Timing of clade divergence and discordant estimates of genetic and ...

R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 3Fig. 1. Localities examined for all gene-based analyses of Noturus exilis. Localities examined for the morphological data comparisons are provided in Section 5.2.2.1. Genetic variationPairwise sequence divergences and average pairwise divergenceswithin and between recovered clades were generated usingMega v. 4 (Tamura et al., 2007) for both markers to examine patternsof sequence variation. Base compositional analysis was conductedto determine the average frequency of bases in both datasets using PAUP 4.0b10 (Swofford, 2003).2.2.2. Gene tree reconstructionIndependent phylogenetic hypotheses were generated for eachdataset using the maximum parsimony criterion implemented inPAUP 4.0b10 (Swofford, 2003). The most parsimonious trees werefound using the heuristic search option and 100 random additionsequence replicates with the tree-bisection-reconnection optionof branch-swapping and save multiple trees, branches of lengthzero collapsed, and retention of minimal length trees options. Nodesupport was assessed using non-parametric bootstrap re-samplingprocedure with 1000 pseudoreplicates.Bayesian maximum likelihood analyses (BML) were used togenerate phylogenetic hypotheses for each dataset independentlyand for a concatenated data set that included both the Cyt b andRAG2 genes. In all BML analyses, each gene was partitioned bycodon position. The optimal model of sequence evolution foreach data partition was determined from 56 progressivelycomplex models tested in Modeltest v.3.7 (Posada and Crandall,1998). The selected model under the Akaike InformationCriterion (AIC) and its parameters were implemented in therespective BML analyses in Mr. Bayes 3.1.1 (Ronquist and Huelsenbeck,2003) using the LSET and APPLYTO commands. Modelparameter values were estimated for each partition independentlyusing the ULINK command. Four Markov chains with defaultpriors were used in all analyses and random trees wereused to start each chain. Runs consisted of 5 million generationsof Markov Chain Monte Carlo (MCMC) simulations. Four replicateruns were conducted to ensure the MCMC went through asufficient number of iterations to allow convergence in the estimationsof tree topology with the best maximum likelihood posteriorprobability. The burn-in of the MCMC analysis wasdetermined by graphically examining the ML scores at each ofthe sampled generations to find where values converged. Alltrees recorded prior to the burn-in were discarded. The posteriorprobability or frequency at which a clade occurred in theremaining trees was used as an indication of node support. Inall analyses, N. gyrinus (Cyt b GenBank No.: AY327295; RAG2GenBank No.: AY327090), N. flavus (Cyt b: AY327287; RAG2:AY327086), and N. insignis (Cyt b: AY327301; RAG2:AYDQ492400) were used as outgroups. In the Cyt b dataset, N.albater (AY327268), N. baileyi (AY327273), N. eleutherus(AY327278), N. hildebrandi (AY327298), N. miurus (AY327307),and N. stigmosus (AY327319) also were used as outgroups; andN. lachneri (AY327090) and N. leptocanthus (AY327094) also wereused as outgroups in the RAG2 analysis.Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 5and Rambaut 2007). Three fossil-determined minimum bound lognormalage estimates that represented prior distributions, and thebest-fit models of sequence evolution for the two-gene data setwere used to estimate the posterior probability density of divergencetimes.A separate alignment of the Cyt b + RAG2 sequences that includedthe same N. exilis, N. gyrinus, N. flavus, and N. insignis sequencesas in the combined phylogenetic analysis describedpreviously, plus two species of Ameiurus (A. brunniens; A. catus),two species of Ictalurus (I. furcatus; I. punctatus), and Cranoglanisbouderius (Cyt b: AF16879; RAG2: DQ492401), was generated. Cranoglanisis member of the family Cranoglanididae, which is sister tocatfishes of the family Ictaluridae (Sullivan et al., 2006; Lundberget al., 2007), to which Noturus, Ameiurus, and Ictalurus belong. Thisalignment was subjected to an independent BML analysis using thesame parameters as described previously for the two-gene analysisof the more limited data set to estimate a tree that included taxaencompassing selected fossils for estimating divergence times ofNoturus exilis clades and to serve as a target tree in the BEAST v.1.6.1 analyses.Node age constraints were set using lognormal priors