members that occur in the Ozark Highlands, including the eyelash, fringehead, and grotto sculpins and the Black river and midlands races (Figure 2; Table 2). Each taxon was sampled from two or more localities within its known range. The data matrix also contained representatives of the banded sculpin complex from outside the Ozark Highlands, including samples of the midlands race, Sequatchie River race, and Kanawha sculpin C. kanawhae. Taxa hypothesized to be closely related to the banded sculpin complex were also included: four undescribed taxa belonging to the genus Cottus (Bluestone, Clinch, Holston, and smoky sculpins), pygmy sculpin C. paulus, and black sculpin C. baileyi (Kinziger et al. 2005). The phylogenetic analysis was rooted with mottled sculpin C. bairdii (Kinziger et al. 2005). Specimens were preserved in 95% ethanol or frozen at 858C. The DNA was extracted from muscle or fin tissue using the QIAGEN QIAamp DNA minikit according to the manufacturer’s instructions or using chelex methods (Miller and Kapuscinski 1993). The ATPase-8 and ATPase-6 genes were amplified in a single segment using the polymerase chain reaction (PCR), primers L8933 and H9795 (Kontula et al. 2003), and cycling conditions of 35 cycles of 918C for 1 min, 588C for 1 min, and 608C for 2 min. The PCR products were purified using the QIAGEN QIAquick gel extraction kit following manufacturer instructions. The DNA sequence data were generated using dye terminator cycle sequencing chemistry (Beckman Coulter DTCS Quick Start Kit)

Kinziger 2003 Kinziger 2003 Robins 1954 Burr et al. 2001 Robins 1954 1954 1954 1954 1954

at 30 cycles of 968C for 20 s, 508C for 20 s, and 608C for 4 min. The ATPase-8 and ATPase-6 genes were sequenced using two nested primers. The reverse primer was NLATPR1 (5 0 -GTGGGCTGGTTTCGTATYCCAATA-3 0 ) or NLATPR2 (5 0 -TGCAAGGCC[T or C]AGGTTBAGGGAAAG-3 0 ), and the forward primer was NLATP1 (5 0 -AGCARCCCTCCTAACCTCTCTT3 0 ). Sequencing products were purified according to Beckman Coulter instructions and run on a Beckman Coulter CEQ 8000 automated DNA sequencer. Sequences are deposited in GenBank EU181372– EU181420, AY833311, AY833319–AY833322, AY833277, AY833278, AY833290, and AY833291. Sequence alignment was performed using ClustalX (Thompson et al. 1997) and was unambiguous, containing no insertions or deletions. Phylogenetic hypotheses were generated using both parsimony and likelihood methods using PAUP* (Swofford 2000). The maximum parsimony analyses were conducted using a heuristic search with 10,000 replications of random addition sequences. Branch support for parsimony analysis was assessed using bootstrap analysis with 10,000 pseudoreplicates of a simple heuristic search. For the likelihood analysis, the best-fit model of sequence evolution (Tamura–Nei model [TrN] þ gamma distribution parameter [C]; Tamura and Nei 1993) was selected using ModelTest version 3.06 (Posada and Crandall 1998). Maximum likelihood searches were then conducted with the preferred model in PAUP*. Maximum likelihood analysis resulted in trees that were nearly identical to those from parsimony analysis; thus, we discuss the results from the parsimony analysis only. Genetic divergence among taxa was assessed by calculating percent sequence divergence (uncorrected p-distance, or uncorrected number of changes between two sequences) among pairs of samples with PAUP*.

1744

KINZIGER ET AL.

FIGURE 1.—Geographic distribution of five Ozark Highlands members of the banded sculpin complex. Only the midlands race, which also occurs in the Shawnee Hills and Appalachian Highlands, is not endemic to the Ozark Highlands. All members have allopatric distributions. Distributional data are from Kinziger (2003) and Burr et al. (2001). Inset shows the approximate geographic distribution of the entire complex (Lee et al. 1980).

