Submitted 10 October 2019Accepted 27 March 2020Published 4 May 2020

Corresponding authorKen A Sterlingkennethasterlingusdagov

Academic editorJason Bond

Additional Information andDeclarations can be found onpage 14

DOI 107717peerj9014

Distributed underCreative Commons PublicDomain Dedication

OPEN ACCESS

Cryptic diversity among Yazoo Darters(Percidae Etheostoma raneyi) in disjunctwatersheds of northern MississippiKen A Sterling1 Stuart V Nielsen2 Andrew J Brown3 Melvin L Warren Jr1

and Brice P Noonan4

1USDA Forest Service Southern Research Station Stream Ecology Laboratory Oxford MSUnited States of America

2Division of Herpetology Florida Museum of Natural History Gainesville FL United States of America3 Louisiana Purchase Gardens and Zoo Monroe LA United States of America4Department of Biology University of Mississippi University MS United States of America

ABSTRACTThe Yazoo Darter Etheostoma raneyi (Percidae) is an imperiled freshwater fish speciesendemic to tributaries of the Yocona and Little Tallahatchie rivers of the upper YazooRiver basin in northern Mississippi USA The two populations are allopatric isolatedby unsuitable lowland habitat between the two river drainages Relevant literaturesuggests that populations in the Yocona River represent an undescribed species but alack of data prevents a thorough evaluation of possible diversity throughout the rangeof the species Our goals were to estimate phylogenetic relationships of the YazooDarteracross its distribution and identify cryptic diversity for conservation managementpurposes Maximum likelihood (ML) phylogenetic analyses of the mitochondrialcytochrome b (cytb) gene returned two reciprocally monophyletic clades representingthe two river drainages with high support Bayesian analysis of cytb was consistentwith the ML analysis but with low support for the Yocona River clade Analyses ofthe nuclear S7 gene yielded unresolved relationships among individuals in the LittleTallahatchie River drainagewithmostly low support but returned amonophyletic cladefor individuals from the Yocona River drainage with high support No haplotypes wereshared between the drainages for either gene Additional cryptic diversity within the twodrainages was not indicated Estimated divergence between Yazoo Darters in the twodrainages occurred during the Pleistocene (lt1 million years ago) and was likely linkedto repeated spatial shifts in suitable habitat and changes in watershed configurationsduring glacial cycles Individuals from the Yocona River drainage had lower geneticdiversity consistent with the literature Our results indicate that Yazoo Darters in theYocona River drainage are genetically distinct and that there is support for recognizingYazoo Darter populations in the Yocona River drainage as a new species under theunified species concept

Subjects Conservation Biology Evolutionary Studies Freshwater BiologyKeywords Cryptic diversity Phylogenetics Recent divergence Yazoo Darter Etheostoma

INTRODUCTIONThe southeastern United States has a globally significant amount of diversity among itsfreshwater fishes (Abell et al 2008) A large portion of this diversity is contained within

How to cite this article Sterling KA Nielsen SV Brown AJ Warren Jr ML Noonan BP 2020 Cryptic diversity among Yazoo Darters(Percidae Etheostoma raneyi) in disjunct watersheds of northern Mississippi PeerJ 8e9014 httpdoiorg107717peerj9014

Etheostomatinae (Percidae) the darters (Jelks et al 2008 Page amp Burr 2011) Thoughthe group shows a wide variety of life history strategies and associated distributionalpatterns (Fluker Kuhajda amp Harris 2014) many species of darters are range-limited(microendemics) (Page 1983 Page amp Burr 2011) and share a suite of life historycharacteristics that are associated with limited dispersal (Turner amp Trexler 1998 Turner2001) including niche conservatism (Keck amp Near 2010) The discovery ofmicroendemismin darters is occurring more frequently because at least in part the routine use of genetictools is increasingly uncovering cryptic diversity (Hollingsworth Jr amp Near 2009 April etal 2011 Echelle et al 2015 Kozal et al 2017Matthews amp Turner 2019)

The Yazoo Darter (Etheostoma raneyi Suttkus and Bart 1994) is a snubnose darter (cladeAdonia sensu Near et al 2011) distributed in the upper Yazoo River basin in north-centralMississippi (Figs 1 and 2 Figs S1ndashS3) Surface geology mostly comprises highly erodibleunconsolidated sands and clays with resulting fine substrates within streams Topography isrelatively flat compared with upland regions but is more variable compared with the LowerGulf Coastal Plain and Mississippi Alluvial Plain to the west (Ross 2001 Keck amp Etnier2005 Powers amp Warren Jr 2009) (Fig 1) Yazoo Darters occur in headwater tributariesof the Little Tallahatchie (LTR) and Yocona (YR) rivers whose confluence lies inbottomland habitat of the Mississippi Alluvial Plain which is unfavorable for the darterIn common with other snubnose darters Yazoo Darters are small (lt65 mm StandardLength) benthic insectivores lacking a swim bladder (Page 1983 Johnston amp Haag 1996Sterling Warren Jr amp Henderson 2013) Long distance movements for spawning or feedingare not documented for snubnose darter species Larvae of snubnose darters includingthe Yazoo Darter are pelagic but active swimmers upon hatching and select for shelteredareas out of direct current immediately downstream of spawning areas passive drift oflarvae is not documented (Simon ampWallus 2006 Ruble Sterling amp Warren Jr 2019) Apopulation genetic study of the Yazoo Darter using microsatellite data indicated limitedhistorical dispersal among tributary streams and virtually no contemporary dispersal likelybecause of anthropogenic habitat destruction (Warren Jr Haag amp Adams 2002 Sterlinget al 2012) Genetic structure was high across small spatial scales among some tributarypopulations (Fst = 003ndash017) within each major drainage where the species occurs (LTRand YR) and was also high between drainages (Fst = 017ndash029) (Sterling et al 2012)

