Molecular systematics and historical biogeography of the Nocomis biguttatus species group (Teleostei: Cyprinidae): Nuclear and mitochondrial introgression and a cryptic Ozark species

Molecular Phylogenetics and Evolution 81 (2014) 109–119

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Molecular systematics and historical biogeography of the Nocomis biguttatus species group (Teleostei: Cyprinidae): Nuclear and mitochondrial introgression and a cryptic Ozark species Anthony A. Echelle a,⇑, Michael R. Schwemm a,1, Nicholas J. Lang a, Brett C. Nagle b,c, Andrew M. Simons b, Peter J. Unmack d, William L. Fisher e,2, Christopher W. Hoagstrom f a

Zoology Department, Oklahoma State University, United States Department of Fisheries, Wildlife, and Conservation Biology, Bell Museum of Natural History, University of Minnesota, United States c Minnesota Pollution Control Agency, United States d Institute for Applied Ecology and Collaborative Research Network for Murray-Darling Basin Futures, University of Canberra, Australia e Oklahoma Cooperative Fish and Wildlife Research Unit, Oklahoma State University, United States f Department of Zoology, Weber State University, United States b

a r t i c l e

i n f o

Article history: Received 20 May 2014 Revised 9 September 2014 Accepted 13 September 2014 Available online 22 September 2014 Keywords: Nocomis Phylogenetics Systematics Cytochrome b S7 Growth hormone

a b s t r a c t The Nocomis biguttatus species group ranges widely across North America from the Red River in Oklahoma and Arkansas north to Minnesota and east–west from Wyoming to Ontario. The group includes three traditionally recognized allopatric species: the wide-ranging N. biguttatus and two geographically more restricted species, N. asper from the western Ozarks (Arkansas River system) and two disjunct locations in the Red River system, and N. effusus from the Green, Cumberland, and lower Tennessee rivers. Separate analyses of the mitochondrial cytb gene and two nuclear genes (S7 intron 1 and a portion of the gene for growth hormone, GH), each resolved a cryptic species previously treated as N. biguttatus from the southern Ozarks (White River). Relationships among the four species were unresolved because of conflicts between cytb and S7 and a lack of resolution for GH. A previously indicated N. biguttatus–N. effusus sister-relationship appears to reflect past hybridization and mtDNA capture by N. effusus. Nocomis biguttatus includes four primary cytb clades with unresolved inter-relationships. A Northern Ozarks– Great Plains–Upper Midwest Clade and an Ohio River–Eastern Great Lakes Clade presumably represent late Quaternary dispersal from glacial refugia in, respectively, the northern Ozarks and an unglaciated portion of the Ohio River system. Other clades include one from the Meramec River and a Black River– St. Francis River Clade. There was evidence in N. effusus for a phylogeographic break between the lower Tennessee River and the Green-Cumberland basins. Geographic structure is weak in N. asper, indicating relatively recent contact between now disjunct populations in the Arkansas and Red river basins. The Blue River population of N. asper appears to reflect late Pleistocene or Holocene hybridization and genetic swamping of a resident native population of N. biguttatus by an invading population of N. asper. This postulates past occurrence of N. biguttatus far south of its present range. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The North American genus Nocomis includes nine formally described taxa of medium sized (up to about 20 cm, SL) cyprinid species occurring primarily in clear, gravel-bottomed upland streams. Three morphologically defined species groups are ⇑ Corresponding author. E-mail address: [email protected] (A.A. Echelle). Present address: Department of Biology, University of New Mexico, United States. Present address: College of Natural Resources, University of Wisconsin-Stevens Point, United States. 1 2

http://dx.doi.org/10.1016/j.ympev.2014.09.011 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

traditionally recognized (Lachner and Jenkins, 1971a,b; Lachner and Wiley, 1971): the N. leptocephalus group with three described subspecies in southeastern United States, the N. micropogon group of three species in northeastern United States, and the N. biguttatus group of three species in drainages of the Great Lakes and the Mississippi and Ohio rivers. A phylogenetic analysis of morphology supported recognition of the three species groups (Mayden, 1987), but more recent molecular results indicated paraphyly for the N. micropogon group (Nagle and Simons, 2012). The latter study also uncovered deeply divergent clades within the N. leptocephalus group, suggesting the presence of at least five species in upland reaches of coastal streams in southeastern United States (Nagle

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and Simons, 2012). The wide-ranging N. biguttatus group was not sampled as thoroughly as the other groups, leaving the possibility of undetected diversity. In this paper, we present a geographically rather fine-grained analysis of the molecular systematics of the N. biguttatus species group. Nagle and Simons’s (2012) tree-based analysis of divergence time indicated that the species group arose at the Miocene– Pliocene boundary (5.3 mya) and diversified beginning in the Middle Pleistocene (1 mya; Nagle and Simons, 2012). The wideranging N. biguttatus occurs from the Ozarks into the upper Mississippi and Ohio rivers to Minnesota and New York with a disjunct population in Wyoming (Lachner and Jenkins, 1971a,b). Disjunct historical populations in Colorado and western Nebraska and Kansas have been extirpated (Propst and Carlson, 1986; Miller et al., 2005). Nocomis effusus is restricted to the Lower Tennessee and upper Green and Barren rivers of Kentucky and Tennessee. The third species, N. asper, is, with two exceptions, restricted to the western Ozark region in the Arkansas River basin. The exceptions are disjunct populations in the Red River basin in Oklahoma and Arkansas (Lachner and Jenkins, 1971b; Robison and Buchanan, 1988). Previous studies have supported monophyly of the N. biguttatus species group, but they disagree regarding relationships among the three traditionally recognized species. Lachner and Jenkins (1971b) suggested that N. asper and N. effusus are sister species based on coloration and the distribution and size of nuptial tubercles. In explicitly phylogenetic assessments, Mayden’s (1987) morphological analysis and Nagle and Simons’s (2012) analysis of mtDNA and protein-coding nuclear genes recovered, respectively, the N. asper– N. biguttatus and N. effusus–N. biguttatus sister pairs. We used mtDNA and nuclear DNA sequences to re-examine relationships among the members of the species group. The results indicate a previously undetected cryptic species in the southern Ozarks. Our relatively dense sampling of populations also provides perspective on within-species phylogeography and reveals instances of past interspecific genetic introgression, including an example of both mitochondrial and nuclear introgression. 2. Methods 2.1. Data collection We obtained specimens (or Genbank sequences) representing 202 fish from 76 localities across the range of the N. biguttatus species group (Appendix A; Fig. 1). Collections were made with seine and backpack electroshocking and whole specimens or fin clips were preserved in 100% ethanol in the field. For outgroups we used one specimen from each of the three primary clades of the sistergroup to the N. biguttatus group. For mitochondrial cytochrome b (cytb), these included sequences deposited in Genbank by Nagle and Simons (2012) for N. micropogon, N. leptocephalus, and N. raneyi (Appendix A). Archived tissues from the specimens producing these cytb sequences were used to generate outgroup sequences for two nuclear genes, intron 1 for the S7 ribosomal protein gene (S7) and a section of the growth hormone gene (GH). DNeasy kits (Qiagen, Valencia, California) were used to extract genomic DNA from the specimens collected in this study. We sequenced 184 fish for cytb (1140 bp) and obtained an additional 18 sequences from Genbank (Appendix A). Subsets of these fish were sequenced for S7 (829 aligned bp; n = 93) and a section of GH that included introns 3 and 4, exon 4 and portions of exons 3 and 5 (1147 aligned bp, n = 17). The small GH subset was chosen in an attempt to capture the variation detected in cytb and S7. For cytb, PCR amplification employed primers HA and LA (Schmidt et al., 1998) in 10-lL reactions containing 5 lL (2X) Multiplex PCR Mastermix, 3.5 lL ddH20, 0.5 lL (0.5 lM) of each