Results and Discussion The data matrix contained 150 variable characters, 86 of which were phylogenetically informative. Including only members of the banded sculpin complex, the data set contained 83 variable characters, 63 of which were phylogenetically informative. Mean nucleotide frequencies were 0.249 for A, 0.268 for T, 0.359 for C, and 0.124 for G, and there was no significant base compositional bias among taxa (v2180 ¼ 5.81; P ¼ 1.00). Uncorrected p-distances for the entire data matrix ranged from 0.000 to 0.089. The banded sculpin complex was resolved as nonmonophyletic in the ATPase gene tree due to the placement of Holston sculpin (Table 1; Figure 3). Jenkins and Burkhead (1994) indicated that the Holston sculpin was morphologically similar to both the banded sculpin complex and the mottled sculpin complex; those authors were unsure to which group the Holston sculpin should be assigned. Because the

FIGURE 2.—Map of the Ozark Highlands, indicating sites at which five members of the banded sculpin complex were collected. Other samples included in the analysis are described in Table 2. For delineation of Ozark Highlands, Appalachian Highlands, Shawnee Hills, and Mobile Basin regions, see Mayden (1988).

mtDNA tree nests Holston sculpin deeply among other members of the banded sculpin complex, we assign it to this group. Addition of Holston sculpin to the banded sculpin complex brings the total number of taxa in the complex to 15. Although the change makes the complex monophyletic, support for this clade is weakly supported (bootstrap [BS] ¼ 58). Four clades were resolved within the banded sculpin complex. Two clades were composed of taxa from the Ozarks, and two clades included taxa from the Appalachians: (1) Ozark A was composed of the Ozarks midlands race and the fringehead and eyelash sculpins (BS ¼ 92); (2) Ozark B was composed of the Black River race (BS ¼ 60); (3) Appalachian A was composed of Shawnee Hills and Appalachian Highlands midlands race and the grotto sculpin, Sequatchie River race, and Holston sculpin (BS ¼ 87); and (4) Appalachian B included the Appalachians midlands race, Bluestone sculpin, and Kanawha sculpin (Figure 3). The Appalachian B clade was supported as basal;

GENETIC VARIATION IN THE BANDED SCULPIN COMPLEX

1745

may be in the initial stages of divergence. Despite the low levels of genetic differentiation observed, the genetic and morphological data are consistent; therefore, recognition of the fringehead and eyelash sculpins as distinct forms within the banded sculpin complex should continue (Kinziger 2003). Black River Race

however, relationships among the remaining three clades were unresolved.

The ATPase gene tree resolved the Black River race as a weakly supported monophyletic group (BS ¼ 61) differing from all other taxa included in the analysis by at least 1.4% (Figure 3). Like the fringehead and eyelash sculpins, the Black River race exhibits relatively low levels of genetic differentiation from all other members of the banded sculpin complex. Nevertheless, these genetic differences are concordant with the morphological findings supporting continued recognition of this taxon (Kinziger 2003). Nested within the Black River race are two wellsupported clades, one including individuals from the Black River drainage (BS ¼ 96) and the other containing individuals from the Current, Eleven Point, Spring, and Strawberry River drainages (BS ¼ 98; Figure 3). These two lineages are genetically distinct, differing by 1% sequence divergence. These data suggest low gene flow between these geographically proximate lineages. The barrier to gene flow is probably the low-gradient, sand–silt-bottom habitats present in the downstream portions of the Black River drainage (Pflieger 1997). To date, no morphological characters have been identified that separate these two clades; however, the divergence observed in mtDNA between these clades suggests that additional investigation is warranted.

Fringehead and Eyelash Sculpins

Grotto Sculpin

The mtDNA gene tree resolved the fringehead and eyelash sculpins as a monophyletic group but with fairly low support (BS ¼ 60; Figure 3). Divergence between the clade containing the fringehead and eyelash sculpins and all other taxa was at least 0.2%. The close relationship and monophyly of the fringehead and eyelash sculpins as suggested by the mtDNA tree are consistent with morphology, as these two taxa are the only Cottus members known to have cirri (Kinziger 2003). The eyelash and fringehead sculpins differ by two fixed nucleotide substitutions. The fringehead sculpin was recovered as monophyletic (BS ¼ 87), and the eyelash sculpin was resolved as nonmonophyletic. Although the fringehead and eyelash sculpins are genetically different from other members of the banded sculpin complex and from each other, the differences are slight, which indicates that these taxa

The grotto sculpin was resolved as sister to the midlands race from the Shawnee Hills and differs by three nucleotide substitutions from the Shawnee Hills sample (Figure 3). Like the other members of the banded sculpin complex evaluated herein, the grotto sculpin exhibits relatively low levels of genetic divergence. Nevertheless, due to the concordant differences in mtDNA and morphological data, we suggest continued recognition of the grotto sculpin (Burr et al. 2001). Additional studies including all three phenotypes of grotto sculpin discussed by Burr et al. (2001) are needed for a full evaluation of concordance between genetic and morphological divergence in the grotto sculpin.

