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2010 SOUTHEASTERN NATURALIST 9(2):373–384
Multiple Nuclear Gene Analysis of the Divergence Between
Populations of the Tallapoosa Darter,
Leos G. Kral*
Abstract - Populations of Etheostoma tallapoosae (Tallapoosa Darter) have previously
been shown to be genetically divergent for mitochondrial DNA. In this study,
PCR primers were developed to amplify portions of six nuclear genes, and sequences
of these genes were assessed in three of the most divergent Tallapoosa Darter populations.
This analysis shows that these populations are also highly divergent for nuclear
gene sequences and thus any adaptive or potentially adaptive variation is likely to be
partitioned among the populations. Sequences of the six nuclear genes have also been
determined in the closely related species E. coosae and E. brevirostrum, and the utility
of these nuclear gene sequences to the elucidation of darter phylogeny is discussed.
Etheostoma tallapoosae Suttkus and Etnier (Tallapoosa Darter) is endemic
to the Tallapoosa River system above the fall line (Suttkus and Etnier
1991). Analysis of mitochondrial DNA in a previous study (Brogdon et al.
2003) has shown that this species is subdivided into at least four genetically
divergent reproductively isolated populations that were designated as management
units (MU) according to the definition of Moritz (1994a, 1994b).
These MUs do not share any mitochondrial haplotypes. The upper Tallapoosa
River MU and the Little Tallapoosa MU are the most divergent, with
a 1.6% mitochondrial sequence divergence. The mitochondrial sequence
divergence between the Enitachopco Creek MU and the upper Tallapoosa
River and Little Tallpapoosa River MUs is 1.04% and 1.10%, respectively.
The Jay Bird Creek MU is very closely related to the upper Tallapoosa River
MU, with a mitochondrial sequence divergence of 0.26%, and was not included
in this study. The maximum mitochondrial sequence divergence of
1.6% between the most genetically diverged populations is fairly low, and no
obvious morphological differences have been noted between those populations
(Suttkus and Etnier 1991). Furthermore, since mitochondrial genomes
sort faster than nuclear genomes, it is not certain that these populations have
significantly different allelic frequencies of nuclear genes such that actual
or potential adaptive diversity associated with the nuclear genome is partitioned
among the various populations.
To help ascertain the degree of nuclear gene divergence in these
populations, PCR primers were developed to amplify portions of six singlecopy
protein-coding nuclear genes. These primers are complementary to
*Department of Biology, University of West Georgia, Carrollton, GA 30118; lkral@
374 Southeastern Naturalist Vol. 9, No. 2
conserved exon sequences and also amplify the targeted gene segments from
other darter species (both Etheostoma and Percina). As such, these primers
can be used to assess the nuclear genetic heterogeneity of other darter
species and may have utility in the estimation of darter phylogeny. Current
estimation of darter phylogeny is mainly based on morphological and mitochondrial
DNA sequence analysis (e.g., Near 2002, Porter et al. 2002),
although some recent studies utilize both mitochondrial sequences and intron
1 of the nuclear S7 ribosomal protein gene (e.g., Keck and Near 2008).
The problem with estimating phylogeny with mitochondrial sequences alone
is that incorrect phylogenies may be inferred if hybridization has resulted in
mitochondrial introgression with subsequent replacement by drift or selective
sweep in one of the species (Alves et al. 2003, Fredsted et al. 2006, Ray
et al. 2008). Basing phylogenies on single nuclear genes is also less accurate
than basing phylogenies on multiple nuclear genes (Edwards et al. 2007,
Gadagkar et al. 2005, Gontcharov et al. 2004, Liu et al. 2008).
In this study, the nuclear sequence heterogeneity of six genes was determined
in three Tallapoosa Darter populations, previously designated as the
most divergent MUs, to ascertain if nuclear gene divergence exists in these
populations. Using the six nuclear genes, relationships of these populations
were also assessed to the closely related species Etheostoma coosae Fowler
(Coosa Darter) and Etheostoma brevirostrum Suttkus and Etnier (Holiday
Darter) to determine the congruence of nuclear gene phylogenies and previously
determined mitochondrial phylogenies (Brogdon et al. 2003, Porter et
Materials and Methods
PCR primers were designed to amplify portions of six protein-coding
genes for a total of about 10,900 base pairs of nuclear DNA, of which 3900
base pairs represent protein-coding exon sequences. The following gene
segments were amplified: Glyceraldehyde-3-phosphate dehydrogenase,
Ribosomal Protein S6, Lysophospholipase II, Phospholipase C-gamma-1,
Kelch Repeat and BTB (POZ) Domain Containing Protein, and an open
reading frame of about 1050 base pairs corresponding to an exon within a
gene of unknown function (designated the uORF gene). The PCR primers
are anchored within conserved exon sequences and amplify across distantly
related darter species (data not shown).