with minimumage bounds determined from a subset of dated catfish fossilspreviously used by Lundberg (2007) to estimate lacantuniid catfishdivergence and by Hardman and Hardman (2008) to estimateAmeiurus (Bullhead Catfish) diversification rates, and two Noturusfossils (Lundberg, 1975) not used in previous studies. The minimumage (Ma) estimates for fossils used as calibration points were:(1) Astephus sp., 65 mya (lognormal prior mean and standard deviationof 1.1 and root prior offset of 65); (2) Ictalurus, 19 mya (lognormalprior mean and standard deviation of 1.0 and offset valueof 19) and (3) Noturus sp./N. furiosus, 2.6 mya (lognormal priormean and standard deviation of 1.0 and offset 2.6). Astephus sp. isthe oldest known ictalurid fossil (63–65 Ma; Lundberg, 1975) andwas applied to the node representing the divergence of Ictaluridaefollowing Hardman and Hardman (2008; Fig. 5, node 1). The Ictalurusfossil date (19 Ma; Lundberg 1975, 1992) was applied to thenode representing the Ictalurus clade, also following Hardmanand Hardman (2008; Fig. 5 node 2). The application of these fossilsand their corresponding dates as minimum age estimates for thedescribed nodes have been shown to be good predictors of catfishdivergence times (Lundberg et al., 2007; Hardman and Hardman,2008). The third calibration point is based on two fossils of Noturusdescribed from the Pliocene (Fig. 5, node 3; Lundberg et al., 2007).The use of 2.6 my as a minimum age estimate for the genus Noturuswas supported by estimates of Pliocene divergence times for otherspecies of Noturus (Egge and Simons, 2006). Other fossils wereavailable, but could not be placed phylogenetically given the estimatedphylogeny.The birth–death process speciation prior was used to estimatebranching rates and a total of four independent runs of 60 milliongenerations each were conducted in BEAST v. 1.6.1. Resulting logfiles were viewed in Tracer v. 1.4 (Drummond and Rambaut,2007) to examine marginal probabilities of each run to ensureconvergence of parameter values and estimated node ages andto ensure effective sample sizes (>1000). Samples recorded priorto stationarity were discarded as burn-in. LogCombiner v. 1.4.6was used to combine the resulting tree and log files of each run.TreeAnnotater v. 1.4.6 (Drummond and Rambaut, 2007) was usedto summarize the posterior probability density of the combinedtree and log files. FigTree v. 1.1 (Drummond and Rambaut,2007) was used to visualize the mean and 95% highest posteriorprobability density estimates of divergence times for Noturus exilisclades. A separate BEAST analysis was conducted without alignmentdata, using the same parameters as previously described,to determine the effect of the calibration priors on divergencetime estimates.2.4. Morphological variationExamination of morphological variation focused on the samegeographic regions and river systems as that examined for geneticvariation; however, several populations for which tissues for geneticdata were unavailable were included in the morphologicalcomparisons. These included extirpated populations from theGreen River (Ohio River) system and Kentucky River system (OhioRiver) and a possibly extirpated population from the Licking River(Ohio R.) of Kentucky. A list of the populations examined for morphologyis provided in Section 5.2.4.1. MorphometricsMeasurements were taken on 281 specimens from 58 localitiesdistributed across the range of the species. Variation in body shapewas assessed using a truss network (Bookstein et al. 1985) of16interlandmark distances and an additional 16 standard measurementsincluding: (1) dorsal-fin base length, (2) dorsal-fin insertionto adipose-fin insertion, (3) adipose-fin insertion to anal-fin insertion,(4) anal-fin base length, (5) anal-fin origin to pelvic-fin origin,(6) pelvic-fin origin to pectoral-fin origin, (7) dorsal-fin insertion topectoral-fin origin, (8) dorsal-fin insertion to pelvic-fin origin, (9)dorsal-fin insertion to anal-fin origin, (10) body depth at the posterioreye margin, (11) body width at the posterior eye margin(12)body depth at dorsal-fin origin, (13) body width at dorsal-fin origin,(14) body width at pectoral-fin origin, (15) body width atanal-fin insertion, (16) snout to posterior eye margin, (17) snoutto dorsal-fin insertion, (18) snout to adipose-fin insertion, (19)snout to anal-fin origin, (20) snout to pelvic-fin origin, (21) snoutto pectoral-fin origin, (22) inter-orbital distance, (23) head length,(24) dorsal-fin spine