FIGURE 3.—Parsimony phylogram of 1 of 15 equally parsimonious trees for 58 ATPase-8 and ATPase-6 sequences examined in members of the banded sculpin complex and related taxa. Terminal labels correspond to sample numbers in Figure 2 and Table 2. Bootstrap values are shown above branches.

Midlands Race The midlands race was resolved as nonmonophyletic, as samples were resolved within the Ozark A,

1746

KINZIGER ET AL.

TABLE 2.—Sample number, taxon, locality, museum voucher number, and number of individuals examined in an analysis of mitochondrial DNA variation in members of the banded sculpin complex and related taxa. Abbreviations follow Leviton et al. (1985) and Leviton and Gibbs (1988). Sample number 1

Locality

16

Ha Ha Tonka Spring at Ha Ha Tonka State Park, 2 mi south of Camdenton Eyelash sculpin Wet Auglaize Creek along County Road A, about 8 mi south of Brumley Eyelash sculpin Osage River at Tuscumbia Eyelash sculpin Big Piney Creek at County Road. BB, about 8 mi southwest of Licking Eyelash sculpin Roubidoux Road at Business 44, Waynesville Fringehead sculpin Meramec River at Scotts Ford; access at Thurman Lake Road about 4 mi west of Steelville Fringehead sculpin Huzzah Creek at Highway E, Scotia Black River race Black River at Lesterville; access off Peola Road Black River race Markham Spring at Markham Spring Recreation Area, north of Browns Crossing Black River race Current River at County Road KK, Akers Ferry Black River race Current River at Highway 160, west of Doniphan Black River race Eleven Point River at Highway 160, Riverton Black River race Eleven Point River at Highway 93, Dalton Black River race Tributary to Warm Fork of Spring River, 1.5 mi north of Thayer Black River race Mill Creek 0.1 mile east-southeast of Evening Shade at Highway 56 Grotto sculpin Tom Moore Cave

17

Midlands race

18

Midlands race

19

Midlands race

20 21

Midlands race Midlands race

22

Midlands race

23

Midlands race

24

Midlands race

25

Midlands race

26

Midlands race

27

Midlands race

28 29 30 31

Midlands Midlands Midlands Midlands

32

Sequatchie River race Smoky sculpin Elk River at Elk Mills, off of US Highway 321 Bluestone sculpina Wrights Valley Creek along Route 650, northeast of Bailey Clinch River at U.S. Highway 460 in Richlands Clinch sculpina a Holston sculpin Middle Fork Holston River at U.S. Highway 11 on eastern edge of Marion Kanawha sculpin East River at County Road 30–1 in Willowton Pygmy sculpin Coldwater Spring at Coldwater Spring Black sculpin Liberty Creek along Route 608, 3.5 airmi southeast of Pounding Mill Mottled sculpin Poletown Brook at East Lee Highway, east of Fort Chiswell

2 3 4 5 6 7 8 9 10 11 12 13 14 15

33 34 35 36 37 38 39 40 a

Taxon Eyelash sculpin

race race race race

Whitewater River at Highway 51, 1 mi north of Alliance Dry Creek, 8 mi north of Batesville at Cold Creek Lane North Fork White River at County Road CC, about 10 mi west of West Plains Beaver Creek at Highway 76, Bradleyville Calton Creek at Highway VV, 1.5 mi south-southwest of Pleasant Ridge Little Flat Creek at Highway C, 0.5 mi southeast of McDowell War Eagle Creek, 10 mi north U.S. Highway 412 at County Road 45 Spring River at County Road V, about 3 mi west-northwest of Mt. Vernon Whitewater Creek at unnamed road, 4 mi south-southeast of Grove Goose Creek, 4.5 mi north of Rosiclare at County Road 600N Trammel Fork at old State Road, 1 mi northwest of Red Hill West Fork Stones R at Sulpher Spring Road Hinson Creek at Highway 79, 3 mi west of Dover Trace Creek at Highway 13, Waverly Liberty Creek along Route 608, 3.5 airmi southeast of Pounding Mill Sequatchie River, 1 mi east of State Route 28

Undescribed members of Cottus.

