Phylogenetic and Morphological Diversity of the ...

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Logan C. Kozal1, 2, Jeffrey W. Simmons3, Jon Michael Mollish3, Daniel J. MacGuigan1,. Edgar Benavides1, Benjamin P. Keck4, and Thomas J. Near5.
Phylogenetic and Morphological Diversity of the Etheostoma zonistium Species Complex with the Description of a New Species Endemic to the Cumberland Plateau of Alabama Logan C. Kozal1, 2, Jeffrey W. Simmons3, Jon Michael Mollish3, Daniel J. MacGuigan1, Edgar Benavides1, Benjamin P. Keck4, and Thomas J. Near5 1

Department of Ecology and Evolutionary Biology, Osborn Memorial Labs, Yale University, New Haven CT 06520-8106 USA 2

3 4

Berkeley College, Yale University, New Haven CT 06520-8106 USA

Resources and River Management, Tennessee Valley Authority, Chattanooga, TN 37402-2881 USA

Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996 USA

5 Corresponding author: Department of Ecology and Evolutionary Biology, Osborn Memorial Labs, Yale University, New Haven CT 06520-8106 USA; Peabody Museum of Natural History, Yale University, New Haven, CT 06520-8106 USA —email: [email protected]

ABSTRACT We provide a description of the Blueface Darter, Etheostoma cyanoprosopum, which is distributed in the upper Sipsey Fork of the Mobile Basin and the upper portion of the Bear Creek system in the Tennessee River Drainage. The distinctiveness of Etheostoma cyanoprosopum is assessed through analysis of morphological variation and molecular phylogenetic diversity within the Etheostoma zonistium species complex. In addition to analyzing disparity of morphometric and meristic traits, we present phylogenetic analyses of a mitochondrial gene and two nuclear genes and identify genetic clusters through analysis of 25 microsatellite loci. In the mitochondrial DNA (mtDNA) gene tree, Etheostoma cyanoprosopum is resolved as the sister lineage to a clade containing all other species of the Etheostoma zonistium complex. Etheostoma zonistium is paraphyletic with respect to both Etheostoma pyrrhogaster and Etheostoma cervus, which do not resolve as sister species in the mtDNA gene tree. The two nuclear gene trees are much less resolved, but the S7 ribosomal protein intron 1 (S7) gene tree resolves Etheostoma cyanoprosopum and all sampled populations of Etheostoma zonistium as a clade with strong Bayesian posterior node support. Etheostoma cyanoprosopum is morphologically differentiated from Etheostoma zonistium by a shallower body, a more elongate nape, a higher number of lateral line scales, a higher number of transverse scale rows, and differences in coloration.

KEYWORDS Species delimitation, new species, Etheostomatine, Percidae, Perciformes, Teleostei, Actinopterygii

Introduction The three closely related darter species Etheostoma zonistium, E. pyrrhogaster, and E. cervus are distributed in the Coastal Plain and Western Valley of the Tennessee River in Kentucky, Mississippi, Tennessee, and Alabama, USA (Bailey and Etnier 1988; Powers and Mayden 2003; Powers and Warren 2009). Etheostoma pyrrhogaster and

E. cervus are distributed in the Obion and Forked Deer river systems, respectively (Figure 1), which drain into the Mississippi River (Bailey and Etnier 1988; Powers and Mayden 2003). Etheostoma zonistium is distributed throughout the lower Tennessee River drainage from the lower Bear Creek system in Mississippi and Alabama to the Clarks River system and the Spring Creek system of the Hatchie River (Figure 1), which drains into

Bulletin of the Peabody Museum of Natural History 58(2):263–286, October 2017. © 2017 Peabody Museum of Natural History, Yale University. All rights reserved. • http://peabody.yale.edu

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FIGURE 1. Map detailing the geographic distribution of Etheostoma cyanoprosopum, E. zonistium, E. pyrrhogaster, and E. cervus. Inset map of the upper Bear Creek system focuses on the distribution of E. cyanoprosopum. Species occurrences labeled as “records only” come from museum collections (FishNet 2 2012) and Tennessee Valley Authority Index of Biotic Integrity Database.