All but one of the gene segments were initially characterized from
randomly selected cDNA clones of Tallapoosa Darter muscle/skin mRNA.
Orthologous genes and likely intron/exon structure of those genes were identified by alignments of the cDNA sequences with genomes of Denio rerio
Hamilton (Zebra Danio), Takifugu rubipres Temminck and Schlegel (Japanese
Pufferfish), and Tetraodon nigroviridis de Proce (Green Puffer Fish) on
the Ensembl Blast server. Exon-anchored PCR primers were then designed
with Primer Premier (Biosoft) to amplify across each intron, and sizes of the
Tallapoosa Darter introns of these gene segments were determined. Final
2010 L.G. Kral 375
exon-anchored PCR primer pairs were designed that amplified about 1000
base pair segments, except in those cases where a single intron exceeded that
length. Intron/exon structures of gene segments and amplification products
of these gene segments are diagramed in Figure 1. PCR primers that amplify
the six genes in twelve fragments are listed in Table 1.
Figure 1. Intron/exon structure of gene fragments utilized in this study. Boxes represent
exons and connecting lines represent introns. Gene fragments greater than
1000 nucleotides are amplified in segments, and these are indicated below each gene
fragment. Exons and portions of exons are labeled A, B, C, etc. in the downstream
direction and relative to the gene fragment characterized. Sizes of gene fragments are
specific for the Tallapoosa Darter population in the Tallapoosa River.
376 Southeastern Naturalist Vol. 9, No. 2
The uORF gene was not identified from a cDNA clone, but rather was
identified from a clone of RAPD-PCR amplified genomic DNA. This
sequence is homologous to an exon of a gene present in T. rubripes and
T. nigroviridis, but it is possible that this uORF gene fragment is an amplification of a portion of a pseudo gene. However, since all indel mutations
of this uORF gene segment in the Tallapoosa Darter and the Coosa Darter
maintain an open reading frame (data not shown), it is likely that this gene
is functional in the darters.
Some of the genomic DNA samples previously isolated for analysis of
Tallapoosa Darter population structure (Brogdon et al. 2003) were utilized
in this study. Specifically, DNA was utilized from the three most genetically
divergent sets of populations previously identified as MUs by mitochondrial
DNA sequence analysis: MU 1—the population in the upper Tallapoosa
River system (8 individuals), MU 2—the population in the Little Tallapoosa
River system (10 individuals) and the population in the portion of the Tallapoosa
River system just south of Harris reservoir (8 individuals), and MU
3—the population in Enitachopco Creek (5 individuals).
To minimize costs of identifying nucleotide differences fixed in each MU
sample, PCR amplifications were performed on combined genomic DNA
from multiple individuals. Equal amounts of genomic DNA from individuals
Table 1. Primers that amplify gene segments diagramed in Figure 1. Letters that follow gene
designation in the primer name correspond to exons as labeled in Figure 1.
Gene Primer name Primer sequence 5’ → 3’
uORF uorf-sense CTCTTCTATTTTGCAGCACC
Kelch kelch-sense GCAAGAAACCAGGCTAAACA
GAPDH gapdh-sense GAGTCCACAGGTGTATTCACA
LPL lplAC-sense CCCGATTCCCGTCACTCT
PLC plcAC-sense CAGCAGCCAAGAAGAACTCA
S6 s6AB-sense CAAGGGTCACTCCTGCTACCG
2010 L.G. Kral 377
of each population were combined to form four population samples, and each
sample was amplified in 50-ul reactions containing 100 ng of the combined
DNA, 25 ul of Qiagen HotStarTaq Master Mix, and 0.5 uM concentration of
a sense primer and 0.5 uM concentration of the matching antisense primer.
The PCR conditions were identical for all twelve primer pair reactions:
95 °C for 15 minutes to activate the Taq polymerase, followed by 35 cycles
of 30 seconds at 94 °C, 1 minute at 55 °C, and 2 minutes at 72 °C. The final
cycle was followed by a 10-minute incubation at 72 °C.