Drainage

County

State

Osage

Camden

Missouri

Osage

Camden

Missouri

Osage Gasconade River

Miller Texas

Missouri Missouri

Gasconade River Meramec River

Pulaski Crawford

Missouri Missouri

Meramec River Black River Black River

Crawford Reynolds Wayne

Missouri Missouri Missouri

Current River Current River Eleven Point River Eleven Point River Spring River

Shannon Ripley Oregon Randolf Oregon

Missouri Missouri Missouri Arkansas Missouri

Strawberry River

Sharp

Arkansas

Direct to Mississippi Perry Missouri River Castor River to Mississippi Bollinger Missouri River White River Independence Arkansas White River

Ozark

Missouri

White River White River

Taney Barry

Missouri Missouri

White River

Barry

Missouri

White River

Madison

Arkansas

Arkansas River

Lawrence

Missouri

Arkansas River

Delaware

Oklahoma

Big Creek to Ohio River

Hardin

Illinois

Green River

Allen

Kentucky

Cumberland River Cumberland River Tennessee River Tennessee River

Rutherford Stewart Humphreys Tazewell

Tennessee Tennessee Tennessee Virginia

Tennessee River

Marion

Tennessee

Tennessee River New River

Carter Tazewell

Tennessee Virginia

Clinch River Holston River

Tazewell Smyth

Virginia Virginia

New River Alabama River Tennessee River

Mercer Calhoun Tazewell

West Virginia Alabama Virginia

New River

Wythe

Virginia

GENETIC VARIATION IN THE BANDED SCULPIN COMPLEX

TABLE 2.—Extended.