primer, and 0.5 lL (1–4 ng) DNA. The PCR thermal profile was 95 °C, 15 min; 25–30 cycles of 94 °C for 40 s, 52 °C for 90 s, and 72 °C for 60 s; 72 °C, 10 min. Amplification of S7 was done with nested reactions. The reaction mixture and products from an initial amplification with external primers (S7RPEX1F and S7RPEX3R; Chow and Hazama, 1998) was diluted with ddH2O (1:49) and 1 lL of the dilution was used in a second amplification with internal primers (1F.2 and 2R.67; Unmack et al., 2012). Both amplifications employed 25-lL PCR reactions: 0.125 lL (5 U/lL) Promega (Madison, Wisconsin) GoTaqÒ Flexi DNA polymerase; 5 lL (5X) Promega PCR buffer; 2.5 lL (25 mM) MgCl2; 1.25 lL (0.5 lM) each, forward and reverse primers; 0.2 lL (25 mM) dNTPs; 13.7 lL ddH2O; 1 lL (1–4 ng) DNA. The thermal profile used in both reactions was as follows: 94 °C for 60 s; 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 90 s; 72 °C for 7 min. Nested reactions for GH employed three sets of primer pairs developed by P. Unmack (Moyer et al., 2009) for cyprinids. Using the conditions and reaction-mixture concentrations described for S7, we performed an initial PCR with external primers E3.3F and E5.183R, diluted the resulting mixture 1:49 with ddH2O, and used 1 lL of the dilution in each of two separate reactions with internal primer pairs E3.61F–E4.133R and E4.11F–E5.173R. This amplified two overlapping segments extending from a portion of exon 3 into exon 5 and encompassing introns 3 and 4. Final PCR products were cleaned for sequencing with either the Wizard SV PCR cleanup kit or EXOSAP (USB Corp., Cleveland, Ohio) with a modified temperature profile of 37 °C for 30 min, 80 °C for 15 min and 12 °C for 5 min. We used the amplification primers in sequencing reactions and resolved the sequences with an ABI model 3130 sequencer (Applied Biosystems, Foster City, California). We used Geneious ver. 5.6.4 (Biomatters Ltd., New Zealand) for sequence editing and alignment. 2.2. Data analysis Maximum parsimony and Bayesian analyses were performed separately for cytb (n = 198 fish), S7 (n = 93), GH (n = 17), and the concatenated S7-GH sequences (n = 17). Indelligent v. 1.2 (http:// imperialis.inhs.illinois.edu/dmitriev/indel.asp; Dmitriev and Rakitov, 2008) was used to obtain sequences from insertion– deletion heterozygotes for S7 and GH introns. We used Geneious ver. 5.6.4 (Biomatters Ltd., New Zealand) for sequence alignment and editing, including a search for stop codons in protein coding sequences. The penultimate codon (number 379) was a stop codon (DNA = TAA) in both N. biguttatus and N. effusus but specified the amino acid tryptophan in all others; codon 380 specified alanine in all Nocomis examined. GapCoder (Young and Healy, 2003) was used to identify gaps from the aligned database and to assign character-states for presence (= 1) and absence (= 0) of identified gaps. PAUP⁄ v. 4.0.b10 (Swofford, 2001) was used for maximum-parsimony phylogenetic analyses (heuristic search with TBR branchswapping, 10 random addition sequence replicates) and bootstrap assessments of nodal support (10 random addition sequences; 1000 pseudoreplicates). Bayesian phylogenetic analyses employed MrBayes v.3.2 (Ronquist and Huelsenbeck, 2003) with the data partitioned by codon position separately for cytb and the concatenated GH exons 3–5. A version of Modeltest (Posada and Crandall, 1998), MrModeltest v. 2.3 (https://github.com/nylander/ MrModeltest2), was used to choose a substitution model for each of the following data partitions (chosen model in parentheses): cytb positions 1 (K80 + G), 2 (HKY + I), and 3 (GTR + I + G), concatenated GH exons 3–5 positions 1 (JC), 2 (JC), and 3 (F81), S7 intron 1 (HKY + G), and concatenated GH introns 3 and 4 (F81). For the S7 and GH gap characters, we followed the MrBayes manual and used the JC substitution model with the command lset coding = variable. The cytb and S7 analyses ran for 5 million generations

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Fig. 1. Upper: Species ranges and northern collection localities for the N. biguttatus species group. Unshaded area outlined in black = distribution of N. cf. biguttatus (upper White River). Dotted line shows the approximate maximum extent of ice during glacial maxima of the Pleistocene. Lower: southern collection localities. Unlabeled streams mentioned in the text include the following (by species): N. asper, Blue River (1) and South Fork of the Ouachita River (2–3) of the Red River drainage; Illinois (4–8), Elk (12– 14), and Spring (15–17) rivers and Spring (9–10) and Spavinaw (11) creeks of the Neosho River system; N. cf. biguttatus, upper White River (18–24); N. biguttatus, upper Black River (25–32), St. Francis River (33), Castor River (34), Little Saline River (35), Meramec River (36), Ozark tributaries of the Missouri River (37–41); N. effusus, Barren River (70) of the Green River system, Cumberland River (71–75), Big Richland Creek (76) of the lower Tennessee River.

(burnin = 1.25 million) and those for GH and the S7-GH matrix ran for 3 million generations (burnin = 0.75 million). To assure stationarity and convergence, we used Tracer 1.5 (http://beast.bio.ed. ac.uk/) to examine log-likelihoods across generations and monitored the standard deviation of the split frequencies, assuming that stationarity was achieved at values <0.01. Percent sequence divergence (uncorrected) was computed with MEGA6 (Tamura et al., 2013).

mative characters for the entire gene): cytb, 1140 bp (60, 168); S7, 829 bp with 21 gap characters (29, 65); GH (18, 56) intron 3 and 4, 315 bp with 27 gap characters; GH exons 3 and 5, 332 bp. The sequences generated in this study are deposited in Genbank under the following accession numbers: cytb, KM281536–KM281563, KM281565–KM281590; S7, KM281591–KM281619; and GH, KM281620–KM281632, KM281634, KM281636–KM281637. 3.2. Systematics

3. Results 3.1. Data The aligned sequences included the following for each of the three loci surveyed (parentheses: number of haplotypes for mtDNA, or alleles for S7 and GH, and number of parsimony-infor-

The Bayesian and maximum parsimony analyses for each of the three genes separately (Figs. 2–4) and the S7-GH concatenation (not shown) all supported monophyly for the N. biguttatus species group. With two exceptions, they all resolved the following primary clades: one each for N. asper and N. effusus, one for the White River population (herein considered an undescribed species,

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N. cf. biguttatus) and one for the remaining populations referred to as N. biguttatus. The exceptions occurred in Blue River, where, for cytb, four of the 24 N. asper examined (16.6%) had a haplotype (T) derived from N. biguttatus. For S7, one of 15 fish (6.6%) was heterozygous for the common allele (s5) in N. asper and an allele (s18) from the N. biguttatus clade. Relationships of the s18 allele are unresolved within N. biguttatus, but cytb haplotype T is sister to one of the four primary clades in N. biguttatus (Fig. 2). Neither of the two heterospecific elements in Blue River N. asper was detected in N. biguttatus. The results for cytb and S7 both indicate paraphyly for the traditionally recognized N. biguttatus. The White River species (N. cf. biguttatus) is sister to the N. effusus–N. biguttatus clade (Fig. 2) for cytb, whereas, for S7, it is sister to N. asper (Fig. 3). The GH data provided little support for relationships among the four primary clades (Fig. 4). With one exception, relationships inferred among populations used in the analysis of the combined nuclear genes (S7 and GH; not shown) were identical to those in the analysis of S7 alone. The exception is maximum parsimony bootstrap support (62%) with the combined S7-GH data for a sister relationship between N. effusus and the remainder of the N. biguttatus species group. This relationship appeared (with no statistical support) in

the optimal Bayesian tree for the analysis of GH alone, but in none of the other maximum parsimony or Bayesian trees. 3.3. Phylogeography This section primarily focuses on cytb. The results for S7 and GH provided little evidence of geographic structure within the four primary clades of the species group. The cytb haplotypes from the 44 populations of N. biguttatus (not including the White River species) fell into four statistically supported, geographically defined clades with un-resolved interrelationships (Fig. 2): (1) a Black River–St. Francis River Clade in the Black, St. Francis, and Castor rivers; (2) a Meramec River Clade comprising the one individual examined from the drainage (site 36); (3) a Northern Ozark–Great Plains–Upper Midwest Clade extending from the northern Ozarks (Missouri River and Little Saline Creek) northwest to the Platte River in Wyoming and north to the Hudson Bay drainage and the western Great Lakes as far east as a western tributary of Lake Huron (site 64); and (4) an Ohio River– Eastern Great Lakes Clade occurring from the Wabash River in Indiana (sites 59–61) to the Scioto River (site 67) in Ohio, and in the Great Lakes region from tributaries of eastern Lake Michigan (site

Fig. 2. Bayesian cytb tree for the Nocomis biguttatus species group. Outgroups not included. Terminal nodes labeled with haplotype designation followed by site numbers (Fig. 1) of occurrence and (in parentheses) number of individuals carrying the haplotype. Solid dots = Bayesian probability >0.95; open dot = P = 0.91; numbers below nodes = maximum parsimony bootstrap support.