A phylogenetic analysis of Upper Gulf Coastal Plain snubnose darters (Etheostomapyrrhogaster E cervus and E raneyi see Fig 1) in western Kentucky Tennessee andnorthern Mississippi indicated that Yazoo Darters inhabiting the LTR and YR weregenetically distinct and reciprocally monophyletic with high posterior support Powers ampWarren Jr (2009) suggested that the same vicariant events isolated all forms of darters theyexamined in the Upper Gulf Coastal Plain However the study was limited to six YazooDarters from only a few streams in each drainage (n= 12) (Powers amp Warren Jr 2009)

The Yazoo Darter is categorized as vulnerable by the American Fisheries Society (Jelks etal 2008) and the Southeastern Fishes Council (Warren Jr et al 2000) as globally imperiledby the Nature Conservancy (NatureServe 2019) as sensitive by the USDA Forest Service(USDA Forest Service 2013) and as a Tier 1 species of greatest conservation need bythe Mississippi State Wildlife Action Plan (Mississippi Museum of Natural Science 2015)

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Figure 1 Distribution of snubnose darters among lower Mississippi River drainages of Kentucky Ten-nessee andMississippi (southeastern United States)Major river systems and physiographic provincesdiscussed in the text are shown abbreviations are defined as LT= Little Tallahatchie MAP=Missis-sippi Alluvial Plain LGCP= Lower Gulf Coastal Plain UGCP= Upper Gulf Coastal Plain

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Human-assisted gene flow among tributaries within each drainage was recommended as aconservationmanagement action (Sterling et al 2012) Even so an investigation of possiblecryptic diversity across the speciesrsquo distribution within each drainage as well as estimatesof genetic structure using markers reflecting deeper evolutionary relationships is neededto better inform such an action We used genetic sequences from mitochondrial cytb and

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Figure 2 Map showing genetic tissue sample sites for Yazoo Darters in the Little Tallahatchie Riverdrainage (blue) and Yocona River drainage (red)Names of watersheds used for genetic distance esti-mates (see Table 3) and discussed in the text are also shown Numbers correspond to data in Table 1 YR= Yocona River LTR= Little Tallahatchie River

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nuclear S7 genes to (1) investigate possible cryptic diversity within and between eachmajor drainage (2) estimate phylogenetic relationships among populations within eachdrainage to inform discussion of human-assisted gene flow for conservation managementand (3) to assess the results from Powers amp Warren Jr (2009) using larger sample sizes fromsites across the distribution of the species

MATERIAL AND METHODSWe sampled 117 individuals from 20 streams representative of the entire range of the YazooDarter via single-pass backpack electrofishing dip nets and seines Collecting localitiesincluded nine streams in the YR drainage and 11 streams in the LTR drainage (Fig 2Table 1 Tables S1 and S2) We obtained tissue samples by either taking pelvic fin clips or bycollecting voucher specimens which we stored in 95 ethanol at minus74 C This study wasconducted with the approval of the University of Mississippi IACUC Committee (protocol09-027) using annual collection permits issued to us from the Mississippi Museum ofNatural Science (2009ndash2017 0604091 0513101 0624112 0622122 0602132 06101420624151 0715163 1010173)

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Table 1 Genetic tissue sample data for each of the twomajor drainages within the distribution of the Yazoo Darter Sample locations drainageand sample sizes for genetic analyses are shown Site ID numbers correspond to Fig 2 see Tables S1 and S2 UT unnamed tributary

Site ID Drainage Stream Cytb n S7 n Latitude Longitude

1 Yocona River Pumpkin Creek 4 1 34327 minus893982 Yocona River Yellow Leaf Creek 2 1 34348 minus894553 Yocona River Morris Creek 4 4 34283 minus895444 Yocona River Taylor Creek 5 5 34293 minus895895 Yocona River Splinter Creek 3 4 34251 minus896426 Yocona River Mill Creek 6 6 34167 minus89527 Yocona River Gordon Branch 3 2 3414 minus895498 Yocona River UT Otoucalofa Creek 4 3 34125 minus896119 Yocona River Johnston Creek 6 6 34124 minus8964110 Little Tallahatchie River Big Spring Creek 10 13 34664 minus8941311 Little Tallahatchie River GrahamMill Creek 3 3 34503 minus8949112 Little Tallahatchie River Hurricane Creek 2 3 34425 minus8949613 Little Tallahatchie River Deer Creek 6 6 34316 minus8978514 Little Tallahatchie River Yellow Rabbit Creek 5 4 34819 minus8910615 Little Tallahatchie River Chilli Creek 5 4 34682 minus8917316 Little Tallahatchie River UT Tippah River 2 2 34709 minus8925617 Little Tallahatchie River Chewalla Creek 4 3 34725 minus8930518 Little Tallahatchie River Cypress Creek 7 6 34382 minus8929819 Little Tallahatchie River Puskus Creek 12 6 34443 minus8934120 Little Tallahatchie River Bay Springs Branch 2 1 34429 minus89396