Sample number

Latitude

Longitude

1

37.9739

92.7676

HSU 3633

1

2

37.9966

92.5209

HSU 3472

2

3 4

38.2326 37.4595

92.4587 91.9890

HSU 3634 HSU 3444

2 2

5 6

37.8282 37.98 0 73

92.2056 91.4562

HSU 3644 HSU 3471

2 2

7 8 9

38.0290

91.2137

36.9778

90.6019

HSU 3643 HSU 3632 HSU 3628

2 2 2

10 11 12 13 14

37.3756 36.6160 36.6488 36.4210 36.5316

91.5524 90.8397 91.2011 91.1394 91.5435

HSU HSU HSU HSU HSU

3641 3645 3638 3637 3446

2 2 1 2 1

15

36.0657

91.6104

HSU 3642

1

SIUC 20916

1

16

Collection number

Number sequenced

17

37.5791

90.0016

HSU 3626

2

18

35.9014

91.5964

HSU 3627

1

19

36.7582

92.1546

HSU 3630

2

20 21

36.7798 36.8805

92.9077 93.7565

HSU 3646 HSU 3449

1 1

22

36.8221

93.7946

HSU 3447

2

23

36.2055

93.8803

HSU 3640

1

24

37.1157

93.8942

HSU 3629

2

25

36.5144

94.7638

HSU 3445

2

26

37.49679

88.33173

HSU 3625

1

27

36.7400

86.2900

HSU 3635

1

28 29 30 31

35.9401 36.4813 36.0860 37.0491

86.4650 87.8820 87.7932 81.6566

KU 32225 HSU 3459 HSU 3455 HSU 3463

1 1 1 2

32

35.12325

85.29766

HSU 3636

2

33 34

36.2767 37.2260

81.9902 81.3852

KU 32240 KU 32239

1 1

35 36

37.0870 36.8508

81.7687 81.4857

UAIC 12726.01 UAIC 11013.06

1 1

37 38 39

37.3483 33.6036 37.0491

80.8864 85.9261 81.6566

UAIC 12505.01 UAIC 11253.01 KU 32218

1 1 1

40

36.9493

80.9118

KU 32219

1

1747

Appalachians A, and Appalachians B clades (Figure 3). The range of sequence divergence within the midlands race ranged from 0% to 5%. The nonmonophyletic structure and relatively large among-individual sequence divergence within the midlands race illustrates that this taxon is poorly understood and contains undiscovered diversity. Additional studies are needed to evaluate the relationship between genetic lineages described herein and their morphological characteristics. Spring River samples (Table 2, sample 14) were not examined in the morphological study by Kinziger (2003) and thus were not assigned to a taxonomic group at that time. Based on the close relationship between the Spring and Black River samples in our genetic analysis, we assign this sample to the Black River race (Table 2; Figure 3). Kinziger (2003) assigned the Strawberry River collections (Table 2, sample 15) to the midlands race due to this sample’s low pectoral fin ray count, which distinguished it from the Black River race. However, the genetic data suggest a close relationship between the Strawberry River and Black River samples, indicating that this sample may belong to the Black River race. Conflict between the morphological and molecular data and the geographic intermediacy of this sample between the midlands and Black River races suggest that the Strawberry River may represent a zone of contact between these two taxa. Populations of the midlands race in the White River (samples 18–23) and Arkansas River (samples 24 and 25) drainages are geographically disjunct (Figure 2); however, these populations are closely related to one another (Figure 3). The close relationship is probably best explained by stream capture, wherein the upper Arkansas River drainage captured portions of the White River (Bretz 1965). These hypothesized captures would have resulted in transfer of biota from the White River to the Arkansas River drainage. The stream capture hypothesis is further supported by the restriction of the midlands race to upper portions of the Arkansas River drainage and absence of this race from lower portions of the drainage (Robison and Buchanan 1988). Timing of Ozark Cottus Divergence The level of mtDNA sequence divergence resolved among members of the Ozark Highlands members of the banded sculpin complex ranged from 0.2% to 1.4%. In contrast, a similar study of taxa assigned to the mottled sculpin complex from many of the same drainages in the Ozark Highlands showed much higher levels of mtDNA sequence divergence ranging from 3.5% to 6.55% between taxa and from 0.5% to 2.84%

1748

KINZIGER ET AL.

among drainages within taxa (Kinziger and Wood 2003). Assuming a crude molecular clock, these data suggest that members of the banded sculpin complex have diverged much more recently than members of the Ozark Highlands mottled sculpin complex. It is postulated further that the difference in the timing of divergence in these two lineages is related to temperature tolerances and habitat preferences; the banded sculpin complex generally has a tolerance for warmer temperatures in more downstream areas, whereas members of the mottled sculpin complex prefer colder water in more upstream areas (see review in Jenkins and Burkhead 1994). Thus, as temperatures gradually increased after the Pleistocene glaciation, members of the mottled sculpin complex became geographically isolated more quickly, resulting in their higher levels of divergence relative to the more recently isolated banded sculpin complex. Bluestone River Stream Capture Bluestone sculpin from the Bluestone River (New River drainage, Virginia; sample 34) occur in a different drainage than the midlands race in Liberty Creek (Clinch River, Virginia; sample 31); however, these two populations were resolved as sister taxa (Figure 3). The close relationship is probably a result of stream capture. Ross (1972) hypothesized that the Bluestone River once drained into the Clinch River and was later captured by the New River drainage (see also Jenkins and Burkhead 1994). Acknowledgments For help with tissue collection, we thank J. Switzer, J. Ray, and N. Lang. We thank D. Neely, B. Burr, J. Adams, and D. Keeney for donating tissue. R. Nakamoto, B. Crouch, C. Justice, and M. Thomas helped with DNA sequence data collection. Collecting permits were obtained from Missouri Department of Conservation, Missouri Department of Natural Resources, Arkansas State Game and Fish Commission, and the National Park Service. References Berg, L. S. 1965. Freshwater fishes of the USSR and adjacent countries, volume 3. Israel Program for Scientific Translations, Jerusalem. Bretz, J. H. 1965. Geomorphic history of the Ozarks of Missouri. Missouri Geological Survey and Water Resources, Rolla. Burr, B. M., G. L. Adams, J. K. Krejca, R. J. Paul, and M. L. Warren, Jr. 2001. Troglomorphic sculpins of the Cottus carolinae species group in Perry County, Missouri: distribution, external morphology, and conservation status. Environmental Biology of Fishes 62:279–276.