length, (25) pectoral-fin spine length, (26)predorsal length, (27) adipose-fin base length, (28) dorsal-finheight, measured from dorsal-fin origin to end of longest ray,(29) anal-fin height, measured from anal-fin origin to end of longestray (30) pectoral-fin height, measured from pectoral-fin originto end of longest ray (31) caudal-fin height, measured along medialray, and (32) standard length (SL). Measurements were made onthe left side of the body on specimens >35 mm SL. Unless notedotherwise, methods followed those described by Hubbs and Lagler(1958) or Taylor (1969).Measurements were analyzed using a multivariate PrincipalComponents Analysis (SYSTAT v.8.0) of log-transformed data. Thefirst principal component recovered was considered to be a sizecomponent and was not used in population comparisons. Variableswith high component loadings were considered potentially informativetaxonomically and to have contributed most to any separationamong taxa or populations in the PCA scatterplots.To further identify potentially diagnostic characters, ranges ofmeasurements standardized by standard length were alsocompared to identify populations with non-overlapping or marginally-overlappingranges. Measurements are presented as thousandthsof SL.2.4.2. PigmentationCharacteristics were described from 164 specimens and 34localities from across the range of the species. The width or depthof the distal black band was measured for caudal, anal and dorsalfins; measurements were made at the middle of the fin, and valueswere standardized for comparison by dividing the width or depthof the band by the respective fin height or length. Notes were madeon the distribution of black coloration in the fin (e.g., covering allrays, all rays except two, etc.). In the caudal fins of some individuals,the fin was black or dusky throughout and the exact width ofthe distal black band could not be determined. In such cases, finswere noted as ‘overall dusky’.Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 9Fig. 4. An 85% majority rule consensus phylogram of the post burn-in trees resulting from the Bayesian analysis of the Cytochrome b and RAG2 genes. Numbers on branchesreflect posterior probabilities. Site numbers used in Fig. 1 and Table 1 identify members of each clade, followed by the number of sequences from each site recovered withinthat clade (if more than one).Cytochrome b. With this notable exception, the general observationof conserved morphology among closely related species ofmadtom catfishes and the low levels observed among geneticallydivergent clades of N. exilis (Taylor, 1969; Egge and Simons,2006, 2009) indicate that morphology alone may commonly under-estimatediversity for this group of fishes.Given the estimated early Miocene to late Oligocene age of thegenus, the low-levels of morphological divergence in madtom catfishesis interesting considering the degree of morphological variationobserved for other similarly-aged groups of small-bodied,benthic, Central Highlands stream fishes such as darters (Percidae).The relatively low levels of morphological divergence observed inmadtom catfishes may be due, at least in part, to reduced levelsof selection acting on morphological features. For example, dartersrely on external morphological features such as color and fin shapefor mate identification, selection, and antagonistic displays for nestsites. Thus, considerable variation in external morphology,particularly in color and pigmentation pattern, is observed amongclosely related species as a result of sexual selection (Page, 1983).Catfishes, however, rely less on visual cues, using other senses suchas smell and taste to interact with the biotic and abiotic environmentand are primarily nocturnal (Lundberg, 1970).4.2. Timing of clade diversificationThe Central Highlands Vicariance Hypothesis (Mayden, 1988)predicts a widespread diverse pre-Pleistocene fish fauna andnumerous studies have observed patterns of diversity and cladeage estimates that are consistent with this hypothesis (e.g., Berendzenet al., 2008; Hollingsworth and Near, 2009; Keck and Near,2010). Based on fossil evidence the genus Noturus arose prior to thePleistocene (Lundberg, 1975), a result also supported herein by thelate Oligocene age estimate for the MRCA of the genus. Noturus exiliswas estimated to have originated in the Miocene, indicating thatPlease cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