We isolated whole genomic DNA (MacManes 2013) and used previously publishedPCR primers to amplify the entire mitochondrial cytb gene (1140 bp Song Near amp Page1998) and the forward sequence of intron 1 of the nuclear S7 ribosomal gene (599 bpChow amp Hazama 1998) PCR components were as follows 98 microl ddH2O 02 microl dNTP04 microl MgCl2 2 microl 5x reaction buffer 02 microl each 10 nM primer 015 microl PhireTM Taqand 15 microl of template DNA (sim15 microl total reaction volume) We set conditions for PCRreactions as 98 C for 30 s followed by 30 cycles of 98 C for 6 s 531minus56 C for 30 s and72 C for 60 s We purified and sequenced PCR products using ExoSAP-IT (ThermoFisherScientific) and Big Dye (ver 31 ThermoFisher Scientific) according to manufacturerrsquosrecommendations Arizona State University DNA sequencing facility processed thesamples (httpsasucorefacilitiesorgservice_centershow_external3900asu-dna-lab)using an automated ABI 3730 sequencer We assembled all resulting forward and reversesequences into contigs for each individual and aligned them using MEGA (ver 7026Kumar Stecher amp Tamura 2016) We obtained outgroup sequence data and sequencesfor two additional Yazoo Darters (Near et al 2011) one from each major drainage fromGenBank for use in our analyses Sequence data for this study are available from GenBank(httpswwwncbinlmnihgovnuccore) and Dryad (Kozal et al 2017) (Tables S1ndashS3)

Data from cytb and S7 could not be combined into a single concatenated analysisbecause the data were not derived from the same set of individuals (Matthews amp Turner2019) We used PartitionFinder V 111 (Lanfear et al 2012) to find the best-fit model for

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each locus The cytb dataset was partitioned by 1st 2nd and 3rd codon positions andthe S7 dataset was analyzed as a single partition We analyzed partitioned datasets foreach gene (Tables S1 and S2) using Bayesian Inference (BI) implemented in MrBayes ver326 (Ronquist et al 2012) via CIPRES Science Gateway ver 33 (httpswwwphyloorg)(Miller Pfeiffer amp Schwartz 2010) Each partitionanalysis included the most appropriatesubstitution models for the two loci as suggested by PartitionFinder We used two runsof MrBayes for 106 generations four Markov chains sampled every 10000 steps andTracer (ver 171 Rambaut et al 2018) removed 25 of the posterior trees as burn-in We then generated a 50 majority rule consensus tree in MrBayes We used thesame data to construct Maximum Likelihood (ML) trees using RAxML-HPC ver 80(httpscmeh-itsorgexelixiswebsoftwareraxml) (Stamatakis 2014) also using theCIPRES Science Gateway ver 33 (Miller Pfeiffer amp Schwartz 2010) We used the defaultGTRmodel and performed 100 bootstrap replicates to assess nodal support We considerednodes with posterior probabilities ge95 as strongly supported (Huelsenbeck amp Ronquist2001)

We visualized relationships among individuals using haplotype networks (TCS v 121Clement Posada amp Crandall 2000) for each gene We estimated uncorrected pairwisegenetic distances (p-distances) using MEGA ver 7026 (Kumar Stecher amp Tamura 2016)between drainages and among watersheds within drainages for each gene For comparisonwe also generated p-distances among all snubnose darters (clade Adonia sensu Near et al2011) using our data and publicly available cytb genetic sequences (see Table S4 for geneticsequence data)

We calculated the number of haplotypes and haplotype diversity for both loci usingDNAsp V 510 (Librado amp Rozas 2009) between drainages and among watersheds withindrainages We calculated estimates of divergence times using rates of molecular evolutionfor the cytb (18my) and S7 (034my) genes reported by Near et al (2011) and ourobserved genetic distance values produced by MEGA