Amplified PCR products were gel purified using the Qiagen Qiaquick Gel
Extraction kit or the Zymogen Zymoclean Gel DNA Recovery kit. The purified PCR products were then sequenced directly utilizing both the sense and
antisense PCR primers as sequencing primers. The purified PCR products
were also cloned into pSMART GC HK plasmids utilizing the Lucigen GC
Cloning and Amplification Kit according to manufacturers instructions, and
clones of the PCR products were sequenced utilizing the flanking SL1 and
SR2 primers. Sequencing was carried out by Functional Biosciences, Inc.,
Madison, WI (www.functionalbio.com).
Initial contig assembly from sequence alignments of direct PCR product
reads as well as two to three clones of each gene fragment were carried out in
GeneJockey II DNA analysis software (Biosoft). All variable site differences
that were fixed in the population samples were identified by aligning gene
contigs of the four population samples with the direct PCR product sequence
traces in Geneious Pro bioinformatics software (Biomatters, Ltd.), and chromatogram
peaks at each variable site were examined to ascertain lack of
nucleotide heterogeneity within each population. Eight independent clones
of each of the twelve PCR-amplified gene fragments from the combined Enitachopco
Creek genomic DNA samples were also sequenced, and sequence
heterogeneity observed among the cloned sequences was compared to that
observed in sequence traces obtained from a population mixture of PCRamplified DNA.
Sequences of these six genes were also determined from individual Coosa
Darter and Holiday Darter samples by sequencing of both the direct PCR
products as well as two to three clones of those PCR products as described
above. Phylogenetic analyses were conducted using MEGA 4.0 software
(Kumar et al. 2008, Tamura et al. 2007).
Exact test of population differentiation was carried out with ARLEQUIN
population genetics software version 3.1 (Excoffier et al. 2005, Raymond
and Rousset 1995).
A total of about 10,900 base pairs of single-copy genomic DNA was sequenced
in each of three previously defined Tallapoosa Darter MUs, and 95
sites that are variable among these MUs were identified (Table 2). Of these
95 sites, 75 are single-nucleotide polymorphisms (SNPs), 18 are indels, and
2 are GT dinucleotide simple tandem repeats (STRs). Of the 93 sites that are
378 Southeastern Naturalist Vol. 9, No. 2
either SNPs or indels, 89 of the variants have been verified as being fixed in
each of the MU samples by examination of sequence traces. Four SNP sites,
identified as variable among the populations from sequences of cloned fragments,
could not be verified as being fixed among the population samples
because the sequences of that portion of the gene fragment in combined
population samples were not of sufficient quality. Verification that heterogeneous
sites would be detectable in sequence traces obtained from combined
DNA samples was obtained by a comparison of the sequence data from combined
Enitachopco Creek DNA samples and eight individually sequenced
clones of those DNA samples. The sites that were heterogeneous within the
direct PCR product sequence chromatograms of the combined population
sample proved to also be variable among the individual cloned sequences.
The approximately 10,900 base pairs of DNA sequenced in this study are
composed of about 7000 base pairs of intron sequence and about 3900 base
pairs of exon sequence. The two STR loci, 17 of the 18 indels, and all but 13
of the 75 SNPs are located in the intron regions. All but one of the 13 SNPs
in the exon regions result in synonymous substitutions. The one SNP that
resulted in a non-synonymous substitution results in an arginine to a serine
substitution in the uORF gene. The one indel located in an exon region is a
deletion of two amino acids from the uORF sequence present in the upper
Tallapoosa River and Enitachopco Creek populations that maintains the same
Table 2. Nuclear gene sequence variation among populations. Only variable sites are shown
at their respective base numbers. Abbreviations within reference sequence: I = indel (+ indicates
sequence present and – indicates sequence absent), S = STR (L, M, and S indicate
relative length). Abbreviations of populations: TR = Tallapoosa River, EC = Enitachopco
Creek, and LT = identical sequence in both Little Tallapoosa River and south of Harris
Population uOR FK GAPDH LPL PLC
0000000 00 0000000 00000000000 000000000000000000
0000001 11 1222222 23333333444 555666666666667777
3466790 14 9112677 90223359036 148113466788991345
8223322 83 5385606 30691458103 090586857925066407
1003813 50 0715139 24717666398 921378107141218060
GAITTAT TA TGTTTTC ACCACACGIST GTIASCAGAATTCAIIGG
TR ..-.... .. ....... ........+M. ..+TL..C....A.-+.A
EC C.-.... A. .T..... ........+L. ..+.S........G--..