the Mississippi (Burr and Warren 1986; Bailey and Etnier 1988; Boschung et al. 1992; Etnier and Starnes 1993; Ross 2001). Bailey and Etnier (1988) identified specimens distributed on the Cumberland Plateau in Hubbard Creek of the Sipsey Fork (Mobile Basin) as E. zonistium but noted that these populations have higher counts of lateral line scales and more deeply embedded scales on the cheeks, breast, and prepectoral areas. The populations in Hubbard Creek and in the upper Bear Creek system of the Tennessee River drainage in Alabama were recognized as a distinct, but undescribed, species based on meristics and pigmentation patterns (Kuhajda and Mayden 1995). Boschung and Mayden (2004:568–569) refer to this undescribed species as the Blueface Darter. Phylogenetic analyses using nuclear and mitochondrial DNA (mtDNA) resolve the Blueface Darter as the sister lineage of a clade containing

Etheostoma pyrrhogaster, E. cervus, and a paraphyletic E. zonistium (Near et al. 2011, fig. 3). The molecular phylogeny supports the hypothesis that the Blueface Darter is a distinct species, but the paraphyly of E. zonistium highlights the potential for additional undescribed species diversity in this lineage. In this study, we expand the molecular phylogenetic analysis to include specimens of E. zonistium sampled throughout its geographic distribution, assess genetic divergence at 25 microsatellite loci, and determine if lineages identified in the phylogenetic and population genetic analyses show morphometric disparity and divergence of meristic traits used to delimit and identify species of darters. Our analyses support the conclusion that the Blueface Darter is a distinct species, which is formally described here, and that the E. zonistium complex contains diversity that may represent additional distinct species.

Phylogenetic and Morphological Diversity of Etheostoma • Kozal et al.

Materials and Methods Morphological Analyses Comparative meristic morphological data was collected from more than 400 specimens of Etheostoma zonistium, E. cervus, E. pyrrhogaster, E. cf. zonistium (Blueface Darter), and Etheostoma raneyi (Table 1). E. raneyi was included to provide a deeper evolutionary perspective on morphological divergence, as the species is resolved as the sister lineage of the E. zonistium complex (Near et al. 2011). Specimens were obtained from field sampling and the following museum collections: Auburn University Museum of Natural History (AUM), Auburn, Alabama, USA; Field Museum of Natural History (FMNH), Chicago, Illinois, USA; Illinois Natural History Survey (INHS), Champaign, Illinois, USA; Mississippi Museum of Natural Science (MMNS), Jackson, Mississippi, USA; University of Tennessee (UT), Knoxville, Tennessee, USA; Division of Vertebrate Zoology Ichthyology Collection (YPM ICH), Peabody Museum of Natural History, Yale University, New Haven, Connecticut, USA. Institutional abbreviations follow Sabaj (2014), except that YFTC refers to the Yale Fish Tissue Collection. The sampling locations of specimens examined for morphological comparisons are shown in Figure 1. The numbers of scale rows and fin elements were determined from each specimen as outlined in Hubbs and Lagler (1958) and Page (1981), with the exception of the number of transverse scale rows, which was counted as described by Page (1983:16, fig. 2), and all rays of the second dorsal fin were counted. Terminology for body pigmentation follows Bailey and Etnier (1988), Powers and Mayden (2003), and Etnier and Starnes (1993:518–519, 552–554). We analyzed variation in meristic traits with cross-validation linear discriminate analysis (LDA) as applied in the MASS package (Venables and Ripley 2002) of the statistical program (R Core Team 2008). For each individual, a Bayesian posterior probability of assignment to each group was calculated, and then the individual was assigned to the group with the highest probability. The meristic characters analyzed with LDA included the following: number of lateral line scales, number of scale rows above the lateral line, number of scale rows below the lateral line, number of transverse scale rows, number of scales