10 R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxxFig. 5. Maxiumum credibility chronogram for catfish species estimated from the combined independent BEAST analyses using the mitochondrial cytochrome b and nuclearRAG2 genes. Bars show uncertainty in the estimated divergence with the length representing the 95% highest posterior density (HDP) of node ages. Numbers on nodeshighlight the phylogenetic placement of fossil calibrations described in the methods: (1) Astephus, 65 Ma; (2) Ictalurus, 19 Ma; and (3) Noturus, 2.6 Ma. Italicized numbers atnodes are mean node age estimates. Posterior probability and bootstrap support values for nodes are given in Figs. 2–4.Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 11Table 3Average between clade and within clade RAG2 sequence divergences. Cladescorrespond to those recovered in Figs. 2 and 4.Average between clade divergencesClade A (%) Clade B (%) Clade CClade A 0.00Clade B 0.17 0.09Clade C 0.23 0.16 0.16Average within cladedivergence (%)Table 4Divergence times of Noturus exilis reported as posterior mean ages in millions of years(mya). Ages estimated using the uncorrelated lognormal relaxed clock method.Credibility intervals for age estimates are given as 95% highest posterior density(HPD). Nodes (A–D) correspond to those in Fig. 6.Node Posterior mean age (mya) 95% HPDA 0.36 0.05, 1.00B 3.68 1.78, 6.13C 5.47 3.16, 8.73D 9.67 5.32, 14.93Fig. 6. Maxiumum credibility chronogram for Noturus exilis estimated from thecombined independent BEAST analyses using the mitochondrial cytochrome b andnuclear RAG2 genes. Italicized numbers at nodes are mean node ages. Bars showuncertainty in the estimated divergence with the length representing the 95%highest posterior density (HDP) of node ages. Age estimates and the 95% HPD forfocal nodes (A–D) are provided in Table 4. In the Neogene geologic time scale shown’Ps’ = Pleistocene, ’Pl’ = Pliocene, and ’Mi’ = Miocene (in part). Posterior probabilityand bootstrap support values for nodes are given in Figs. 2–4.both the genus and species lineages were part of the diverse pre-Pleistocene fish fauna, lending further support to Mayden’s(1988) Central Highlands Vicariance Hypothesis.Diversification within Noturus exilis occurred more recently,spanning the pre-Pleistocene–Pleistocene boundary. Hardy et al.(2002) argued the degree of genetic structure and divergence withinNoturus exilis arose before or very early in the Pleistocene. Usingthe poikilotherm mt DNA clock they estimated that N. exilis cladesdiverged 0.77–7.6 mya. Using multiple catfish fossils to calibraterates of molecular divergence, we find on average younger age estimatesfor clades of N. exilis, which had mean ages of 0.36–5.47. Theestimated age of Clade A, from the mid-Pleistocene is consistentwith recent diversification within the Central Highlands associatedwith events of the Pleistocene glacial cycles (Hocutt et al., 1986).Contradictory to predictions of the CHVH, Hocutt et al. (1986)hypothesized that the Pleistocene glacial cycles were the primarygenerator of the diversity and distribution of fishes in the CentralHighlands region and that this diversity was relatively young.Diversification of the two other clades (B and C) of N. exilis, however,occurred prior to the Pleistocene (Pliocene and Miocene),demonstrating that both ancient and recent events have playedprominent roles in the evolutionary history of the species.Divergence of the clade-rich and widespread Clade C (5.47 mya,95% HPD: 3.16, 8.73; Fig. 6) corresponds to a drastic drop in sea-leveloccurring at the Miocene–Pliocene boundary. Near et al. (2003)found a period of rapid diversification in Micropterus and Nagle andSimons (2012) observed divergence among the eastern and westernclades of Nocomis (Cyprinidae) associated with this drop insea-level. The congruent timing of diversification among these distantlyrelated groups of fishes indicates that common geologicalevents or climate shifts likely influenced their diversification.Although, the estimated diversification time of genus Noturus(Miocene to late Oligocene) is younger than the estimate recoveredby Hardman and Hardman (2008; late Eocene–early Oligocene), itsupports their speculation of climate change of the Oligocene andMiocene potentially playing a more prominent role in North Americanfish species diversification than the Eocene–Oligocene climate-shift.Near et al. (2005) emphasized the impact of thedramatic cooling event of the latter on North American freshwaterfishes in general, and particularly for the Centrarchidae (basses andsunfishes) for which the age of the root node was estimated at theEocene–Oligocene boundary. However, Hardman and Hardman(2008) noted that most of the extant genera of Centrarchidae andmuch of the diversification within Ictaluridae was Oligocene to Pliocenein age. The latter was also observed based on the clade agesestimated herein for diversification within each of the catfish genera(Ictalurus, Ameiurus, and Noturus) examined. DiversificationPlease cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