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Fig. 4. Bayesian GH tree for the N. biguttatus species group. Outgroups not included. See Fig. 3 for remainder of legend.

Fig. 3. Bayesian S7 tree for the Nocomis biguttatus species group. Outgroups not included. Terminal nodes labeled with the allele designation followed site numbers (Fig. 1) of occurrence and (in parentheses) number of individuals carrying the allele and number of heterozygotes. Absence of parentheses = one individual and asterisks signify heterozygotes; e.g., haplotype s9 occurred in 1 heterozygote at locality 7, two individuals at locality 9 (a heterozygote and a homozygote), and one heterozygote at locality 17. Solid dots = Bayesian probability >0.95; numbers below nodes = maximum parsimony bootstrap support.

62) and western Lake Huron (site 65) to the Lake Ontario drainage (site 69). Levels of sequence divergence were 6 0.2% within the four clades and 0.4–0.9% between clades. Haplotypes from both the Northern Ozarks–Great Plains–Upper Midwest Clade and the Ohio River–Eastern Great Lakes Clade were detected in the Kalamazoo River system (sites 62 and 63) in the eastern Lake Michigan drainage and in western tributaries of Lake Huron (sites 64 and 65). Potential geographic overlap between these clades also occurs in the Wabash River drainage. Haplotype BB of the Northern Ozarks–Great Plains–Upper Midwest Clade was detected in a western Wabash River tributary (Vermilion River, site 58) and haplotypes of the Ohio River–Eastern Great Lakes Clade were detected in a northern tributary (haplotypes P and P1; Tippecanoe River, sites 59 and 60) and an eastern tributary (haplotype P; White River, site 61). Another example of potential geographic overlap involves haplotype U of the Northern Ozarks– Great Plains–Upper Midwest Clade. This haplotype was detected in the northern Ozarks (Gasconade River, sites 37 and 38) and in the range of the Black River–St. Francis River Clade (Current River,

site 28). A haplotype of the latter clade was detected elsewhere in the Current River (site 29). The one statistically supported cytb clade within N. asper (Fig. 2) comprised all haplotypes (A, B, B1, and C) detected in the 27 fish examined from the Illinois River (sites 4–8). This clade was restricted to the Illinois River except for the presence of haplotype A in Spring Creek (site 9) of the Neosho River drainage and Blue River (site 1) of the Red River system and the presence of haplotype B1 in Blue River and the other Red River sites sampled (Ouachita River, sites 2 and 3). Other cytb haplotypes in the relatively large Blue River collection (n = 24) were widespread in the Neosho River (haplotype A7) or they were unique to Blue River and either weakly divergent from Neosho River haplotypes or, as previously mentioned, belonged to a clade otherwise comprising haplotypes from N. biguttatus. Congruent geographic structure for cytb and S7 variation is indicated for N. effusus. For both loci, the one individual sampled from the lower Tennessee River (site 76) was the statistically supported sister to a well-supported clade comprising all fish examined from the Green and Cumberland rivers (Figs. 2 and 3). The samples of N. cf. biguttatus (White River species) showed no evidence of geographic structure.

4. Discussion 4.1. Systematics and speciation Each of the three genes examined (cytb, S7, GH) resolved four primary clades in the N. biguttatus species group: N. effusus, N. asper, the White River species (N. cf. biguttatus), and a clade comprising the remainder of the traditionally recognized N. biguttatus. Based on cytb, Nagle and Simons (2012) found N. effusus to be the recently divergent (400,000 yr) sister to N. biguttatus (Nagle and Simons, 2012). This agrees with our cytb results and with Nagle and Simons’s (2012) analysis of concatenated sequences from cytb and two protein-coding nuclear genes. In contrast, the nuclear sequences examined herein support N. effusus as one of three (S7) or four (GH) primary lineages with unresolved inter-relationships.

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Branch lengths among the four primary clades in the cytb and nuclear trees from this study suggest that the relatively young cytb sister relationship between N. effusus and N. biguttatus reflects past N. effusus  N. biguttatus hybridization and mtDNA capture, an often noted phenomenon among fishes (Carson and Dowling, 2006; Broughton et al., 2011; Near et al., 2011; Unmack et al., 2014). The nuclear protein-coding genes assayed by Nagle and Simons (2012) gave little resolution of relationships in the species group, and any signal in the combined nuclear dataset likely was masked by the mtDNA. It is likely that the geographically restricted N. effusus captured, and became fixed for, mtDNA from a population of the widespread N. biguttatus, rather than the reverse. This represents deep introgression, with diversification and reciprocal monophyly evolving subsequent to mtDNA capture (Near et al., 2011). The cytb and S7 trees differ in placement of the White River species as sister, respectively, to the N. biguttatus–N. effusus clade or to N. asper. The concatenated nuclear sequences (S7 and GH) also supported the N. asper-White River species relationship, but this might reflect an over-riding S7 signal, because GH alone gave no resolution. The S7-cytb conflict could represent lineage sorting from a polymorphic ancestor or it might reflect another instance of genetic introgression. The apparent youthfulness of the members of the N. biguttatus species group (61 my) makes either hypothesis tenable. Resolution of relationships among the four species of the N. biguttatus species group will require analysis of additional nuclear markers. Monophyly for independent DNA sequences (mtDNA and two nuclear genes) qualifies the White River form as a species under the phylogenetic and evolutionary species concepts (Mayden, 1997). Further, S7 and GH divergences of the White River form are similar to or greater than those for the three traditionally recognized species of the N. biguttatus species group. Lachner and Jenkins (1971b) treated the White River as a separate geographic unit to allow assessment of intergradation between N. asper and N. biguttatus. There was no evidence of intergradation and no trait was diagnostic of the White River population. However, Nocomis from the White River apparently reaches larger body sizes (>200 mm SL) than other populations of the traditionally recognized N. biguttatus (<165 mm SL), and it showed modal shifts toward larger scale counts than all members of the N. biguttatus species group except N. effusus (Lachner and Jenkins, 1971b). Further, head length in the White River species is shorter than in N. asper and other populations traditionally grouped under N. biguttatus. Lachner and Jenkins (1971b) noted that N. asper and the White River form represent the extremes of head length in the N. biguttatus species group. This is of particular interest because of the indication from S7 of a possible sister relationship between N. asper and the White River species. Although showing considerable overlap, regressions of head length (% SL) against SL for the two forms differed significantly in slope and Y-intercept. The two forms also differ in the characters traditionally used to separate N. biguttatus and N. asper: tubercles on the head and nape and laterally on the body (N. asper) versus tubercles only on the head and nape and red postocular spot well developed in adults and subadults (P100 mm SL) of both sexes (N. asper) versus well developed only in adult males (Lachner and Jenkins, 1971b). The White River species contributes to a general pattern of high endemicity among southern Ozark species in tributaries of the White River system (including the Black River basin). These include cyprinids (Mayden, 1988; this study), ictalurids (Egge and Simons, 2006), centrarchids (Cashner and Suttkus, 1977), and percids (Ceas and Page, 1997; Mayden, 2010). Each of these groups includes one to two described species endemic to the upper White River (or White River and adjacent southern Ozark drainages). Other endemics include the undescribed Ozark Darter, Etheostoma cf.