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around the caudal peduncle, number of first dorsal fin spines, number of second dorsal fin rays, and number of pectoral fin rays. We ran LDA with a grouping scenario that included Etheostoma cf. zonistium, E. cervus, E. pyrrhogaster, E. zonistium from the lower Tennessee River system, E. zonistium from the Hatchie River system, and E. raneyi. A principal component analysis (PCA) of the meristic traits was executed using R version 3.2.0 (R Core Team 2002). We explored the data for normal distributions with the function “hist” and obvious outliers with box plots with the function “boxplot.” We also used the function “lofactor” in the R package DMwR version 0.4.1 (Torgo 2011) that implements the local outlier factor (LOF) algorithm to identify density-based local outliers (Breunig et al. 2000). LOF is a clustering method that identifies samples in less dense areas as potential outliers. LOF compares the local density of each point to the local density of a specified number (k) of neighboring points. We used multiple values for k, initially 5 and then ranging from 10 to 100 in increments of 10, to determine any change in identified outliers. Each point is assigned a score based on the differences in local density and we recorded the top five outliers identified for each run of LOF with a different k. We removed the five outliers that were identified most frequently from the dataset used in the PCA. We preformed PCA using the “prcomp” function in R. We used the “ggbiplot” function in the R package ggplot2 (Wickham 2016) to plot the data points in principal component (PC) space. Additionally, we performed a landmark morphometric analysis to compare disparity in body shape between specimens of the Etheostoma cf. zonistium and E. zonistium. Digital images of the left lateral side of 20 specimens (ten males and ten females from each of the two species) were acquired by photographing in a glass squeeze tank (Table 1). We digitized a series of 28 fixed landmarks on each image using tpsDIG v. 2.21 (Figure 2; http://life.bio.sunysb.edu/morph/). Because specimens of darters often contort during or after preservation, we used the unbend module in tpsUtil v. 1.63 (http://life.bio.sunysb.edu/morph/) to correct curvature due to preservation. The thin plate spline interpolation function, relative warps, was applied to the landmark coordinate data using tpsRelW v. 1.62 (http://life.bio.sunysb.edu/ morph/). Analysis and discussion are restricted to

Etheostoma zonistium

Etheostoma cyanoprosopum

Species

West Fork Clarks River, Calloway Co., Kentucky Dabbs Creek, Henderson Co., Tennessee West Fork Clarks River, Calloway Co., Kentucky

YPM ICH 016224

No voucher YPM ICH 023574

Wildcat Creek, Calloway Co., Kentucky

Bear Creek, Franklin Co., Alabama Little Bear Creek, Franklin Co., Alabama Hubbard Creek, Lawrence Co., Alabama Hubbard Creek, Lawrence Co., Alabama McNair Creek, Franklin Co., Alabama

YPM ICH 030309 YPM ICH 030310 YPM ICH 027015 YPM ICH 027130 YPM ICH 027168

YPM ICH 026986

Hubbard Creek, Lawrence Co., Alabama Bear Creek, Franklin Co., Alabama

YPM ICH 020211 YPM ICH 020333

Wildcat Creek, Calloway Co., Kentucky

Bear Creek, Franklin Co., Alabama Bear Creek, Franklin Co., Alabama Bear Creek, Franklin Co., Alabama Little Bear Creek, Franklin Co., Alabama McNair Creek, Franklin Co., Alabama Hubbard Creek, Lawrence Co., Alabama

AUM 30634 AUM 41954 AUM 65493 AUM 41966 AUM 65128 YPM ICH 016152

YPM ICH 022963

Hubbard Creek, Lawrence Co., Alabama

Locality

UT 91.6969

Catalog

35.805520 36.6147

36.741

36.60939

36.608272

34.33965 34.417553 34.3074 34.3074 34.39273

34.3074 34.33965

34.33965 34.33965 34.33965 34.399322 34.39273 34.3074

34.3074

Latitude

–88.334179 –88.4328

–88.46164

–88.17128

–88.170317

–87.54721 –87.603128 –87.5034 –87.5034 –87.55398

–87.5034 –87.54721

–87.54721 –87.54721 –87.54721 –87.626512 –87.55398 –87.5034

–87.5034

Longitude

NA Meristic

Meristic

Meristic

Meristic

Meristic Meristic Meristic-morphometric Meristic-morphometric Meristic-morphometric

Meristic-morphometric Meristic

Meristic Meristic Meristic Meristic Meristic Meristic-morphometric

Meristic

Morphology

Continued

mtDNA, nDNA, msat NA NA NA NA NA mtDNA, nDNA, msat nDNA, msat mtDNA, nDNA, msat NA NA NA NA mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, msat mtDNA, nDNA, msat

Genetics

TABLE 1. Information on specimens used in morphological and genetic analyses. Abbreviations: mtDNA, mitochondrial DNA; nDNA, nuclear genes; msat, microsatellites; NA, not applicable.

266 Bulletin of the Peabody Museum of Natural History 58(2) • October 2017

Species

TABLE 1 CONTINUED.