12 R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxxTable 5Frequency distribution of preopercular mandibular pores (POM) for Noturus exilisfrom clades A–C of Figs. 2 and 4. Numbers in bold highlight modal POM counts foreach clade.Species/clade POM pores # Ind. Mean9 10 11Noturus exilis (Clade A) 7 50 57 10.9Noturus exilis (Clades B + C) 1 101 2 104 10.0Fig. 7. Scatterplot of factor scores generated from the Principle ComponentsAnalysis of all morphometric characters and individuals of Noturus exilis examined.Individuals/populations are grouped by clades as recovered in Figs. 2 and 4.Component loadings for variables measured are provided in the text.among genera and for the family predated the Eocene–Oligocenecooling event, indicating that, as suggested by Hardman and Hardman(2008), this event had minimal impact on North Americancatfish divergence and that climate shifts or geological events, suchas the warming event of the Oligocene or sea level fluctuations ofthe Miocene–Pliocene, likely played a more influential role in theirdiversification.4.3. Phylogeographic relationshipsGeographic variation in N. exilis displays a number of interestingbiogeographic features. Data from both Cyt b and RAG2 indicatethat populations in the Illinois and Skunk river systems in the glaciatedregions of Illinois and Iowa are more closely related to thosein the Ozarks, particularly the Missouri River and its tributaries andthe St. Francis River, than they are to populations in the EasternHighlands. This suggests that northern populations are the resultof dispersal from populations in the Ozarks up the Mississippi Riverfollowing retreat of the Pleistocene glaciers, which is furthersupported by the estimated mid-Pleistocene (mean nodeage = 0.57, 95% HDP: 0.24–1.05) diversification for this clade of N.exilis (Fig. 6). This finding is consistent with the Leading Edge Modelof population expansion (Hewitt, 1996), which states that aquatictaxa in northern glaciated portions of North America arecomprised of lineages persisting and/or differentiating in southernrefugia during the Pleistocene. These lineages then expandednorthward into newly available habitats as glaciers receded. Similarresults have been found for other taxa in the upper MississippiRiver system, including Etheostoma caeruleum, Percina evides, andNoturus flavus (Near et al., 2001; Ray et al., 2006; Faber et al.,2009). Bay Creek, a tributary of the Ohio River in southern Illinois,also seems to have been populated by Ozarkian fishes, rather thanby invasions from closer tributaries of the Ohio River, viz., the lowerTennessee and Cumberland rivers.The phylogeographic position of Clear Creek, a small direct tributaryof the Mississippi River in southern Illinois and adjacent toBay Creek, was of particular interest. Hardy et al. (2002) foundshared haplotypes between the Clear Creek system and the LittleRed River (White River) of Arkansas. Although our sample sizeswere smaller, we found no shared haplotypes between these regionsand both the Cyt b and RAG2 data indicate that the LittleRed River population is genetically distinct and divergent fromall others examined. As with Bay Creek, Clear Creek is allied withOzarkian fishes, but those from the Missouri and St. Francis riversand not the White River system as previously proposed.The Cyt b data suggest that populations of Noturus exilis in theBlack River system (Current, Black, and Strawberry rivers) of thelower White River drainage are more closely related to populationsin the Eastern Highlands, Missouri River drainage, and previouslyglaciated regions than they are to those in the upper White riversystem. The RAG2 data did not resolve these relationships, andwe know of no other fish that shows this exact pattern. However,distributions of other upland Ozarkian fishes do support a phylogeographicbreak between the upper White River and the Black River(lower White River). For example, populations of the RainbowDarter, Etheostoma caeruleum, from the Black