LT .C+CCTC .G C.ACCCT TTTGTCTT-SC AG-.MTG.TGCG..++A.
2010 L.G. Kral 379
reading frame as the sequence present in the Little Tallapoosa River population
as well as in the Coosa Darter and the Holiday Darter (data not shown).
Variants of 89 SNP/indel sites appear to be fixed in each of the three
MU samples. These 89 variable sites define three ancestral alleles. At least
another 20 variable sites are not fixed among the population samples (data
not shown) and represent variations in alleles derived from the three ancestral
alleles. Thus, the three ancestral alleles and their derivatives are fixed
among the Tallapoosa Darter population samples of the three separate MUs.
No attempt has been made to reconstruct the derived alleles since the intra
population variable sites were primarily identified in sequence traces of
mixed-population PCR-amplified gene fragments and three of the complete
gene sequences were reconstructed from multiple fragments.
Statistical tests of population divergence can not be performed directly
because population sequence data was obtained from combined DNA samples
and not from individuals. A single ancestral allele type was detected in the
combined sequence data from 5 to 10 individuals of a population (potentially
10 to 20 different alleles), but it is possible that a few copies of the other
ancestral alleles may be present in the sample and these escaped detection.
In an attempt to determine if the combined data of this small sample size
could support nuclear genetic divergence among populations, an exact test
of population differentiation (Raymond and Rousset 1995) was performed
utilizing worst-case scenario where each population sample contained only
80% of the detected allele and 10% of each of the other two alleles. The
populations previously defined as MUs show significant differences under
these simulated conditions (Table 3). Note that the Little Tallapoosa River
and south of Harris Reservoir populations are part of the same MU and show
no significant differentiation.
Maximum parsimony (MP), neighbor joining, minimum evolution,
and UPGMA analyses of individual genes and concatenated sequences
of all genes were performed for all three Tallapoosa Darter MUs as well
as the Holiday Darter and the Coosa Darter. All analyses of concatenated
sequences resulted in the same tree topologies as the MP tree shown in
Figure 2. Twenty-one of the 327 variable sites in the concatenated gene
sequences among the three species were parsimony-informative. Topologies
of trees obtained for individual genes were essentially concordant with the
concatenated dataset except for the uORF gene, where the Holiday Darter
Table 3. Nuclear genetic differentiation of Tallapoosa Darter populations under simulated
condition of 80% population specific allele frequency. Significance level (P-value) of nondifferentiation
test. Abbreviations: NS = not significant, TR = Tallapoosa River, LT = Little
Tallapoosa River, HR = south of Harris Reservoir, and EC = Enitachopco Creek.
TR LT HR
LT 0.00319 ± 0.0005
HR 0.00558 ± 0.0006 1.0 (NS)
EC 0.02853 ± 0.0014 0.01790 ± 0.0013 0.02555 ± 0.0016
380 Southeastern Naturalist Vol. 9, No. 2
partitions within the Tallapoosa Darter clade (data not shown). The uncorrected
pairwise sequence divergence of the concatenated genes between the
Tallapoosa Darter populations ranged from 0.17% to 0.55% (Table 4), and
the average distance between Tallapoosa Darter populations and the Holiday
Darter averaged 0.84% (0.80% to 0.88%, Table 4).
Analysis of nuclear DNA sequences showed that the Tallapoosa Darter
populations previously identified as being genetically diverged for mitochondrial
haplotypes (Brogdon et al. 2003) are also genetically diverged
for single-copy nuclear protein-coding genes. Of the approximately 10,900
nucleotides sequenced, 89 variable sites have been confirmed as fixed among
the population samples analyzed. These fixed differences show that the MU
samples are each homogeneous for one major ancestral allele type at each
of the six loci examined, and heterogeneity within each MU sample is comprised
of variants of those ancestral allele types. The sample size is sufficient
to determine that significant nuclear gene divergence is partitioned among
the MUs (Table 3), but the total number of individuals examined is not great
enough to conclude that these allele-frequency differences are fixed in the
actual populations from which the samples were analyzed.