West Fork Clarks River, Calloway Co., Kentucky East Fork Clarks River, Calloway Co., Kentucky East Fork Clarks River, Calloway Co., Kentucky East Fork Clarks River, Calloway Co., Kentucky East Fork Clarks River, Calloway Co., Kentucky Trace Creek, Graves Co., Kentucky Duncan Creek, Marshall Co., Kentucky West Fork Spring Creek, Hardeman Co., Tennessee West Fork Spring Creek, Hardeman Co., Tennessee Marshall Creek, Hardeman Co., Tennessee

Jonathan Creek, Calloway Co., Kentucky

Turnbo Creek, Decatur Co., Tennessee

Sycamore Creek, Benton Co., Tennessee

Turkey Creek, Benton Co., Tennessee Leath Creek, Hardin Co., Tennessee

Horny Head Creek, Decatur Co., Tennessee

YPM ICH 023793

YPM ICH 027106

YPM ICH 026975

YPM ICH 027322

YPM ICH 027341

No voucher YPM ICH 027305

YPM ICH 027262

No voucher

YPM ICH 023553 YPM ICH 023584 MMNS 41010

YPM ICH 023010

YPM ICH 023001

YPM ICH 022423

YPM ICH 022248

Locality

Catalog

35.7319

35.916588 35.060014

35.871833

35.46814

36.69478

35.16197

35.081523

36.824433 36.758283 35.101731

36.503017

36.65375

36.5028

36.55185

36.6147

Latitude

–88.13464

–88.113867 –88.303971

–88.13525

–88.13478

–88.22422

–89.06675

–89.111898

–88.550567 –88.4489 –89.083017

–88.310433

–88.27925

–88.31085

–88.316633

–88.4328

Longitude

Meristic

NA Meristic-morphometric

Meristic

Meristic-morphometric

Meristic

Meristic

NA

Meristic Meristic Meristic

Meristic

Meristic

Meristic

Meristic

Meristic

Morphology

Continued

mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, nDNA, msat mtDNA, msat mtDNA, nDNA, msat mtDNA, nDNA, msat

mtDNA, msat

NA NA NA

NA

NA

mtDNA, nDNA, msat NA

NA

Genetics

Phylogenetic and Morphological Diversity of Etheostoma • Kozal et al.

267

Etheostoma raneyi

Etheostoma pyrrhogaster

Etheostoma cervus

Species

TABLE 1 CONTINUED.

YPM ICH 018264

INHS 38700 INHS 63889 UT 91.3496 UT 91.3565

UT 91.2324 UT 91.4252

Clear Creek, Henry Co., Tennessee North Fork Obion River, Henry Co., Tennessee Terrapin Creek, Weakley Co., Tennessee Terrapin Creek, Graves Co., Kentucky Pumpkin Creek, Lafayette Co., Mississippi Graham Mill Creek, Lafayette Co., Mississippi Pumpkin Creek, Lafayette Co., Mississippi

Jacks Creek, Chester Co., Tennessee

UT 91.1283

AUM 28302

AUM 64804

AUM 28258

AUM 65197 UT 91.6212 UT 91.6678

Tributary to Indian Creek, Toshmingo Co., Mississippi Stewman Creek, Decatur Co., Tennessee

Locality

Pennywinkle Creek, Tishomingo Co., Mississippi Pennywinkle Creek, Tishomingo Co., Mississippi Pennywinkle Creek, Tishomingo Co., Mississippi Little Cripple Deer Creek, Tishomingo Co., Mississippi Cripple Deer Creek, Colbert Co., Alabama Clarks Creek, Chester Co., Tennessee Clarks Creek, Chester Co., Tennessee

AUM 28266

YPM ICH 027302

YPM ICH 020989

Catalog

34.32694

36.508546 36.498644 34.32694 34.50277

36.42511 36.42493

35.48225

34.71325 35.49832 35.49832

34.73444

34.74222

34.74222

34.7375

35.4281

34.857083

Latitude

–89.39739

–88.498928 –88.49009 –89.39739 –89.49083

–88.38182 –88.41156

–88.51849

–88.10945 –88.58758 –88.58758

–88.1975

–88.15528

–88.15528

–88.13556

–88.17503

–88.190883

Longitude

NA

NA NA Meristic Meristic

Meristic Meristic

Meristic

Meristic Meristic Meristic

Meristic

Meristic

Meristic

Meristic

Meristic-morphometric

Meristic

Morphology

mtDNA, nDNA

mtDNA, nDNA mtDNA, nDNA NA NA

NA NA

NA NA mtDNA, nDNA, msat NA

NA

NA

NA

mtDNA, nDNA, msat mtDNA, nDNA, msat NA

Genetics

268 Bulletin of the Peabody Museum of Natural History 58(2) • October 2017

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269

FIGURE 2. Landmarks used in morphometric analysis shown on a specimen of Etheostoma zonistium. Photograph by MR Thomas.

relative warp axes explaining more than 5% of variation.