spectabile (Ceas and Burr, 2002) and a divergent clade of slender madtom, Noturus exilis (Blanton et al., 2013). No simple vicariance hypothesis appears to explain the White River endemics because the estimated times of divergence vary considerably for the species that have been examined. For example, mtDNA estimates for three White River endemics, the Ozark darter, a White River clade of N. exilis, and the Ozark Bass, Ambloplites constellatus, have been diverging from their sister groups for, respectively, about 3, 5, and 10 my (Roe et al., 2008; Blanton et al., 2013; Bossu et al., 2013). In contrast, age estimates for nodes in the cytb tree for the N. biguttatus species group (Nagle and Simons, 2012), along with the interspecies similarity in the nuclear tree branch lengths, imply a divergence time of only about one million years for the White River species. Endemic species of mixed ages seem characteristic of North American highland systems and likely reflect both vicariance and taxon-specific dispersal abilities and opportunities (Strange and Burr, 1997; Bossu et al., 2013; Hoagstrom et al., 2014). The geographic pattern of diversity in the N. biguttatus species group and the cytb estimate of time to the most recent common ancestor (mrca) of the group (1 my) suggests Pleistocene speciation via isolation from a once more widespread ancestor. The cytb estimate suggests that the ancestor of the species group originated about 5 mya (Nagle and Simons, 2012), and early Pleistocene (late Blancan) fossils in a Republican River paleovalley (Missouri River basin) show that Nocomis (referred to as N. biguttatus) was present in the Osage Plains region of northern Kansas at least as early as 2 mya (Eshelman, 1975). These observations, together with present distributions, suggest that the mrca might have ranged from what is now the western plains of North America into the Interior Low Plateau region presently occupied by N. effusus. Events associated with Pleistocene glaciation (Blum et al., 2000; Anthony and Granger, 2007; Rutter et al., 2012) would have isolated ancestral N. effusus from western populations. Branch lengths in the S7 and GH trees suggest that the isolation of N. effusus was roughly contemporaneous with the origins of the three remaining species of the N. biguttatus species group, origins that likely mirror development of the modern topography of the Ozark Plateau. In the Late Miocene and Pliocene, the area was part of an expansive alluvial plain covered with late Tertiary sediments and drained by meandering, northeast trending streams that were part of the pre-glacial drainage of the Missouri River (Aber, 1985; Elfrink and Siemens, 1998; Hoagstrom et al., 2014). The present radial arrangement of upland streams around the plateau reflects (1) a phase of tectonic uplift that began in the Pliocene or early Pleistocene and likely continues to the present (McKeown et al., 1988), and (2) erosion and entrenchment of streams into the plateau (Bretz, 1965). These events, together with the Pleistocene development of the surrounding lowlands on the west (Osage Plains), south (modern Arkansas River floodplain), and east (Mississippi alluvial plain), would have promoted allopatric speciation in taxa associated with upland habitats on the Ozark Plateau.

4.2. Interspecific introgressive hybridization Evidence of past contact and mitochondrial introgression between now allopatric forms can provide useful insights into history. For example, the presence of the mtDNA of common shiner, Luxilus cornutus, in allopatric populations of the striped shiner, L. chrysocephalus, was explained by Quaternary climatic oscillations and associated shifts of L. cornutus to the south of its present distribution (Dowling and Hoeh, 1991; Duvernell and Aspinwall, 1995). A similar shift for N. biguttatus east of the Mississippi River would explain the evidence of contact and hybridization between N. effusus and N. biguttatus. This would have

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occurred about 400,000 yrs ago based on cytb divergence time between the two species (Nagle and Simons, 2012). The presence of genetic elements from N. biguttatus in Blue River N. asper appears to reflect natural contact rather than anthropogenic introduction of N. biguttatus into Blue River. Our results indicate 17% mtDNA introgression and 3% S7 introgression in Blue River and no introgression in other populations of N. asper. The heterospecific elements in Blue River N. asper were represented by a single haplotype/allele for both genes. Natural contact is consistent with our failure to detect either the heterospecific cytb haplotype (T) or the heterospecific S7 allele outside of Blue River, suggesting they were not recently introduced into Blue River. This argument is especially convincing for haplotype T, which is both restricted to Blue River and sister to the widespread Northern Ozarks–Great Plains–Upper Midwest cytb clade of N. biguttatus. 4.3. Phylogeography This section is restricted to the relatively widespread and well-sampled N. biguttatus and N. asper. The potential sister relationship, for both cytb and S7, between the lower Tennessee River and Green-Cumberland populations of N. effusus requires corroboration with larger sample sizes and more thorough geographic coverage. The White River species showed no evidence of phylogeographic structure. The geographic structure of cytb variation in N. biguttatus has evolved in the last 400,000 years based on the estimated time since capture of mtDNA from N. biguttatus by N. effusus. The cytb tree for N. biguttatus includes four geographically defined clades with unresolved relationships: (1) a Northern Ozarks–Great Plains– Upper Midwest Clade, (2) an Ohio River–Eastern Great Lakes Clade, (3) a Black River–St. Francis River Clade, and (4) a Meramec River Clade. Levels of divergence were low, suggesting late Pleistocene origins of these clades (uncorrected divergence 6 0.2% within and 0.4–0.9% between clades). The Northern Ozarks–Great Plains–Upper Midwest Clade reflects relatively recent dispersal from a southern glacial refugium, likely the northern Ozarks, into previously glaciated regions as far north as the upper Great Lakes and Hudson Bay drainages. Recent dispersal is indicated by the shallow structure of the clade and individual haplotype distributions. For example, haplotype X was detected in two Missouri River tributaries (Ozarks) and it occurs as far north as Lake Superior and northeastern Lake Huron. Late Pleistocene or Holocene dispersal northward from southern glacial refugia has been suggested for similarly distributed clades in a variety of fishes (Near et al., 2001; Berendzen et al., 2003, 2010; Ray et al., 2006; Borden and Krebs, 2009; Bossu et al., 2013). The Ohio River–Eastern Great Lakes Clade of N. biguttatus is almost entirely restricted to regions that were under glacial ice in the late Pleistocene. Similarly distributed mitochondrial clades are found in the rainbow darter, Etheostoma caeruleum (Ray et al., 2006), orangethroat darter, E. spectabile (Bossu et al., 2013), and least darter, E. microperca (A. Echelle et al., unpubl.). This might represent late or post-Pleistocene dispersal from glacial refugia east of the Mississippi River as suggested for the northeastern clades of E. caeruleum and E. spectabile, the similarly distributed rosyface shiner, Notropis rubellus (Berendzen et al., 2008), and northeastern populations of smallmouth bass, Micropterus dolomieu (Borden and Krebs, 2009). Possibilities for N. biguttatus include unglaciated southern tributaries of the Ohio River such as the Kanawha River in West Virginia and the Kentucky River in Kentucky, both of which have records of now extirpated, potentially native populations of N. biguttatus (Welsh et al., 2013). Particularly strong support for a Kentucky River refugium exists for the northeastern clade of E. spectabile (Bossu et al., 2013). An earlier, more southern, Pleistocene refugium for eastern populations of N. bigutt-

115

atus is indicated by the estimated 400,000 yrs (Nagle and Simons, 2012) since the postulated contact and mtDNA capture involving N. biguttatus and N. effusus in Kentucky and Tennessee. The region encompassing Michigan’s Lower Peninsula and the Wabash River likely represents a zone of post-Pleistocene secondary contact between the Northern Ozark–Great Plains–Upper Midwest Clade and the Ohio River–Eastern Great Lakes Clade. The sampling in this region (n = 1–3 per site) allowed little opportunity to detect polymorphic populations, but haplotypes representing both clades were found separately in two tributaries of the Kalamazoo River (eastern Lake Michigan tributary), two closely spaced tributaries of northeastern Lake Huron, and two rivers (Vermilion and Tippicanoe) of the Wabash River basin. Evidence of secondary contact between eastern and western clades in this region has been attributed to postglacial northward and east–west dispersal aided by proglacial lakes and temporary outlets to the Mississippi, Illinois, and Ohio River basins (Gach, 1996; April and Turgeon, 2006; Borden and Krebs, 2009). The Current River of the Black River system in the southeastern Ozarks is an area of likely secondary contact between the Northern Ozarks–Great Plains–Upper Midwest and Black River–St. Francis River clades of N. biguttatus. A haplotype of the former clade was detected only in the Gasconade River drainage of the northern Ozarks and the headwaters of the Current River. The indicated headwater transfer into the Black River system via dispersal or stream capture from the Gasconade River might also explain the Ozark distribution of the golden crayfish, Orconectes luteus. This species is widespread in Missouri River tributaries of the northern Ozarks and absent in all other Ozark streams except the Current River (Pflieger, 1996). Phylogeographic studies that have included Meramec River fishes highlight the role of taxon-specific dispersal abilities and opportunities in accounting for faunal assemblages. Meramec River populations typically are in shallow clades with populations from Ozark tributaries of the Missouri River (Near et al., 2001; Hardy et al., 2002; Berendzen et al., 2003, 2010), but the E. caeruleum population is in an equally shallow clade (Ray et al., 2006) with populations far to the south (Black and St. Francis rivers, lower and middle Mississippi River basin). On the other hand, the potential for longterm isolation is illustrated by the Meramec populations of N. biguttatus and E. spectabile (Bossu et al., 2013) and the sister relationship between the Missouri saddled darter E. tetrazonum and the Meramec saddled darter, E. erythrozonum, endemics of, respectively, the Missouri and Meramec rivers (Switzer and Wood, 2002, 2009). Additional sampling is needed to clarify connectedness between Meramec River N. biguttatus and other populations because the Meramec is located between streams carrying the Northern Ozark–Great Plains–Upper Midwest Clade: the Missouri River and Little Saline Creek, a direct tributary of the Mississippi River situated where headwater transfer is not likely to explain the observed pattern. The genetic structure of N. asper appears to reflect relatively recent gene flow between southwestern Ozark streams (Arkansas River system) and the Red River system (Blue and Ouachita rivers). In Ozark populations, cytb diversity is partitioned between the Neosho and Illinois rivers, with little sharing of haplotypes, whereas haplotypes in both of these systems are identical to or weakly divergent from Red River haplotypes. A variety of other fishes occurring in springfed Ozark streams are known to share identical haplotypes with disjunct populations in the Red River drainage. These include Ozark logperch Percina caprodes fulvitaenia (Lynch, 2010), southern redbelly dace, Chrosomus erythrogaster (B. Krieser, pers. comm.), and northern studfish, Fundulus catenatus (Strange and Burr, 1997). This pattern conforms to the hypothesis (Cross, 1970; Burr, 1978) that presently disjunct, southern populations of typically more northern, cooler-water fishes were more widespread