RESULTSThe most appropriate substitution models for the 1st 2nd and 3rd codon positions of thecytb (1139 nucleotides (nt) in length) were F81 GTR+G and K80+I and for the S7 gene(530 nt) F81+G Results fromBayesian andML analyses for cytb indicate twomonophyleticclades congruent with the two river drainages (Figs 3 and 4 Figs S4 and S5) Supportfor reciprocally monophyletic clades was high for the ML analysis (bootstrap supportYR 95 LTR 100) but only weakly supported for the Bayesian analysis (posteriorprobabilities YR 012 LTR 10) Results from the S7 data (Figs 5 and 6 Figs S6and S7) indicated weakly supported and inconsistent phylogenetic relationships amongindividuals from the LTR drainage though samples from the YR drainage composeda single clade with high support (95 bootstrap support and 097 posterior probability)Haplotype networks for S7 and cytb indicate that no haplotypes were shared betweendrainages (Fig 7) A total of fifteen genetic characters from both genes are diagnostic ofYazoo Darters in the two major river drainages (Table 2)

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Figure 3 Phylogenetic tree of the partitioned cytb dataset using Bayesian estimation (MrBayes ver326) Bayesian posterior probabilities ge095 are shown (except for the Yocona River clade) at the nodes(see Table S1 for sequence data) bubble sizes for the pruned nodes are proportional to sample size LTR= Little Tallahatchie River drainage YR= Yocona River drainage

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Figure 4 Phylogenetic tree of the partitioned cytb dataset using maximum likelihood estimation(RAxML-HPC ver 80) Bootstrap values ge95 are shown at the nodes (see Table S1 for sequence data)bubble sizes for the pruned nodes are proportional to sample size LTR= Little Tallahatchie Riverdrainage YR= Yocona River drainage

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Figure 5 Phylogenetic tree of the partitioned S7 dataset using Bayesian estimation (MrBayes ver326) Bayesian posterior probabilities ge095 are shown at the nodes (see Table S2 for sequence data)bubble sizes for the pruned nodes are proportional to sample size LTR= Little Tallahatchie Riverdrainage YR= Yocona River drainage

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Figure 6 Phylogenetic tree of the partitioned S7 dataset using maximum likelihood estimation(RAxML-HPC ver 80) Bootstrap values ge95 are shown at the nodes (see Table S2 for sequence data)bubble sizes for the pruned nodes are proportional to sample size LTR= Little Tallahatchie Riverdrainage YR= Yocona River drainage

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Figure 7 S7 (A) and cytb (B) haplotype networks for samples among the Little Tallahatchie andYocona River drainages and watersheds (egCypress Creek) within drainages (see Fig 2) Red andblue indicates the Yocona and Little Tallahatchie river drainages respectively

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Uncorrected p-distances for Yazoo Darters (cytb) between drainages were 08 andamong watersheds within drainages was 001 in the YR drainage and 01 and 011 inthe LTR drainage (Table 3) For comparison p-distances (cytb) among other snubnosedarters ranged from 05ndash1453 (Tables S4ndashS5) P-distances for Yazoo Darters (S7)between drainages were 03 and among watersheds within drainages were 001 in theYR drainage and ranged from 007ndash017 in the LTR drainage (Table 3)

Haplotype diversity was higher in the LTR drainage (cytb Hd = 066 11 haplotypesS7 Hd = 048 2 haplotypes) than in the YR drainage (cytb Hd = 011 3 haplotypesS7 Hd = 006 2 haplotypes) Estimated times of divergence were 088 (S7) and 044 my(cytb)

DISCUSSIONOur results indicate (1) there is no evidence of cryptic diversity within each major riverdrainage (2) genetic diversity is lower in the YR drainage relative to the LTR drainage (3)consistent with the results from Powers amp Warren Jr (2009) there is support for recognizingYazoo Darter populations in the YR drainage as a distinct species under the unified speciesconcept (de Queiroz 2007) (4) Our estimates of time of divergence are similar to estimatesfor closely related snubnose darter species in Tennessee and Kentucky (Kozal et al 2017)which supports the proposal in Powers amp Warren Jr (2009) that the same vicariant eventsled to a late Pleistocene species radiation among snubnose darters in western Tennesseeand Kentucky and northern Mississippi

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Table 2 Genetic characters that diagnose allopatric populations of Yazoo Darters in the Little Tal-lahatchie (LTR) and Yocona rivers (YR) using mitochondrial cytochrome b (cytb) and nuclear S7genes Numbers indicate the location of the character along the genetic sequence A adenine C cytosineT thymine G guanine

Character (cytb) LTR YR

147 A G165 C T585 C T588 T C654 A G876 G A897 A G930 G A1056 A G1090 G A1107 G A1113 G ACharacter (S7 ) LTR YR286 G Ainsertion 478 G ndashinsertion 479 C ndash

Table 3 Uncorrected pairwise genetic distances (p-distance ) among watersheds for Yazoo DartersValues for cytb are below the diagonal and for S7 are above the diagonal abbreviations are defined in thetable

Otoucalofa Cr YR LTR Tippah R Cypress Cr

Otoucalofa Creek 001 035 024 041Yocona River 001 036 025 043Little Tallahatchie River 082 081 013 007Tippah River 076 075 011 017Cypress Creek 083 082 010 010