Molecular Analyses: Phylogenetics, Divergence Times, and Population Structure We investigated the phylogenetic relationships of Etheostoma zonistium, E. cervus, E. pyrrhogaster, E. cf. zonistium, and E. raneyi through analyses of DNA sequences of the mitochondrial cytochrome b (cytb) gene and two nuclear genes: S7 ribosomal protein intron 1 (S7) and exon 2 of the recombination activating gene 1 (rag1). Sampling locations of specimens used for genetic analyses are listed in Table 1 and shown in Figure 1. Specimens for genetic analyses were preserved either through the freezing of whole specimens in liquid nitrogen or through placement of a tissue biopsy in 95% ethanol. Genomic DNA was extracted using either a standard phenol-chloroform protocol or the DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA, USA). Each of the three genes was polymerase chain reaction (PCR) amplified using previously published primer sequences and cycling conditions (Chow and Hazama 1998; Near et al. 2000; Lopez et al. 2004). Amplification products were prepared for sequencing using a polyethylene glycol precipitation. Both strands of each gene were sequenced and contiguous sequences were assembled from individual sequencing reactions using the computer program Geneious v. 7.2 (Kearse et al. 2012). The DNA sequences were aligned by eye. Etheostoma etnieri was treated as the outgroup species in the phylogenetic analyses because in previous studies it was resolved as

the sister lineage of all other species of Adonia (Near et al. 2011, fig. 3), which includes the E. zonistium complex and E. raneyi. The optimal data partitioning scheme among the three codon positions of the cytb and rag1 genes and molecular evolutionary models were determined using the Bayesian information criterion in the computer program Partitionfinder v. 2.1 (Lanfear et al. 2017). The optimal molecular evolutionary model for the S7 gene was determined using jModelTest 2 (Darriba et al. 2012). Gene trees for each of the three sampled loci were inferred using the computer program MrBayes v. 3.2 (Ronquist et al. 2012), in which posterior probabilities for the phylogeny and parameter values were estimated using Metropolis-coupled Markov chain Monte Carlo (MC3) (Larget and Simon 1999; Huelsenbeck et al. 2001). The MrBayes analysis was run for 107 generations with two simultaneous runs, each with four chains. Convergence of the MC3 algorithm and stationarity of the chains was assessed by monitoring the average standard deviation of the split frequencies between the two runs, which was less than 0.005 after 3 ⫻ 106 generations. In addition, the likelihood score and all model parameter estimates were plotted against the generation number to determine when there was no increase relative to the generation number in the computer program Tracer v. 1.5 (Drummond and Rambaut 2007). The first 30% of the sampled generations were discarded as burn-in, and the posterior phylogenies were summarized as 50% majority-rule consensus trees. A TCS haplotype network for E. cf. zonistium was inferred from the cytb sequence data

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TABLE 2. Collection locality and museum voucher information for specimens used in relaxed molecular clock analyses. Species Etheostoma etnieri Etheostoma raneyi Etheostoma cyanoprosopum Etheostoma cervus Etheostoma pyrrhogaster Etheostoma zonistium Etheostoma zonistium Etheostoma zonistium Etheostoma zonistium Etheostoma zonistium

Locality Cherry Creek, White Co., Tennessee Big Spring Creek, Marshall Co., Mississippi Hubbard Creek, Winston Co., Alabama Clarks Creek, Chester Co., Tennessee Terrapin Creek, Weakley Co. Tennessee Leath Creek, Hardin Co., Tennessee, Wildcat Creek, Calloway County Panther Creek, Henry Co. Tennessee West Fork Clarks River, Calloway Co., Kentucky Marshall Creek, Hardeman Co., Tennessee