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in southern regions during glacial periods of the Pleistocene. During interglacials like the present they contracted into springfed upland streams, producing highly fragmented distributions. A novel biogeographic hypothesis is indicated by the previously mentioned genetic markers from N. biguttatus in the Blue River population of N. asper. This seems to reflect genetic swamping of an earlier, resident population of N. biguttatus by an invading population of N. asper. The sister relationship between the heterospecific cytb in Blue River N. asper and the Northern Ozark–Upper Midwest cytb clade of N. biguttatus indicates sufficient time of divergence to permit the evolution of reciprocal monophyly. This requires corroboration with additional sampling because the Blue River collection of N. asper (n = 24) only included four haplotypes from N. biguttatus. The Blue River population is presently separated from the Northern Ozark–Upper Midwest Clade of N. biguttatus by the intervening Neosho and Illinois river populations of N. asper, which, despite relatively large samples, showed no evidence of markers from N. biguttatus. The signal of N. biguttatus in Blue River might be a relict of cooler, Pleistocene times when the species could have been more widely distributed at southern latitudes in western Kansas and Oklahoma. Today, it is northern in distribution or primarily inhabits springfed streams with relatively cool summertime temperatures. Ichthyologists commonly invoke Metcalf’s (1966) hypothetical Ancestral Plains Stream (APS) to explain north–south plains-fish distributions (e.g., Bossu et al., 2013; but see Hoagstrom and Berry, 2006). This postulated stream headed in the ancient Kansas River basin, or perhaps as far north as the ancient Platte River (Metcalf, 1966), from where it traversed the plains southward and west of the Ozark and Ouachita uplifts before emptying into the Gulf of Mexico independently of the lower Red River (Mayden, 1985; Cross et al., 1986). Although favoring the APS, Metcalf (1966) and subsequent authors (Pflieger, 1971; Cross et al., 1986) expressed some uncertainty, including evidence supporting a now better supported (Galloway et al., 2011; Hoagstrom et al., 2014) alternative invoking an ancient Red River that was much more expansive than predicted from the APS hypothesis. Geomorphic evidence supports an ancient Red River that drained most of the proposed APS system and traversed the coastal plain of east Texas, more or less along the course of the present Red River, before emptying into the Gulf of Mexico (Galloway et al., 2011; Hoagstrom et al., 2014). Presence of Nocomis, presumably N. biguttatus, in the Kansas River basin as early as 2 mya (Eshelman, 1975) indicates ample time for it to become widely distributed on the Great Plains. Its presence in the Smoky Hill River valley (near Kanopolis, Kansas) later in the Pleistocene (Neff,

Species Locality number & Stream: State, County Nocomis asper 1 Blue River: OK, Johnston 2 South Fork Ouachita River: AR, Montgomery 3 South Fork Ouachita River: AR, Montgomery 4 Tyner Creek: OK, Adair 5 Flint Creek: OK, Delaware 6 Flint Creek: AR, Benton 7 Clear Creek: AR, Washington 8 Baron Fork: OK, Adair

1975) places the species in the vicinity of the McPherson paleovalley where there was a late-Pleistocene transfer of the Smoky Hill River from the Arkansas River basin to the Missouri River basin (Madole et al., 1991). If N. biguttatus became widespread in the Arkansas River basin, it could have gained access to the Red River basin in relatively close proximity to Blue River where the Canadian River traversed a region (the Gerty Sands) now drained by the headwaters of a Red River tributary (Muddy Boggy River; Hendricks, 1937; Madole et al., 1991). Persistence of a widely disjunct southern population of N. biguttatus in upper Blue River likely is attributable to the springfed nature of the river, and its large size relative to other springfed environments of the region. Over a stream distance of less than 5 km, the headwater reach becomes an almost entirely groundwater-dependent, mid-sized river that supports, along with N. asper, several other disjunct populations of fishes, including C. erythrogaster; black redhorse, Moxostoma duquesnii; E. microperca; and P. c. fulvitaenia (Mayden, 1985; Miller and Robison, 2004). The indicated, relatively recent arrival of N. asper in Blue River might reflect range expansion during the last glacial interval. Results of this study and those of previously mentioned studies showing haplotype sharing among upland fishes of the Ozark and Ouachita highlands indicate that, at times in the relatively recent past, the Arkansas River floodplain was not a severe barrier to dispersal of highland fishes. Acknowledgments We thank D. Lynch, A.F. Echelle, and J. Egge for help in collecting, R. Van Den Bussche for making his laboratory available and R. Mayden, R. Wood, B. Kuhajda, P. Harris, and A. Bentley for tissues from their respective collections. Funding was provided by the U.S. Geological Survey and the Oklahoma chapter of the Nature Conservancy and administered by the Oklahoma Cooperative Fish and Wildlife Research Unit and the Department of Zoology, Oklahoma State University. Additional funding provided by the National Science Foundation EF 0431132 to AMS. Appendix A Species, locality numbers, localities, and number of specimens examined for cytb, S7, and GH. Abbreviations AMS, NJL, PU, and RLM = field numbers from, respectively, A. Simons, J. Egge, N. Lang, P. Unmack, and R. Mayden. Institutional abbreviations follow Leviton et al. (1985). STL = Saint Louis University Ichthyological Collection, St. Louis, Missouri, USA.

Drainage

Voucher

Coordinates

cytb, S7, GH

Red-Mississippi Ouachita-Red

OSUS 27695 OSUS 27564

34.4523, 34.5591,

96.6271 93.7141

24, 16, 1 2, 2, 0

Ouachita-Red

JFBM 46872

34.5578,

93.6950

1a, 0, 0

Illinois-Arkansas-Mississippi Illinois-Arkansas Illinois-Arkansas Illinois-Arkansas Illinois-Arkansas

OSUS 27566 OSUS 27809 JFBM 37877 NJL07-101 OSUS 27565

35.9660, 36.1940, 36.2181, 36.1038, 35.9192,

94.7695 94.7068 94.5442 94.3086 94.6194

1, 1, 0 18, 0, 0 2a, 0, 0 4, 4, 1 1, 1, 0

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A.A. Echelle et al. / Molecular Phylogenetics and Evolution 81 (2014) 109–119 Appendix A (continued)

9 10 11 12 13 14 15 16 17

Species Locality number & Stream: State, County

Drainage

Voucher

Coordinates

cytb, S7, GH

Spring Creek: OK, Cherokee Spring Creek: OK, Mayes Spavinaw Creek; AR, Benton Elk River: AR, Benton Big Sugar Creek: MO, McDonald Elk River: MO, McDonald Fivemile Creek: OK, Ottawa Shoal Creek: MO, Newton Spring River: MO, Lawrence

Neosho-Arkansas Neosho-Arkansas Neosho-Arkansas Elk-Neosho-Arkansas Elk-Neosho-Arkansas

OSUS 27560 PU04-70 OSUS 27604 UAIC 12549 OSUS 27562

36.1442, 36.1646, 36.3965, 36.4933, 36.6218,

94.9078 95.1578 94.4475 94.4708 94.1800

26, 4, 1 0, 0, 1 2, 1, 0 1b, 0, 0 1, 1, 0

Neosho-Arkansas Spring-Neosho-Arkansas Spring -Neosho-Arkansas Neosho-Arkansas

JFBM 46874 OSUS 27559 AMS-01-05 OSUS 27561

36.5603, 36.9881, 37.0275, 37.1159,

94.4239 94.6457 94.5606 93.8944

1a, 0, 0 1, 1, 0 1a, 0, 0 26, 2, 0

White-Arkansas White-Arkansas White-Arkansas White-Arkansas White-Arkansas White-Arkansas