Though the lack of genetically distinct clades within drainages was not a surprise the lackof a clear and consistent link between genetic clades and geography within drainages wasunexpected (Fig 2 Figs S4ndashS7) Though samples from watersheds within drainages show aweak to moderate tendency to be grouped within clades (eg Cypress Creek Tippah RiverOtoucalofa Creek) the low support at most within-drainage nodes (phylogenetic trees)indicates that the only inference that can be made with any confidence is that our data didnot reveal genetically distinct populations or cryptic diversity within either drainage Incontrast microsatellite markers indicated that tributaries do contain genetically distinctpopulations with a strong isolation by distance effect (Sterling et al 2012) This is explainedby higher mutation rates among microsatellite markers and to some extent the effects ofhuman habitat alteration and consequent isolation Taken together patterns of gene flowwithin drainages apparently have not been stable over enough generations to show a clearrelationship between watersheds and genetic subclades in the cytb and S7 data

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Our results show that genetic divergence and diversity is much lower in the YR drainagethan in the LTR drainage (Table 3 Fig 7) Genetic distances between Otoucalofa Creekand the YR are an order of magnitude lower than the distances between Cypress Creekand the Tippah River for example (Fig 2) and the haplotype network results (Fig 7)clearly show less genetic diversity in the YR drainage Lower divergence can be explainedby the difference in area of distribution of the Yazoo Darter between the two drainages(Fig 2) Smaller area of distribution in the YR drainage (smaller watersheds with fewerand smaller streams) would likely result in greater gene flow lower divergence and lessgenetic diversity (Frankham 1996) Another possible explanation for lower diversity inthe YR drainage is a founder effect though our results are not consistent with thisexplanation (eg stochastic processes likely would have resulted in greater divergencebetween drainages than we observed) and do not indicate any mechanism for suchan explanation (eg stream capture) The lower genetic diversity observed in the YRdrainage is consistent with previous genetic studies (Powers amp Warren Jr 2009 Sterlinget al 2012) Our results and the low effective population sizes reported in Sterling et al(2012) indicate that human-assisted gene flow is warranted

Genetic distances between drainages were low but are comparable to other closelyrelated snubnose darters (Tables 3 and 4 Table S5) This is especially true for the BandfinDarter group (Etheostoma zonistium E cervus E pyrrhogaster and E cf zonistium) Similargenetic distances are almost certainly linked to the similar estimates for times of divergenceamong these taxa which are recent (Kozal et al 2017) Our observed distances are alsosimilar to those reported for other sister species pairs of fishes (Johns amp Avise 1998)

The lack of resolution and consistency in phylogenetic clades showing relationshipsin the LTR drainage using the S7 marker (Figs 5 and 6) is not surprising because inyoung clades of darters cytb usually has more power to resolve relationships with highersupport relative to nuclear genetic markers with slower mutation rates and higher effectivepopulation sizes (Avise 2004 Keck amp Near 2008) The lack of resolution for the S7 resultsis consistent with other studies (Keck amp Near 2010 Echelle et al 2015 Kozal et al 2017) Even so the S7 analyses did support a monophyletic clade for samples from the YRdrainage and this might be explained by smaller populations in a smaller watershed asoutlined earlier when discussing relative levels of genetic divergence and diversity

Our results using cytb did produce consistent phylogenetic trees with monophyleticclades for each drainage However support for the monophyly of the YR clade usingBayesian analysis was weak which is odd considering the high support for the ML andS7 analyses The low support for this clade has no apparent biological explanation Evenso the S7 and cytb results indicate recent divergence between Yazoo Darter populationsin the LTR and YR drainages Divergence is supported by the lack of evidence for geneflow between the drainages even for the samples from Deer Creek which are closest to theconfluence of the LTR and YR drainages (Fig 2) No haplotypes were shared betweendrainages and all individuals sorted into clades consistent with the drainages from whichthey were sampled for all phylogenetic trees (Figs S4ndashS7) This is consistent with theliterature (Powers amp Warren Jr 2009 Sterling et al 2012)

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Table 4 Uncorrected pairwise genetic distances (p-distances ) among snubnose darters that are most closely related to the Yazoo Darter(Near et al 2011) Species complexes are grouped following Near et al (2011) labels for undescribed species follows Jelks et al (2008) Bold typeand asterisk= values lt20 LTR Yazoo Darter Little Tallahatchie River drainage YR Yazoo Darter Yocona River drainage Fk Fork see Ta-ble S3 for complete results

E raneyi(Yazoo Darter group)

E zonistium (Bandfin Darter group)

YR LTR E zonistium E cf zonistium E cervus E pyrrhogaster E cyanoprosopum

YRLTR 075E zonistium 761 807E cf zonistium 833 864 129E cervus 845 897 142 050E pyrrhogaster 861 904 144 072 086E cyanoprosopum 884 924 425 475 486 484E bellator 850 899 893 938 948 939 984E chermocki 822 871 874 919 929 920 962lsquolsquoLocust Forkrsquorsquo 970 1018 964 981 980 981 1024lsquolsquoSipseyrsquorsquo 1029 1040 1069 1102 1099 1084 1102lsquolsquoConasaugarsquorsquo 879 871 903 932 941 946 951lsquolsquoAmicalolarsquorsquo 784 795 778 808 817 841 820E brevirostrum 879 871 922 951 960 968 978E simoterum 1433 1424 1503 1501 1504 1542 1501Percina sciera 1691 1681 1681 1730 1746 1747 1711