River system aremore closely related to those from the middle and lower Mississippiriver systems (e.g., Meramac and St. Francis) than to theupper White River system (Ray et al., 2006). The relationship ofthe Black River clade to the clades containing Eastern Highlandsand Missouri River populations was unresolved. Hellbenders, Cryptobranchusalleganiensis, from the upper White River system aremore closely related to those from the Tennessee River of the EasternHighlands and Meramac and Missouri Rivers than to populationsfrom the Black River (e.g., Current and Eleven Point rivers;Routman et al., 1994). In Noturus exilis, the Black River system is alliedwith those from the Eastern Highlands and Missouri River systems.Other madtom catfishes (N. albater and N. maydeni; Egge andSimons, 2006), minnows (Cyprinidae: Luxilus; Mayden, 1988) andbasses (Centrarchidae: Amploblites; Cashner and Suttkus, 1977)also support a Black-White River vicariance.The phylogeographic break observed in many taxa between theupper and lower White River system is often attributed to Mayden’s(1988) pre-Pleistocene drainage hypothesis that states thatthe Mississippi River flowed through the area that is now the BlackRiver. Under this historical drainage pattern scenario, populationsof fishes in each of the Black River system tributaries would havebeen isolated from each other and from the upper White River systemand the other major lower White River tributary, the Little RedRiver system. Hardy et al. (2002) suggested that the observed phylogeneticdivergence between the Black River and upper WhiteRiver was likely due to pre-Pleistocene separation of these drainages,as noted by Mayden (1988). The pattern and timing of cladedivergence observed herein for the clades of N. exilis from theWhite River system also are congruent with this aspect of Mayden’s(1988) pre-Pleistocene drainage hypothesis. Noturus exilisfrom the upper and lower White River (excluding the Little RedRiver of Clade A) were recovered as divergent sub-clades withinthe larger Clade C, which had a diversification age estimate of5.47 mya (95% HPD 3.16, 8.73). This result supports the hypothesisof vicariance associated with pre-Pliestocene drainage patternshifts leading to the observed genetic divergence among the upperand lower White River system populations of N. exilis.Although, the same general phylogeographic break in the WhiteRiver system is observed across multiple taxa, the timing of thebreak has only been estimated for the sister species Noturus albaterfrom the upper White River and Little Red River system, and N.Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 13maydeni from the Black River system (lower White River). The estimateddivergence time for these species is 2.7–3.4 mya (Egge andSimons, 2006). Although calculated under different methods (usingthe cyprinid molecular clock of Dowling et al. (2002)) this impliesisolation leading to divergence of these species was younger thanthat inferred for Noturus exilis. Estimates for the timing of diversificationfor other species that show this geographic break have notbeen generated. Thus, it is unclear if other species are consistentwith the timing of divergence estimated for the Slender Madtom.Given the two different estimates for catfish divergence, it is likelythat the commonly observed pattern of divergence among populationsfrom the upper White River and those from the Black Riversystem, shared by many taxa, is not associated with a single vicariantevent, but may be better explained by vicariance associatedwith multiple of persistent events through time. This finding supportsprevious studies that caution against reliance of shared patternsof clade divergence among taxa, in the absence of temporaldata, to infer historical processes responsible for contemporarypatterns of species or clade diversity (Donoghue and Moore,2003; Keck and Near 2005).At a larger, more general scale, disparity is seen in clade agesestimated for Noturus exilis and those estimated for other small upland,benthic fishes with similar geographic patterns of diversity inthe Central Highlands. For example, populations of the CorrugatedDarter, E. basilare, which is restricted to a small portion of the CumberlandRiver drainage in the Eastern Highlands, diverged between2–8 mya (Hollingsworth and Near, 2009). Although the estimateddivergence time for all Eastern Highland populations (all withinClade C) of N. exilis (at 5.47 mya), is comparable to that estimatedfor