using the computer program POPART v. 1.0 (Leigh and Bryant 2015). A relaxed molecular clock analysis of cytb DNA sequences was performed to estimate the divergence times between Etheostoma cf. zonistium, E. cervus, E. pyrrhogaster, and E. zonistium. With the exception E. zonistium, only one specimen was included from each sampled species (Table 2). The uncorrelated lognormal (UCLN) model of molecular evolutionary rate heterogeneity was implemented using the computer program BEAST v. 1.8 (Drummond et al. 2006; Drummond et al. 2012). The optimal partitioning scheme and molecular evolutionary models were determined using Partitionfinder v. 2.1 (Lanfear et al. 2017). A birth-death diversification prior was used for the branching rates in the phylogeny. Two calibration priors, each with a normal distribution, were based on a previous relaxed clock analysis of darters that estimated the most recent common ancestor (MRCA) of E. etnieri and a sister lineage that contains several species of Adonia at 11.2 million years ago (mya), with a 95% highest posterior density (HPD) of 8.5 to 18.5 mya, and the MRCA of E. raneyi and a clade containing E. cf. zonistium, E. cervus, E. pyrrhogaster, and E. zonistium at 8.4 mya (HPD: 5.8–10.9 mya) (Near et al. 2011). Therefore, in our analysis, the nodes were calibrated using the normal distributions with mean ages of 11.2 and 8.4 mya and a standard deviation of 2.0 and 1.5 mya respectively. The BEAST analysis was run three times, each run consisting of 107 generations. Convergence of model parameter values and estimated node

Voucher UT 91.6694 INHS 64539 YPM ICH 016152 UT 91.6678 INHS 63889 YPM ICH 027305 YPM ICH 022963 INHS 88972 YPM ICH 016224 YPM ICH 027106

ages to their optimal posterior distributions was assessed by plotting the marginal posterior probabilities using the computer program Tracer v. 1.6 (Drummond and Rambaut 2007). The resulting trees and log files from each run were combined using the computer program LogCombiner v. 1.83 (http://beast.bio.ed.ac.uk/ logcombiner). The posterior probability density of the combined tree and log files was summarized using TreeAnnotator v. 1.8.3 (http:// beast.bio.ed.ac.uk/treeannotator). The mean and 95% HPD estimates of divergence times were visualized on the chronogram using the computer program FigTree v. 1.4 (http://beast.bio.ed. ac.uk/figtree). We genotyped specimens of Etheostoma cf. zonistium, E. cervus, and E. zonistium for 25 microsatellite loci using primers developed for E. zonistium by Hereditec (Lansing, NY, USA; Table 3). Many of the targeted loci did not amplify for E. pyrrhogaster; the species was therefore not included in our analyses of the microsatellite loci. Genotyping was performed by a single-reaction nested PCR method (Schuelke 2000). We used a forward primer with a universal M13 [5′TCCCAGTCACGACGT-3′] tail at its 5′ end, a complementary M13 forward primer labeled with one of three fluorescent dyes (6FAM, VIC, or NED) and an unlabeled reverse primer. Microsatellite PCR reactions were performed in a total volume of 12.5 ␮L, containing 1 ␮L of isolated DNA, 1X colorless GoTaq Flexi Buffer (Promega Corporation, Fitchburg, WI, USA), 2.0 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 0.04 mmol/L of both the reverse primer and the M13 tagged

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271

TABLE 3. Microsatellite primer sequences used in genotyping specimens of Etheostoma cyanoprosopum, E. cervus, and E. zonistium.

Locus

Motif

Locus size in base pairs (bp)

Ezni3 Ezni5 Ezni7 Ezni11 Ezni13 Ezni17 Ezni19 Ezni30 Ezni32 Ezni33 Ezni34 Ezni37 Ezni38 Ezni40 Ezni42 Ezni43 Ezni44 Ezni45 Ezni47 Ezni51 Ezni52 Ezni56 Ezni57 Ezni58 Ezni60

AGAT AGAT AAAG AAAG ATCC ACAT AATG ATCC AGAT AGAT ACAT AGAT AGAT AGAT AGAT ATCC ATCC ACAG ACAT ACAG AAAC AAGT AGAT AGAT ACAT

111–179 159–255 132–168 158–170 118–186 157–169 138–166 112–216 111–179 137–261 149–221 149–225 120–232 117–245 280–344 278–410 290–370 313–373 282–366 212–344 300–328 305–325 294–414 304–432 294–370