Eschmeyer, W. N. 1998. Catalog of fishes. California Academy of Sciences, San Francisco. Jenkins, R. E., and N. M. Burkhead. 1994. Freshwater fishes of Virginia. American Fisheries Society, Bethesda, Maryland. Kinziger, A. P. 2003. Evidence supporting two new forms and one previously described race within the Cottus carolinae species complex from the Ozark Highlands. American Midland Naturalist 149:418–424. Kinziger, A. P., and R. M. Wood. 2003. Molecular systematics of the polytypic species Cottus hypselurus (Teleostei: Cottidae). Copeia 2003:624–627. Kinziger, A. P., R. M. Wood, and D. A. Neely. 2005. Molecular systematics of the genus Cottus (Scorpaeniformes: Cottidae). Copeia 2005:303–311. Kontula, T., S. V. Kirilchik, and R. Vainola. 2003. Endemic diversification of the monophyletic cottoid fish species flock in Lake Baikal explored with mtDNA sequencing. Molecular Phylogenetics and Evolution 27:143–155. Lee, D. S., C. R. Gilbert, C. H. Hocutt, R. E. Jenkins, D. E. McAllister, and J. R. Stauffer, Jr. 1980. Atlas of North American freshwater fishes. North Carolina Museum of Natural History, Raleigh. Leviton, A. E., and R. H. Gibbs, Jr. 1988. Standards in herpetology and ichthyology: standard symbolic codes of institution resource collections in herpetology and ichthyology, supplement 1. Additions and corrections. Copeia 1988:280–282. Leviton, A. E., R. H. Gibbs, Jr., E. Heal, and C. E. Dawson. 1985. Standards in herpetology and ichthyology, part I: standard symbolic codes for institutional resource collections in herpetology and ichthyology. Copeia 1985:802–832. Mayden, R. L. 1988. Vicariance biogeography, parsimony, and evolution in North American freshwater fishes. Systematic Zoology 37:331–357. Miller, L. M., and A. R. Kapuscinski. 1993. Genetic variation in northern pike. Transactions of the American Fisheries Society 125:971–977. Page, L. M., and B. M. Burr. 1991. A field guide to freshwater fishes. Houghton Mifflin, New York. Pflieger, W. L. 1997. The fishes of Missouri. Missouri Department of Conservation, Jefferson City. Posada, D., and K. A. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics (Oxford) 14:817–818. Robins, C. R. 1954. A taxonomic revision of the Cottus bairdi and Cottus carolinae species groups in eastern North America (Pisces, Cottidae). Doctoral dissertation. Cornell University, Ithaca, New York. Robins, C. R. 2005. Cottus kanawhae, a new cottid fish from the New River system of Virginia and West Virginia. Zootaxa 987:1–6. Robison, H. W., and T. M Buchanan. 1988. Fishes of Arkansas. University of Arkansas Press, Fayetteville. Ross, R. D. 1972. The drainage history of the Tennessee River. Virginia Polytechnic Institute and State University Research Division Monograph 4:11–42. Swofford, D. L. 2000. PAUP*: phylogenetic analysis using parsimony (* and other methods), version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Tamura, K., and M. Nei. 1993. Estimation of the number of

GENETIC VARIATION IN THE BANDED SCULPIN COMPLEX

nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512–526. Thompson, J. D., T. J. Gibton, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25:4876–4882. Warren, M. L., Jr., B. M. Burr, S. J. Walsh, H. L. Bart, Jr., R. C. Cashner, D. A. Etnier, B. J. Freeman, B. R. Kuhajda, R. L. Mayden, H. W. Robison, S. T. Ross, and W. C. Starnes. 2000. Diversity, distribution, and

1749

conservation status of the native freshwater fishes of the southern United States. Fisheries 25(10):7–31. Watanabe, M. 1960. Fauna Japonica Cottidae (Pisces). Biogeographical Society of Japan, Tokyo. Williams, J. D., and W. M. Howell. 1979. An albino sculpin from a cave in the New River drainage of West Virginia (Pisces: Cottidae). Brimleyana 1:141–146. Williams, J. D., and C. R. Robins. 1970. Variation in populations of the fish Cottus carolinae in the Alabama River system with a description of a new subspecies from below the Fall Line. American Midland Naturalist 83:368–381.