KU 29330 PU04-53 STL 389.01 RLM84-17 RLM85-09 JFBM 47205

35.9670, 35.9834, 35.9398, 36.7760, 36.8350, 36.7580,

93.4000 92.7473 92.1133 93.6861 93.0183 92.1542

1, 0, 1, 1, 1, 7,

White-Arkansas

RLM85-08

36.9817,

92.7054

1, 1, 1

Black-White-Arkansas

STL 180.03

36.6717,

91.6265

1, 1, 1

Black-White-Arkansas

JFBM 44696

36.5508,

91.1941

2, 2, 0

Black-White-Arkansas

JFBM 41842

36.2431,

91.0894

1c, 2, 0

Current-Black-White Black-White-Arkansas Black-White-Arkansas

RLM85-07 JFBM 47209 OSUS 27588

37.4290, 36.6175, 37.5159,

91.6895 90.8397 91.1859

1, 1, 0 3, 3, 0 1, 1, 0

Black-White-Arkansas Black-White-Arkansas St. Francis-Mississippi Castor-St. Francis-Mississippi Mississippi

UAIC 11154 JFBM 46867 OSUS 27589 OSUS 27590 STL 1344.02

37.4508, 37.4167, 37.3587, 37.4278, 37.8464,

90.8272 90.8253 90.7057 90.1707 90.0320

1, 0, 0 3(1)a, 2, 0 1, 1, 0 1, 1, 0 1b, 0, 0

Meramec-Mississippi Gasconade-Missouri Gasconade-Missouri Osage-Missouri

OSUS 27586 RLM83-30 JFBM 40925 OSUS 27587

37.9742, 37.6018, 37.3265, 37.9914,

91.2049 92.2333 92.0029 92.5076

1, 1, 8, 1,

Osage-Missouri Lamine-Missouri Platte-Missouri Ottertail-Red River of the North St. Louis-Lake Superior Saint Croix-Mississippi Mississippi

JFBM 37848 OSUS 27591 MSB 75679 OSUS 27575 OSUS 27577 JFBM 35764 OSUS 27574

37.9450, 38.6155, 41.9333, 46.3793, 46.7064, 45.4817, 45.5191,

93.3091 93.0031 105.383 96.1257 92.3686 92.8742 95.0085

5, 0, 0 1, 0, 0 1, 1, 0 1, 1, 1 1, 1, 0 1a, 0, 0 1, 1, 0

Rum-Mississippi St. Croix-Mississippi Pigeon-Lake Michigan Rock-Mississippi Little Sioux-Missouri Des Moines-Mississippi Rock-Mississippi

JFBM 46873 OSUS 27578 OSUS 27579 STL 592.01 OSUS 27576 UAIC 10149 OSUS 27572

45.3664, 45.8237, 44.8412, 42.5093, 43.1450, 42.5296, 42.4459,

93.3819 91.9335 92.2378 89.1690 95.4866 94.3054 89.0202

1a, 0, 0 1, 1, 0 1, 1, 0 1, 1, 0 1, 1, 0 1, 1, 0 1, 1, 0

Illinois-Mississippi Illinois-Mississippi

STL 809.02 STL 512.01

40.6315, 40.4421,

88.7986 90.8005

1, 1, 0 1, 1, 0

Nocomis cf. biguttatus 18 Buffalo River: AR, Newton 19 Buffalo River: AR, Searcy 20 White River: AR, Izard/Stone 21 Flat Creek: MO, Barry 22 Swan Creek: MO, Christian 23 North Fork White River: MO, Ozark 24 Cowskin Creek: MO, Douglas Nocomis biguttatus 25 Warm Fork Spring River: MO, Oregon 26 Eleven Point River: MO, Oregon 27 Eleven Point River: AR, Randolph 28 Ashley Creek: MO, Dent 29 Current River: MO, Ripley 30 West Fork Black River: MO, Reynolds 31 Black River: MO, Reynolds 32 Black River: MO, Reynolds 33 Big Creek: MO, Iron 34 Castor River: MO, Madison 35 Little Saline Creek: MO, Ste Genevieve 36 Huzzah Creek: MO, Crawford 37 Roubidoux Creek: MO, Texas 38 Big Piney River: MO, Texas 39 Wet Glaize Creek: MO, Camden 40 Pomme de Terre: MO, Hickory 41 Haw Creek: MO, Morgan 42 Laramie River: WY, Albany 43 Pelican River: MN, Otter Tail 44 Midway River: MN, Carlton 45 Sunrise River: MN, Chisago 46 North Fork Crow River: MN, Steams 47 Seelye Brook: MN, Anoka 48 Yellow River: MN, Washburn 49 Meeme River: WI, Manitowoc 50 Dry Creek: WI, Rock 51 Waterman Creek: IA, O’brien 52 Lizard Creek: IA, Webster 53 North Kinnikinnick Creek: IL, Winnebago 54 Rooks Creek: IL, McLean 55 Killjordan Creek: IL,

1, 0, 0, 1, 1, 7,

1, 1, 0, 1,

1 1 0 0 1 0

0 0 0 0

(continued on next page)

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A.A. Echelle et al. / Molecular Phylogenetics and Evolution 81 (2014) 109–119

Appendix A (continued)

Species Locality number & Stream: State, County

Drainage

Voucher

Coordinates

cytb, S7, GH

Sangamon-Illinois

OSUS 27569

40.3594,

88.4225

1, 1, 0

Kaskaskia-Mississippi Vermilion-Wabash Tippecanoe-Wabash Wabash East Fork White-Wabash

OSUS 27570 OSUS 27568 OSUS 27580 JFBM 46868 OSUS 27567

40.0698, 40.1524, 41.0248, 41.1564, 39.4227,

88.3456 87.9019 86.5834 86.1103 85.8456

1, 1, 0 1, 1, 0 1, 1, 0 3a, 0, 0 1, 1, 0

Kalamazoo-Lake Michigan Lake Michigan

OSUS 27594 OSUS 27595

42.3936, 42.2433,

85.2332 84.7504

1, 1, 0 1, 1, 0

Au Sable-Lake Huron Lake Huron

OSUS 27581 OSUS 27596

44.8753, 45.0609,

84.6072 83.5853

1, 1, 0 1, 1, 0

Great Miami-Ohio Scioto-Ohio

OSUS 27585 OSUS 27584

40.3099, 40.0810,

83.8842 82.8906

1, 1, 0 1, 1, 0

Grand-Lake Erie

OSUS 27582

41.4196,

80.8767

1, 1, 0

Lake Ontario

OSUS 27583

43.3368,

77.8112

1, 1, 1

Nocomis effusus 70 Indian Creek: KY, Monroe 71 Whipoorwill Creek: KY, Logan 72 Otter Creek: KY, Wayne 73 Otter Creek: KY, Wayne 74 Red River: KY, Robertson 75 Yellow Creek: TN, Dickson 76 Big Richland Creek: TN, Humphreys

Barren-Green-Ohio Cumberland-Ohio Cumberland-Ohio Cumberland-Ohio Cumberland-Ohio Cumberland-Ohio Tennessee-Ohio

OSUS 27593 UAIC 12500 YPM 18826 JFBM 38254 UF 167942 OSUS 27592 OSUS 27687

36.6945, 36.7964, 36.6660, 36.7058, 36.5961, 36.1500, 36.1570,

85.9568 81.9778 84.9730 84.9644 87.0599 87.5000 87.7723

1, 1, 1 1, 1, 0 1d, 0, 0 6, 0, 0 1, 0, 0 1, 1, 1 1, 1, 1

Nocomis micropogon South Fork Kentucky River: KY, Owsley

Kentucky-Ohio

JFBM 38410

37.3522,

83.7369

1a, 1, 1

Nocomis raneyi Tar River: NC, Vance-Franklin

Tar-Pamlico

JJDE-04-25

36.1688,

78.4493

1a, 1, 1

Nocomis leptocephalus Catawba River: NC, McDowell

Wateree-Santee

JFBM 38760

35.3822,

82.8583

1a, 1, 1

56 57 58 59 60 61 62 63 64 65 66 67 68 69

McDonough Lone Tree Creek: IL, Champaign Copper Slough: IL, Champaign Stony Creek: IL, Vermilion Mill Creek: IN, Pulaski Tippecanoe River: IN, Fulton Eastfork Slash Creek: IN, Shelby Wabascon Creek: MI, Calhoun South Fork Kalamazoo River: MI, Calhoun Guthrie Creek: MI, Otsego South Branch Thunder Bay River: MI, Alpena McKees Creek: OH, Logan Big Walnut Creek: OH, Franklin Baughman Creek: OH, Trumbull West Creek: NY, Monroe

a Genbank accession numbers from Nagle and Simons (2012): N. asper JQ712326, JQ712327, JQ712285, JQ712286, JQ712328; N. biguttatus JQ712283, JQ71232, JQ712324, JQ712323, JQ712284, JQ712325, AY486057; N. effusus JQ712329, JQ712330, JQ712331, JQ712287, JQ712288, JQ712332; N. micropogon JQ712344; N. raneyi JQ712350; N. leptocephalus JQ712362. b Genbank accession numbers GQ275150 and GQ275149 from Schönhuth and Mayden (2010). c Genbank accession number AY486057 (Simons, 2004). d Genbank accession number HQ446750 from Hollingsworth and Hulsey (2011).