E bellator (Warrior Darter group) E brevirostrum (Holiday Darter group)E bellator E chermocki lsquolsquoLocust Fkrsquorsquo lsquolsquoSipseyrsquorsquo lsquolsquoConasaugarsquorsquo lsquolsquoAmicalolarsquorsquo E brevirostrum

E bellatorE chermocki 057lsquolsquoLocust Forkrsquorsquo 511 492lsquolsquoSipseyrsquorsquo 624 605 657lsquolsquoConasaugarsquorsquo 879 860 922 984lsquolsquoAmicalolarsquorsquo 745 707 769 821 315E brevirostrum 888 869 989 1012 105 363E simoterum 1433 1414 1390 1452 1490 1394 1471Percina sciera 1585 1557 1562 1703 1777 1700 1786

The vicariant events and mechanisms that led to isolation of ancestral populationsand ensuing divergence among Upper Gulf Coastal Plain snubnose darters currentlyrecognized as E cervus E pyrrhogaster and E zonistium almost certainly were a factor indivergence of ancestral Yazoo Darters in the upper Yazoo River basin (Powers amp WarrenJr 2009 Kozal et al 2017) (Fig 1) This is based on similar estimated times of divergenceamong these closely related fishes as well as similar surface geology topography andwatershed configurations among them (Warren Jr Haag amp Adams 2002 Keck amp Etnier2005 Powers amp Warren Jr 2009 Smith et al 2009 Near et al 2011 Kozal et al 2017)Though we restrict our discussion to the Yazoo Darter we believe that our interpretationsare generally applicable to these other species We propose that spatial shifts in suitable

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habitat for Yazoo Darters during repeated glacial cycles over the last 2 my led to theisolation of populations in the upper YR and LTR drainages (see Hewitt 1996) Duringglacial periods and low sea levels suitable habitat for Yazoo Darters would have expandeddownstream perhaps into the present Mississippi Alluvial Plain when sea levels were attheir lowest (90ndash140 m below present) Streams were smaller (less precipitation) mayhave been entrenched in bedrock and had higher gradients coarse substrate and coldclear water During interglacial periods sea levels rose streams had more water gradientsmoderated stream valleys filled in with fines and streams were no longer confined tobedrock Suitable habitat for Yazoo Darters would have moved upstream

As suitable habitat shifted up- and downstream in the Yazoo River Basin connectivityamong groups of Yazoo Darters in tributary streams would also have changed Duringinterglacial periods when streams were not confined to bedrock changes in streamconfigurations seemmore likely especially in headwaters However during glacial periodsstreams were smaller (climate was much drier) and confluences lower in the watershedwere less likely to be barriers to dispersal because they were smaller and may have beensuitable habitat for Yazoo Darters Dispersal among tributaries under these conditionsseems more likely (Fisk 1944 Rittenour Blum amp Goble 2007 Past Interglacials WorkingGroup of PAGES 2016)

Spatial changes in the downstream extent of suitable habitat likely interacted withchanges in the location of the ancient confluence of the YR and LTR to isolate YazooDarter populations Reliable data exists for estimating the number duration and timing ofglacial and interglacial periods over about the last 800000 years (Past Interglacials WorkingGroup of PAGES 2016) An estimated 11 cycles between glacial and interglacial periodsare identified Estimated duration of interglacial periods is much shorter (166700 years)than glacial and transitional periods (633300 years) (Fisk 1944 Past Interglacials WorkingGroup of PAGES 2016) Given this setting downstream connectivity among demes wouldhave likely had greater influence structuring Yazoo Darter populations between the twomajor drainages than possible shifts in stream configurations Further changes to theposition of the confluence of the LTR and YR and with the ancient predecessors ofthe Ohio and Yazoo rivers were likely instrumental in the phylogenetic pattern seen inour results (see text and figures in (Fisk 1944) Fig 1) It seems apparent that at somepoint (about 04ndash08 my) suitable habitat for Yazoo Darters no longer encompassed theconfluence of the YR and LTR during glacial periods

Our results help to refine the management actions (ie human-assisted gene flow)suggested in Sterling et al (2012) Phylogenetic trees show a weak to moderate associationbetween watersheds and clades within drainages though we did not find clear evidence ofgenetically distinct groups that were consistent with geography (eg management units toguide human-assisted gene flow) (Figs S4ndashS7) Even so based on our results and those inSterling et al (2012) we recommend relocation of individuals among tributaries that areclosest together within watersheds as categorized in Sterling et al (2012) Within the YRwatershed we recommend restricting movement of individuals to within the OtoucalofaCreek watershed or adjacent tributaries to the mainstem YR Since genetic diversity washigher in the LTR than in the YR and because populations in the YR face greater