5′ primer

3′ primer

CCACTGATTTATCCTCCTGCC TTGTGTGCCTCTAACCTTGC ATGTGGGAAGGAAGATGCTTG AGGATGAAGAACAGGGAGCC GTGACAGCAGCAACACAAATC GTCCAGTCTGTTCAGCATCC TTCTGGTATCTGGTCCCTTGC ATTTCCATTGAACACCGCCC TGCACACTGTACACCAATCG GTTGTCTTGCTACTGGATGCC TTGAAAGCGACATTACGGCC ATCGGTTTAGAATCGAGTCGTC ACTGCATTGTTCAATCCCTGC GTCTGCCCACCCATCTATCC TCGACAATTACAGCACACCC GGCCAGGGTCCAGGTAATAC TCCTCCTCTGATGGTTGTCG GATGCAGCTGGTTGAGTTCC ACAGTTGACATGCACCTACG CAAACACACAGCAGAGGACC CCGGTTTGTGACACTGGTTG TAGAGGAGGAGGAATGCTGC CAGAAATGAGATGTGGGCGG ATAGACCGCTGGCATCTTGG AAAGGGAAGGCTGTGTAGTG

forward primer, 0.64 mmol/L of the fluorescentlabeled M13 primer, and 0.06 mmol/L of GoTaq DNA polymerase (Promega Corporation, Fitchburg, WI, USA). We used an Eppendorf Mastercycler thermo cycler under the following PCR protocol: initial denaturation at 94 °C (5 min), followed by 35 cycles of denaturing (94 °C , 40 s), annealing, and extension (72 °C , 40 s), with the annealing temperature maintained at 57 °C (30 s) for the first 10 cycles and subsequently lowered to 53 °C (30 s) for the final 25 cycles. All microsatellite loci were genotyped on 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) against a LIZ-500 dye size standard (Applied Biosystems, Foster City, CA, USA). We used GENEMAPPER v3.7 (Applied Biosystems, Foster City, CA, USA) to retrieve raw allele sizes and scored them by using the automatic binning function in TANDEM (Matschiner and Salzburger 2009).

CACCTGCAGCCCATACATG ATCCAGTACAAGGCAATGCG CCATCTAACCTTTGCTTTGGAG TGCCCTGCTGAATTTCCTTG AAGCGGATGAAGATGGATGG GAACTGCTGTGGTCGGTTTG GGGAAGAAAGAGCCTCTGTTG CACTCACAGGCAGCAAAGTC AAATCAATCCCTGGCGTCTG CCACCCTCATGATTAGAGTGGG CCTACCACTTTGAGAACACAGC ACAGAATTTAGCGTGACACTGC CATAACGTGGACATGCTCAATC GCTGCATCCTTCAACTGGC GGATAAAGCAAGCACACCCG GCACTCGACACCATTGACAG GTTGATGTGGCTCGGGAAAG CTCTATGAGGGTCAGGGCTG CTGACAACCCTGCAACCATC TCGGACTTCTGGCTAATGGAG ACTGCTAAGCTTTGACACTGC TGCGCTGCTTAGATTGTGTG TCAACGCCCTTGTAAACTGC AGCCAGACCTTTCTCCACTC TAACTCCGCTATCGCTCTGC

Indices of population genetic diversity including observed heterozygosity (Ho), expected heterozygosity (He), mean number of alleles (NA), and allelic richness (AR) were calculated using GENALEX v.6 (Peakall and Smouse 2006). We used HP-RARE (Kalinowski 2005) to determine allelic richness and private allelic richness for each locus while accounting for population variation in sample sizes, and Arlequin v. 3.0 (Excoffier et al. 2005) to calculate observed heterozygosity (Ho) and expected heterozygosity (He) for each locus. We tested for departures from Hardy–Weinberg equilibrium (HWE) in the FSTAT 2.9.3.1 package (http://www2.unil.ch/popgen/softwares/fstat.htm). Statistical significance was determined using a Markov chain method with 5,000 dememorization steps and 100 batches of 5,000 iterations per batch (Guo and Thompson 1992). We assessed if Etheostoma cf. zonistium, E. cervus, and E. zonistium formed distinct genetic

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clusters and if populations within species had structure by using a Bayesian clustering algorithm as implemented in STRUCTURE v2.3.2 (Pritchard et al. 2000; Falush et al. 2003). We used a hierarchical ΔK method to infer the number of genetic clusters by repeating STRUCTURE analyses on each of the K groups inferred in the previous step (Coulon et al. 2008). This was repeated until the log-likelihood for one cluster (K ⫽ 1) was larger than the log-likelihoods for all other values of K or the majority of individuals were not strongly assigned to any cluster (assignment probability