References Aber, J.S., 1985. Quartzite-bearing gravels and drainage development in eastern Kansas. TER-QUA Symposium Series 1, 105–110. Anthony, D.M., Granger, D.E., 2007. A new chronology for the age of Appalachian erosional surfaces determined by cosmogenic nuclides in cave sediments. Earth Surf. Proc. 32, 874–887. April, J., Turgeon, J., 2006. Phylogeography of the banded killifish (Fundulus diaphanus): glacial races and secondary contact. J. Fish Biol. 69, 212–228. Berendzen, P.B., Dugan, J.F., Gamble, T., 2010. Post-glacial expansion into the Paleozoic Plateau: evidence of an Ozarkian refugium for the Ozark minnow Notropis nubilus (Teleostei: Cypriniformes). J. Fish Biol. 77, 1114–1136. Berendzen, P.B., Simons, A.M., Wood, R.M., 2003. Phylogeography of the northern hogsucker, Hypentelium nigricans (Teleostei: Cypriniformes): genetic evidence for the existence of the ancient Teays River. J. Biogeog. 30, 1139–1152. Berendzen, P.B., Simons, A.M., Wood, R.M., Dowling, T.E., Secor, C.L., 2008. Recovering cryptic diversity and ancient drainage patterns in eastern North America: historical biogeography of the Notropis rubellus species group (Teleostei: Cypriniformes). Mol. Phylogenet. Evol. 46, 721–737.

Blanton, R.E., Page, L.M., Hilber, S.A., 2013. Timing of clade divergence and discordant estimates of genetic and morphological diversity in the Slender Madtom, Noturus exilis (Ictaluridae). Mol. Phylo. Evol. 66, 679–693. Blum, M.D., Guccione, M.J., Wysocki, D.A., Robnett, P.C., Rutledge, E.M., 2000. Late Pleistocene evolution of the lower Mississippi River valley, southern Missouri to Arkansas. Geol. Soc. Am. Bull. 112, 221–235. Borden, W.C., Krebs, R.A., 2009. Phylogeography and postglacial dispersal of smallmouth bass (Micropterus dolomieu) into the Great Lakes. Can. J. Fish. Aquat. Sci. 66, 2142–2156. Bossu, C.M., Beaulieu, J.M., Ceas, P.A., Near, T.J., 2013. Explicit tests of palaeodrainage connections of southeastern North America and the historical biogeography of orangethroat darters (Percidae: Etheostoma: Ceasia). Mol. Ecol. 22, 5397–5417. Bretz, J.H., 1965. Geomorphic history of the Ozarks of Missouri. Missouri Geological Survey and Water Resources, Rolla, Missouri. Broughton, R.E., Vedala, K.C., Crowl, T.M., Ritterhouse, L.L., 2011. Current and historical hybridization with differential introgression among three species of cyprinid fishes (genus Cyprinella). Genetica 139, 699-70. Burr, B.M., 1978. Systematics of the percid fishes of the subgenus Microperca, genus Etheostoma. Bull. Alabama Mus. Nat. Hist. 14, 1–53.

A.A. Echelle et al. / Molecular Phylogenetics and Evolution 81 (2014) 109–119 Carson, E.W., Dowling, T.E., 2006. Influence of hydrogeographic history and hybridization on the distribution of genetic variation in the pupfishes Cyprinodon atrorus and C. bifasciatus. Mol. Ecol. 15, 667–679. Cashner, R.C., Suttkus, R.D., 1977. Ambloplites constellatus, a new species of rock bass from the Ozark upland of Arkansas and Missouri with a review of western rock bass populations. Am. Midl. Nat. 98, 147–161. Ceas, P.A., Burr, B.M., 2002. Etheostoma lawrencei, a new species of darter in the E. spectabilie species complex (Percidae: subgenus Oligocephalus), from Kentucky and Tennessee. Ichthyol. Explor. Freshwaters 13, 203–216. Ceas, P.A., Page, L.M., 1997. Systematic studies of the Etheostoma spectabile complex (Percidae; subgenus Oligocephalus), with descriptions of four new species. Copeia 1997, 496–522. Chow, S., Hazama, K., 1998. Universal PCR primers for S7 ribosomal protein gene introns in fish. Mol. Ecol. 7, 1255–1256. Cross, F.B., 1970. Fishes as indicators of Pleistocene and recent environments in the central plains. In: Dort Jr., W., Jones Jr., J.K. (Eds.), Pleistocene and Recent Environments of the Central Great Plains. Department of Geology, University of Kansas, Special Publications 3, pp. 241–257. Cross, F.B., Mayden, R.L., Stewart, J.D., 1986. Fishes in the western Mississippi Basin (Missouri, Arkansas and Red rivers). In: Hocutt, C.H., Wiley, E.O. (Eds.), The Zoogeography of North American Freshwater Fishes. Wiley and Sons, NY, pp. 363–412. Dmitriev, D.A., Rakitov, R.A., 2008. Decoding of superimposed traces produced by direct sequencing of heterozygous indels. PLoS Comput. Biol. 4, e1000113. http://dx.doi.org/10.1371/journal.pcbi.1000113. Dowling, T.E., Hoeh, W.R., 1991. The extent of introgression outside the contact zone between Notropis cornutus and Notropis chrysocephalus (Teleostei: Cyprinidae). Evolution 45, 944–956. Duvernell, D.D., Aspinwall, N., 1995. Introgression of Luxilus cornutus mtDNA into allopatric populations of Luxilus chrysocephalus (Teleostei: Cyprinidae) in Missouri and Arkansas. Mol. Ecol. 4, 173–182. Egge, J.D., Simons, A.M., 2006. The challenge of truly cryptic diversity: diagnosis and description of a new madtom catfish (Ictaluridae: Noturus). Zool. Scripta 35, 581–595. Elfrink, N.M., Siemens, M.A., 1998. Quaternary drainage shifts in Missouri. In: Guidebook for the 45th Annual Field Trip. Association of Missouri Geologists, Farmington, Missouri. pp. 48–49. Eshelman, R.E., 1975. Geology and paleontology of Early Pleistocene (Late Blancan) White Rock Fauna from north-central Kansas. University of Michigan Museum of Paleontology Papers on Paleontology 13, 1–60. Gach, M.H., 1996. Geographic variation in mitochondrial DNA and biogeography of Culaea inconstans (Gasterosteidae). Copeia 1996, 563–575. Galloway, W.E., Whiteaker, T.L., Ganey-Curry, P., 2011. History of Cenozoic North American drainage basin evolution, sediment yield, and accumulation in the Gulf of Mexico basin. Geosphere 7, 938–973. Hardy, M.E., Grady, J.M., Routman, E.J., 2002. Intraspecific phylogeography of the slender madtom: the complex evolutionary history of the Central Highlands of the United States. Mol. Ecol. 11, 2393–2403. Hendricks, T.A., 1937. History of the Canadian River of Oklahoma as indicated by Gerty Sand. Geol. Soc. Am. Bull. 48, 365–372. Hoagstrom, C.W., Berry Jr., C.R., 2006. Island biogeography of native fish faunas among Great Plains drainage basins: basin scale features influence composition. Am. Fish. Soc. Symp. 48, 221–264. Hoagstrom, C.W., Ung, V., Taylor, K., 2014. Miocene rivers and taxon cycles clarify the comparative biogeography of North American highland fishes. J. Biogeog. 41, 644–658. Hollingsworth Jr., R., Hulsey, C.D., 2011. Reconciling gene trees of eastern North American minnows. Mol. Phylogenet. Evol. 61, 149–156. Lachner, E.A., Jenkins, R.E., 1971a. Systematics, distribution, and evolution of the chub genus Nocomis Girard (Pisces: Cyprinidae) of eastern US with a description of a new species. Smithsonian Contr. Zool. 85, 1–97. Lachner, E.A., Jenkins, R.E., 1971b. Systematics, distribution, and evolution of the Nocomis biguttatus species group (Family Cyprinidae: Pisces) with description of a new species from the Ozark Upland. Smithsonian Contrib. Zool. 91, 1–28. Lachner, E.A., Wiley, M.L., 1971. Populations of the polytypic species of Nocomis leptocephalus (Girard) with a description of a new subspecies. Smithsonian Contrib. Zool. 92, 1–35. Leviton, A.E., Gibbs Jr., R.H., Heal, E., Dawson, C.E., 1985. Standards in herpetology and ichthyology: Part 1. Standard symbolic codes for institution resource collections in herpetology and ichthyology. Copeia 1985, 802–832. Lynch, D., 2010. Phylogeography of the Ozark logperch, Percina caprodes fulvitaenia, in the Red and Arkansas river basins. Unpublished M.S. thesis, Oklahoma State University, Stillwater. Madole, R.F., Ferring, C.R., Guccione, M.J., Hall, S.A., Johnson, W.C., Sorenson, C.J., 1991. Quaternary geology of the Osage Plains and Interior Highlands. In: Morrison, R.B. (Ed.), Quaternary Nonglacial Geology: Conterminous U.S. Geological Society of America, Boulder, Colorado, pp. 503–546. Mayden, R.L., 1985. Biogeography of Ouachita Highland fishes. Southwest Nat. 30, 195–211. Mayden, R.L., 1987. Historical ecology and North American highland fishes: a research program in community ecology. In: Matthews, W.J., Heins, D.C. (Eds.), Community and Evolutionary Ecology of North American Stream Fishes. University of Oklahoma Press, Norman, Oklahoma, pp. 210–222. Mayden, R.L., 1988. Systematics of the Notropis zonatus species group, with description of a new species from the Interior Highlands of North America. Copeia 1988, 153–173.