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risks (ie area of distribution is much smaller estimates of effective population sizes areextremely low there is no evidence of contemporary gene flow among adjacent tributariesstreams yielding Yazoo Darters are nearly all on private lands and there is rapid urbandevelopment in this drainage see Sterling et al 2012 Sterling Warren Jr amp Henderson2013) human-assisted gene flowwithin the Yocona River should be implemented Researchaimed at identifying mechanisms of gene flow is also desperately needed for the YazooDarter which would also help inform management of other imperiled forms of snubnosedarters

CONCLUSIONSOur results indicate that populations of the YazooDarter in the YR drainage are geneticallydistinct and represent a recently diverged and undescribed cryptic species of snubnosedarter However because phylogenetic evidence constitutes only one line of evidence fordivergence we recommend that other lines of evidence for species delimitation under theunified species concept (de Queiroz 2007) be examined Though there are no obviousdifferences in pigment patterns or color between the populations in each drainageSuttkus Bailey amp Bart Jr (1994) noted modal differences in lateral line scale counts andSterling Warren Jr amp Henderson (2013) showed that Yazoo Darters in the YR drainage aresignificantly longer than those in the LTR drainage Further investigation of morphologymeristics and pigment patterns is warranted

ACKNOWLEDGEMENTSWe would like to thank M Bland Z Barnett A Carson C Smith B Sterling W Sterlingand G McWhirter for help collecting samples We are also grateful to J Hubbell and JSchaefer for sharing data and G Henderson for help with figures Any use of trade firmor product names is for descriptive purposes only and does not imply endorsement by theUS Government

ADDITIONAL INFORMATION AND DECLARATIONS

FundingFunding was provided by the USDA Forest Service Southern Research Station Center forBottomland Hardwoods Research The funders had no role in study design data collectionand analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsUSDA Forest Service Southern Research StationCenter for Bottomland Hardwoods Research

Competing InterestsThe authors declare there are no competing interests

Sterling et al (2020) PeerJ DOI 107717peerj9014 1419

Author Contributionsbull KenA Sterling and Stuart VNielsen conceived and designed the experiments performedthe experiments analyzed the data prepared figures andor tables authored or revieweddrafts of the paper and approved the final draftbull Andrew J Brown conceived and designed the experiments performed the experimentsanalyzed the data authored or reviewed drafts of the paper and approved the final draftbull Melvin L Warren Jr conceived and designed the experiments authored or revieweddrafts of the paper and approved the final draftbull Brice P Noonan conceived and designed the experiments performed the experimentsauthored or reviewed drafts of the paper and approved the final draft

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

University of Mississippi IACUC Committee approved the study (IACUC protocol09-027)

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Stream sampling and fish collections were approved by the Mississippi Museum ofNatural Science under a general permit for all our scientists and technicians for all fieldstudies Annual fish collection permits from 2009-2017 0604091 0513101 06241120622122 0602132 0610142 0624151 0715163 1010173

Data AvailabilityThe following information was supplied regarding data availability

The genetic sequences are available at GenBank (see Tables S1ndashS4)

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj9014supplemental-information

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April J Mayden RL Hanner RH Bernatchez L 2011 Genetic calibration of speciesdiversity among North Americarsquos freshwater fishes Proceedings of the Na-tional Academy of Sciences of the United States of America 10810602ndash10607DOI 101073pnas1016437108

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Echelle AA SchwemmMR Lang NJ Baker JS Wood RM Near TJ FisherWL2015Molecular systematics of the Least Darter (Percidae Etheostoma microp-erca) historical biogeography and conservation implications Copeia 10387ndash98DOI 101643CG-14-082

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Jelks HLWalsh SJ Burkhead NM Contreras-Balderas S Diaz-Pardo E HendricksonDA Lyons J Mandrak NE McCormick F Nelson JS Plantania SP Porter BARenaud CB Schmitter-Soto JJ Taylor EBWarren Jr ML 2008 Conservationstatus of imperiled North American freshwater and diadromous fishes Fisheries33372ndash407 DOI 1015771548-8446-338372

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Keck BP Etnier DA 2005 Distributional changes of the fishes of the Hatchie Riversystem in Western Tennessee and Northern Mississippi Southeastern Naturalist4597ndash626 DOI 1016561528-7092(2005)004[0597DCOTFO]20CO2

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Keck BP Near TJ 2010 A young clade repeating an old pattern diversity in Nothonotusdarters (Teleostei Percidae) endemic to the Cumberland RiverMolecular Ecology195030ndash5042 DOI 101111j1365-294X201004866x

Kozal LC Simmons JWMollish JM MacGuigan DJ Benavides E Keck BP NearTJ 2017 Phylogenetic and morphological diversity of the Etheostoma zonistiumspecies complex with the description of a new species endemic to the CumberlandPlateau of Alabama Bulletin of the Peabody Museum of Natural History 58263ndash286DOI 1033740140580202

Kumar S Stecher G Tamura K 2016MEGA7 molecular evolutionary geneticsanalysis ver 70 for bigger data setsMolecular Biology and Evolution 331870ndash1874DOI 101093molbevmsw054

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Librado P Rozas J 2009 DnaSP v5 a software for comprehensive analysis of DNA poly-morphism data Bioinformatics 251451ndash1452 DOI 101093bioinformaticsbtp187