E. basilare, divergence among Cumberland River populations(middle versus lower Cumberland River) of N. exilis, which are geographicallymore widespread than populations of the CorrugatedDarter, was estimated at only 2.5 mya. Such disparity in clade agesfor fishes with similar habitat preferences and geographic rangesprovides further support that multiple events or persistent historicalprocesses, rather than a single shared vicariant event, may oftenbest explain shared biogeographic patterns (Donoghue andMoore, 2003).5. Materials examined5.1. MorphologyNumbers in parentheses are numbers examined for measurements,meristics, and pigmentation, respectively.5.1.1. Mississippi River basin: Arkansas river drainageAR: Pope Co.: McCoy Creek TU 182899 (10); Van Buren Co.:Cadron Creek TU 188831 (5, 5, 5); Washington Co.: Illinois RiverUAIC 12550.09 (5, 5, 5); Yell Co.: Fourche LaFave River TU 98015(6). KS: Cherokee Co.: Spring River-Neosho River UAIC 10460.09(14), USNM 172051 (1). OK: Adair Co.: Tyner Creek UAIC2758.01 (5); Adair-Sequohay Co. Line: Little Lee Creek TU 2254(4); Delaware Co.: Flint Creek TU188767 (10). Illinois RiverDrainage: IL: Bureau Co.: East Bureau Creek INHS 55800 (1, 1,1); McClean Co.: Buck Creek-Mackinaw River INHS 85850 (4);LaSalle Co.: Buck Creek-Fox River INHS 88189 (3). Missouri RiverDrainage: KS: Wabaunsee Co.: Kansas River UAIC 7999.10 (4, 4,4). MO: Dallas Co.: Greasy Creek-Osage River UAIC 10104.09(4); Texas Co.: Piney River-Gasconade River TU 57612 (2), UF172043 (2), USNM 201394 (1, 1, 1); Warren Co.: Massie CreekUAIC 7995.05 (3); Webster Co.: Niangua River USNM 244959 (2,2, 2). Ohio River Drainage: Cumberland River: KY: Trigg Co.: DonaldsonCreek UF 167337 (5, 5, 5); TN: Montgomery Co.: PineyFork-Red River UF 167327 (5, 5, 5); Williamson Co.: MayesCreek-Harpeth River UAIC 13633.02 (3, 3, 3). Green River: KY:Adair Co.: Green River SIUC 61238 (5, 5, 5); Grayson Co.: NolinRiver SIUC (uncataloged SIUC lot, collected 7 July 1959, 4, 4, 4).Kentucky River: KY: Grant Co.: Eagle Creek SIUC 66647 (5, 5, 5).Licking River: KY: Pendleton Co.: South Fork Licking River SIUC19120 (2, 2, 2). Tennessee River: AL: Limestone Co.: Piney CreekUAIC 13287.03 (5, 5, 5); Swan Creek UAIC 13289.03 (5, 5, 5);TN: Coffee Co.: Duck River TU 30304 (4, 4, 4); Lewis Co.: GrindersCreek-Buffalo River UF 172084 (5, 0, 5); Wayne Co.: StokesBranch-Duck River UF 172062 (5, 0, 5); Wayne Co.: Hog CreekUF 172003 (3, 0, 3). Direct Ohio River Tributaries: IL: Pope Co.:Bay Creek UF 167309 (5, 5, 5). Red River Drainage: AR: PolkCo.: Two Mile Creek TU 48515 (8). White River Drainage: BlackRiver: AR: Fulton Co.: South Fork Spring River TU 188810 (8);MO: Shannon Co.: Jacks Fork Current River UAIC 1077.05 (1);Texas Co.: South Prong Current River UF 172072 (7, 0, 5). BuffaloRiver: AR: Baxter Co.: Buffalo River TU 49703 (1). James River:MO: Christian Co.: Finley Creek USNM 263606 (2). Little Red River:AR: Stone Co.: Middle Fork Little Red River UAIC 11370.08 (2, 2,2); Van Buren Co.: Archey Fork UAIC 11368.06 (2, 2,), TU187869 (5, 31, 5); Hartsugg Creek TU 182855 (22, 22). North ForkWhite River: MO: Douglas Co.: Indian Creek UF 171996 (1, 0, 1);Ozark Co.: North Fork White River UAIC 118680.1 (3, 3, 3). UpperWhite River: AR: Benton Co.: War Eagle Creek UAIC 12596.12 (2,2, 2); White River TU 50018 (5). West Fork White River: AR: WashingtonCo.: Hutchins Creek USNM 201395 (6). Other MississippiRiver Systems or Tributaries: Clear Creek: IL:Union Co.: ClearCreek UF 167389 (5, 5, 5); Hutchins Creek UF 172053 (8). MeramecRiver: MO: Franklin Co.: Bourbeuse River UAIC 10321.11 (4,4, 4); Washington Co.: Big River SIUC 25996 (5, 5, 5). Rock River:IL: Ogle Co.: Leaf River INHS 54181 (9). Pecatonica River: IL:Winnebago Co.: Grove Creek Braasch & Smith 1962 (uncataloged,5). Skunk River: IA: Story Co.: Bear Creek UF 170791 (3, 3, 3),170792 (5, 5, 5); Long Dick Creek UF 170793 (3, 3, 3). South HendersonCreek: IL: Henderson Co.: South Henderson Creek INHS90619 (2, 2, 2). St. Francis River: MO: Madison Co.: St. Francis RiverSIUC 28424 (1, 1, 1); Rock Creek SIUC 34926 (5, 5, 5), UF172035 (4).5.2. MolecularMaterials examined arranged by gene and locality number usedin Table 1 and Fig. 1. Following locality numbers are tissue vouchernumber, followed by specimen voucher number in parentheses (ifavailable). Non-institutional abbreviations used are as follows:AMS are collections by A.M. Simons and JJDE are collections byJ.J.D. Egge, loaned through the Bell Museum (University of Minnesota);and