119

Mayden, R.L., 1997. A hierarchy of species concepts: the denouement in the saga of the species problem. In: Claridge, M.F., Dawah, H.A., Wilson, M.R. (Eds.), Species: The Units of Biodiversity. Chapman & Hall Ltd., London, pp. 381–424. Mayden, R.L., 2010. Systematics of the Etheostoma punctulatum species group (Teleostei: Percidae), with descriptions of two new species. Copeia 2010, 716– 734. McKeown, F.A., Jones-Cecil, M., Askew, B.L., McGrath, M.B., 1988. Analysis of stream-profile data and inferred tectonic activity, eastern Ozark Mountains region. US Geol. Surv. Bull. 1807, 1–39. Metcalf, A.L., 1966. Fishes of the Kansas River system in relation to zoogeography of the Great Plains. Publ. Mus. Nat. Hist. Univ. Kansas 17, 23–189. Miller, W.J., Rees, D.E., Carr, R.J. Berube, D.S., 2005. Hornyhead Chub (Nocomis biguttatus): a technical conservation assessment. USDA Forest Service, Rocky Mountain Region. . Miller, R.J., Robison, H.W., 2004. Fishes of Oklahoma. University of Oklahoma Press, Norman. Moyer, G.R., Remington, R.K., Turner, T.F., 2009. Incongruent gene trees, complex evolutionary processes, and the phylogeny of a group of North American minnows (Hybognathus Agassiz 1855). Mol. Phylogenet. Evol. 50, 514–525. Nagle, B.C., Simons, A.M., 2012. Rapid diversification in the North American minnow genus Nocomis. Mol. Phylogenet. Evol. 63, 639–649. Near, T.J., Bossou, C.M., Bradburd, G.S., Carlson, R.L., Harrington, R.C., Hollingsworth Jr., P.R., Keck, B.P., Etnier, D.A., 2011. Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae). Syst. Biol. 60, 565–595. Near, T.J., Page, L.M., Mayden, R.L., 2001. Intraspecific phylogeography of Percina evides (Percidae: Etheostomatinae): an additional test of the Central Highlands pre-Pleistocene vicariance hypothesis. Mol. Ecol. 10, 2235–2240. Neff, N.A., 1975. Fishes of the Kanopolis local fauna (Pleistocene) of Ellsworth County, Kansas. Papers Paleontol. Museum Paleontol. Univ. Michigan 12, 39–48. Pflieger, W.L., 1971. A distributional study of Missouri fishes. Univ. Kansas Pub., Mus. Nat. Hist. 20, 225–570. Pflieger, W.L., 1996. The Crayfishes of Missouri. Missouri Department of Conservation, Jefferson City. Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Propst, D.L., Carlson, C.A., 1986. The distribution and status of warmwater fishes in the Platte River drainage, Colorado. Southwest Nat. 31, 149–167. Ray, J.M., Wood, R.M., Simons, A.M., 2006. Phylogeography and post-glacial colonization patterns of the rainbow darter, Etheostoma caeruleum (Teleostei: Percidae). J. Biogeogr. 33, 1550–1558. Robison, H.W., Buchanan, T.M., 1988. Fishes of Arkansas. University of Arkansas Press, Fayetteville. Roe, K.J., Mayden, R.L., Harris, P.M., 2008. Systematics and zoogeography of the rock basses (Centrarchidae: Ambloplites). Copeia 2008, 858–867. Ronquist, F., Huelsenbeck, J., 2003. Mrbayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rutter, N., Coronato, A., Helmens, K., Rabassa, J., Zárate, M., 2012. Glaciations in North and South America from the Miocene to the Last Glacial Maximum, Comparisons, Linkages and Uncertainties. Springer, Dordrecht. Schmidt, T.R., Bielawski, J.P., Gold, J.R., 1998. Molecular phylogenetics and evolution of the cytochrome b gene in the cyprinid genus Lythrurus (Actinopterygii: Cypriniformes). Copeia 1998, 14–22. Schönhuth, S., Mayden, R.L., 2010. Phylogenetic relationships of the genus Cyprinella based on mitochondrial and nuclear gene sequences. Mol. Phylogenet. Evol. 55, 77–98. Simons, A.M., 2004. Phylogenetic relationships in the genus Erimystax (Actinopterygii: Cyprinidae) based on the cytochrome b gene. Copeia 2004, 351–356. Strange, R.M., Burr, B.M., 1997. Intraspecific phylogeography of North American highland fishes: a test of the Pleistocene vicariance hypothesis. Evolution 51, 885–897. Switzer, J.F., Wood, R.M., 2002. Molecular systematics and historical biogeography of the Missouri saddled darter Etheostoma tetrazonum (Actinopterygii: Percidae). Copeia 2002, 450–455. Switzer, J.F., Wood, R.M., 2009. Etheostoma erythrozonum, a new species of darter (Teleostei: Percidae) from the Meramec River drainage, Missouri. Zootaxa 2095, 1–7. Swofford, D.L., 2001. PAUP⁄ Vers. 4.0b Phylogenetic analysis using parsimony (and other methods). Sinauer Associates, Sunderland, Massachusetts. Tamura, K., Peterson, S.G., Filipski, D., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 12, 2725–2729. Unmack, P.J., Bagley, J.C., Adams, M., Hammer, M.P., Johnson, J.B., 2012. Molecular phylogeny and phylogeography of the Australian freshwater fish genus Galaxiella, with an emphasis on dwarf galaxias (G. pusilla). PLoS ONE 7, e38433. http://dx.doi.org/10.1371/journal.pone.0038433. Unmack, P.J., Dowling, T.E., Laitinen, N.J., Secor, C.L., Mayden, R.L., Shiozawa, D.K., Smith, G.R., 2014. Influence of introgression and geological processes on phylogenetic relationships of western North American mountain suckers (Pantosteus, Catostomidae). PLoS ONE 9. http://dx.doi.org/10.1371/ journal.pone.0090061. Welsh, S.A., Cincotta, D.A., Starnes, W.C., 2013. First Records of Nocomis biguttatus (Hornyhead Chub) from West Virginia Discovered in Museum Voucher Specimens. Northeast Nat. 20, 19–22. Young, N.D., Healy, J., 2003. GapCoder automates the use of indel characters in phylogenetic analysis. BMC Bioinformatics 4, 6.