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Mississippi Museum of Natural Science 2015Mississippirsquos State Wildlife Action Plan2015ndash2025 Mississippi Department of Wildlife Fisheries and Parks JacksonMississippi Available at httpwwwmdwfpcommedia251788mississippi_swap_revised_16_september_2016__reduced_pdf (accessed on October 2018)

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Near TJ Bossu CM Bradburd GS Carlson RL Harrington RC Hollingsworth PRKeck BP Etnier DA 2011 Phylogeny and temporal diversification of darters (Per-cidae Etheostomatinae) Systematic Biology 60565ndash595 DOI 101093sysbiosyr052

Page LM 1983Handbook of Darters Neptune City TFH PublicationsPage LM Burr BM 2011 Peterson field guide to freshwater fishes of North America North

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Powers SL Warren Jr ML 2009 Phylogeography of three snubnose darters (Perci-dae Subgenus Ulocentra) endemic to the southeastern US Coastal Plain Copeia2009523ndash528 DOI 101643CI-08-047

Rambaut A Drummond AJ Xie D Baele G SuchardMA 2018 Posterior summa-rization in Bayesian phylogenetics using Tracer 17 Systematic Biology 67901ndash904DOI 101093sysbiosyy032

Rittenour TM BlumMD Goble RJ 2007 Fluvial evolution of the lower MississippiRiver Valley during the last 100 ky glacial cycle response to glaciation and sea-levelchange GSA Bulletin 119586ndash608 DOI 101130B259341

Ronquist F TeslenkoM Van der Mark P Ayres DL Darling A Houmlhna S Larget BLiu L SuchardMA Huelsenbeck J 2012MrBayes ver 32 efficient Bayesianphylogenetic inference and model choice across a large model space SystematicBiology 61539ndash542 DOI 101093sysbiosys029

Ross ST 2001 Inland fishes of Mississippi Jackson Mississippi University Press ofMississippi 624

Ruble CL Sterling KAWarren Jr ML 2019 Captive propagation and early life historyof the Yazoo Darter (Etheostoma raneyi) Southeastern Naturalist 18525ndash540DOI 1016560580180402

Simon TPWallus R 2006 Reproductive biology and early life history of fishes in the OhioRiver Drainage 4 Percidae- Perch Pikeperch and Darters Boca Raton CRC Press

Smith DP Diehl TH Turrini-Smith LA Maas-Baldwin J Croyle Z 2009 Riverrestoration strategies in channelized low gradient landscapes of West TennesseeUSA Geological Society of America Special Papers 451215ndash229

Song CB Near TJ Page LM 1998 Phylogenetic relations among percid fishes as inferredfrom mitochondrial cytochrome b DNA sequence dataMolecular Phylogenetics andEvolution 10343ndash353 DOI 101006mpev19980542

Stamatakis A 2014 RAxML ver 8 a tool for phylogenetic analysis and post-analysis oflarge phylogenies Bioinformatics 301312ndash1313 DOI 101093bioinformaticsbtu033

Sterling KA Reed DH Noonan BPWarren Jr ML 2012 Genetic effects of habitat frag-mentation and population isolation on Etheostoma raneyi (Percidae) ConservationGenetics 13859ndash872 DOI 101007s10592-012-0335-0

Sterling KAWarren Jr ML Henderson LG 2013 Conservation assessment ofthe Yazoo Darter (Etheostoma raneyi) Southeastern Naturalist 12816ndash842DOI 1016560580120429

Suttkus RD Bailey RM Bart Jr HL 1994 Three new species of Etheostoma subgenusUlocentra from the Gulf Coastal Plain of Southeastern United States Tulane Studiesin Zoology and Botany 2997ndash126

Turner TF 2001 Comparative study of larval transport and gene flow in darters Copeia2001766ndash774 DOI 1016430045-8511(2001)001[0766CSOLTA]20CO2

Turner TF Trexler JC 1998 Ecological and historical associations of gene flow in darters(Teleostei Percidae) Evolution 521781ndash1801DOI 101111j1558-56461998tb02256x

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USDA Forest Service 2013 Forest Service designated sensitive species on the NationalForests and Grasslands Available at httpswwwfsfedusbiology resourcespubs tesfs_ss_310ct05pdf (accessed on October 2018)

Warren Jr ML Burr BMWalsh SJ Bart Jr HL Cashner RC Etnier DA Freeman BJKuhajda BR Mayden RL Robison HW Ross ST StarnesWC 2000 Diversitydistribution and conservation status of the native freshwater fishes of the southernUnited States Fisheries 257ndash29

Warren Jr ML HaagWR Adams SB 2002 Forest linkages to diversity and abundancein lowland stream fish communities In Holland MM Warren Jr ML Stanturf JAeds Proceedings of a conference on sustainability of wetlands and water resources howwell can riverine wetlands continue to support society into the 21st century GeneralTechnical Report SRS-50 Asheville NC USDA Forest Service Southern ResearchStation 168ndash182

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