NJL are collections by N.J. Lang loaned through the Universityof St. Louis.5.2.1. Cytochrome bSite 1: AMS02-01-2, AMS 02-01-3; Site 2: KU 630, 631 (KU23543); Site 3: KU 7892, 7907 (KU 38695); Site 4: FLMNH 2008-0238, 2008-0239 (UF 172043); Site 5: FLMNH 2008-0228, 2008-0229, 2008-0230, 2008-0231, 2008-0232 (UF 172072); Site 6:NAFF 1241 (STL 834.01); Site 7: AMS 02-17-3, 02-17-4, 02-16-1,02-16-2; Site 8: AMS02-04-1, 02-04-2; Site 9: FLMNH 2008-0234, 2008-0235, 2008-0236 (UF 171996); Site: 10: AMS 02-05-1, 02-05-2; Site 11: JJDE 03-13-3, 03-13-4; Site 12: FLMNH 2008-0223, 2008-0224, 2008-0225, 2008-0226 (UF 172035); Site 13:AMS 99-34-1; Site 14: NAFF 1234 (STL 272.04); Site 15: KU8104, 8105 (KU 38748); Site 16: STL 271.02; Site 17: NAFF 1242,1243, 1244 (STL 271.02); Site 18: NAFF 1249 (STL 400.2); Site19: NAFF 1235 (STL 268.01); Site 20: AMS 01-06-1, 01-06-2,JJDE03-11-1, 03-11-2; Site 21: JJDE 03-10-1, 03-10-2; Site 22:NAFF 1240 (STL 458.01); Site 23: FLMNH 2007-0445, 2007-0448,Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022

14 R.E. Blanton et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx2007-0450 (UF 167327); Site 24: FLMNH 2007-0329, 2007-0331,2007-0339 (UF 167337); Site 26: YFTC 7168; Site 27: AMS 02-32-1, 02-32-3; Site 28: NJL07-24; Site 29: FLMNH 2008-0172,2008-0173, 2008-0174 (UF 172062); Site 30: FLMNH 2008-0212,2008-0218, 2008-0221, 2008-0222 (UF 172084); Site 31: FLMNH2008-0186, 2008-0187 (UF 172003); Site 32: FLMNH 2007-0420,2007-0421 (UF 167309); Site 33: FLMNH 2008-0240, 0242, 0242,0243, 0244 (UF 172053); Site 34: FLMNH 2007-0481, 2007-0482(UF 167389); Site 35 INHS 101227-1, 101227-2 (INHS 101227);Site 36: FLMNH 2007-0513, 2007-0514 (UF 17091); Site 37:FLMNH 2007-0515, 2007-0516, 2007-0517 (UF 170792); Site 38:FLMNH 2007-0520, 2007-0521, 2007-0522 (UF 170793); Site 39:AMS 02-22-1, 02-22-2; Site: 40: AMS 02-23-4.5.2.2. RAG2Site 1: AMS02-01-1; Site 2: KU 631 (KU 23543); Site 3: KU 7892,7907 (KU 38695); Site 4: FLMNH 2008-0238, 0239 (UF 172043);Site 5: FLMNH 2008-0228, 0229 (UF 172072); Site 6: NAFF 1241(STL 834.01); Site 7: AMS 02-17-3, 02-17-3-4, 02-16-1, 02-16-2;Site 8: AMS02-04-1, 02-04-2; Site 9: FLMNH 2008-0234, 0235(UF 171996); Site 10: AMS 02-05-1, 02-05-2; Site 11: JJDE 03-13-3; Site 12: FLMNH 2008-0223, 2008-0224 (UF 172035); Site 13:AMS 99-34-1; Site 14: NAFF 1234 (STL 272.04); Site 15: KU8104, 8105 (KU 38748); Site 16: STL 271.02; Site 17: NAFF 1243(STL 271.02); Site 18: NAFF 1249 (STL 400.2); Site 19: NAFF 1235(STL 268.01); Site 20: AMS 01-06-1, JJDE03-11-1; Site 21: JJDE03-10-1; Site 22: NAFF 1240 (STL 458.01); Site 23: FLMNH 2007-0445 (UF 167327); Site 24: FLMNH 2007-0339 (UF 167337); Site25: YFTC 5512; Site 26: YFTC 7168; Site 27: AMS 02-32-1, 02-32-3; Site 28: NJL07-24; Site 29: FLMNH 2008-0172, 2008-0173 (UF172062); Site 30: FLMNH 2008-0212, 2008-0218 (UF 172084); Site31: FLMNH 2008-0186, 2008-0187 (UF172003); Site 32: FLMNH2007-0420, 2007-0421, 2007-0422 (UF 167309); Site 33: FLMNH2008-0240, 0242 (UF 172053); Site 34: FLMNH 2007-0481 (UF167389); Site 35: INHS 101227-1 (INHS 101227); Site 37: FLMNH2007-0515 (UF 170792); Site 39: AMS 02-22-1, 02-22-2; Site 40:AMS 02-23-4; Site 41: YFTC 2923.AcknowledgmentsWe thank B.M. Burr, M. Compton, J. Johansen, N. Lang, M. Thomas,and A. Thomson for assistance collecting specimens, M. Thomasfor photographs of N. exilis, G. Paulay for lab space andequipment, J. Grosso and G. Sheehy for distribution maps, andthe numerous institutions and individuals noted in the MaterialsExamined that provided tissues and specimens. This study wassupported by the All Catfish Species Inventory funded by the NationalScience Foundation (DEB 0315963). Collection of specimensoccurred under state permits to LMP including: Arkansas Permit123020081, Kentucky Permit SC0911011, Missouri Permit 14002,and Tennessee Permit 809.Appendix A. Supplementary materialSupplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2012.10.022.ReferencesAvise, J.C., 2000. Phylogeography: The History and Formation of Species. HarvardUniversity Press, Cambridge, MA.Baric, S., Sturmbauer, C., 1999. 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Rapid allopatric speciation in logperch darters(Percidae: Percina). Evolution 58, 2798–2808.Please cite this article in press as: Blanton, R.E., et al. Timing of clade divergence and discordant estimates of genetic and morphological diversity in theSlender Madtom, Noturus exilis (Ictaluridae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.10.022