The variation observed at the six gene fragments examined in these MU
samples is, with the possible exception of the two codon deletion and nonsynonymous
substitution in the uORF gene, most likely neutral variation.
Table 4. Uncorrected sequence divergence of combined nuclear gene sequences. Abbreviations:
TR = Tallapoosa River, EC = Enitachopco Creek, LT = Little Tallapoosa River, BR = Holiday
Darter, and CS = Coosa Darter.
TR EC LT BR
LT 0.55% 0.51%
BR 0.85% 0.80% 0.88%
CS 2.55% 2.51% 2.61% 2.78%
Figure 2. Maximum parsimony phylogram of combined nuclear gene sequences.
Bootstrap values (1000 replicates) are given at branches. Abbreviations: TR = Tallapoosa
River, EC = Enitachopco Creek, LT = Little Tallapoosa River, BR = Holiday
Darter, and CS = Coosa Darter.
2010 L.G. Kral 381
The high level of allelic divergence present among these MUs suggests that
any non-neutral or potentially non-neutral mutations present in the genome
are likely not homogeneously distributed among the populations. Since no
obvious morphological differences are evident in these genetically divergent
populations, such potentially non-neutral variation would represent cryptic
variation that may be adaptive (potential adaptive variation) under new
environmental conditions or change in genetic background such as future
hybridization between populations or accumulation of new mutations (Le
Rouzic and Carlborg 2007).
Brogdon et al. (2003) designated populations of the Tallapoosa Darter as
MUs based on mitochondrial sequence divergence, and this study characterized
these MUs as showing a high degree of nuclear gene variation. These
results strengthen the need to monitor these populations as MUs since it is
likely that potential adaptive variation is sequestered among these populations.
Maintenance of such variation enhances the evolutionary potential
of the species. Fixation of advantageous traits by drift or selection is more
likely and occurs faster if advantageous alleles are already present with
multiple copies in a population than if such alleles have to arise de novo by
mutation (Barrett and Schluter 2008).
A number of studies have demonstrated that clades defined by mitochondrial
haplotype variation within a darter species are distributed among river
drainages (Krabbenhoft et al. 2008, Piller et al. 2008, Powers et al. 2004,
Ray et al. 2006), as well as allopatrically distributed among tributatries
within major drainages (Hollingsworth and Near 2009, Krabbenhoft et al.
2008). Etheostoma blenniodes Rafinesque (Geenside Darter) clades defined
by intron 1 of the S7 ribosomal protein gene also are distributed among river
drainages (Piller et al. 2008), and populations of Etheostoma mariae Fowler
(Pinewood Darter) showed significant genetic structuring based on AMOVA
of S7 intron 1 sequence analysis (Krabbenhoft et al. 2008). Segregation of
nuclear gene variation is, therefore, likely sequestered among populations
of other darter species as well. It may well be worthwhile to apply the multigenic
analysis presented in this study to populations of other darter species
to more fully define the segregation of nuclear genetic variation among their
populations. As new low-cost, high-throughput sequencing methodologies
are becoming available, a more thorough quantitative study of such variation
should become practical in the near future. The elucidation of darter
evolution will be more complete if an analysis of a significant sampling of
nuclear gene variation complements analysis of mitochondrial and microsatellite
markers as well as morphological variation.
Along with the studies of Krabbenhoft et al. (2008) and Hollingsworth
and Near (2009), this study shows that variation within a darter species does
not only occur among major drainages, but also can occur among tributaries
within a drainage. This result shows that allopatric darter evolution occurs
at much smaller geographic scales than previously thought. This phenomenon
is common across three major eastern US drainages, the Atlantic slope
382 Southeastern Naturalist Vol. 9, No. 2
(Krabbenhoft et al. 2008), the Cumberland (Hollingsworth and Near 2009),
and the Mobile (this study). The relatively low-cost method utilized in this
study is effective in identifying nuclear genomic variation at these small
It has also been demonstrated that multigenic analysis based on the six
genes utilized in this study is suitable for analysis of darter phylogeny.
The cladograms obtained from multigenic analysis (Fig. 2) are concordant
with cladograms of the same species obtained by mitochondrial sequence
analysis (Brogdon et al. 2003, Porter et al. 2002). At the population level,
analysis of the Tallapoosa Darter mtDNA haplotype data from Brogdon et al.
(2003) shows that the Tallapoosa River MU and the Little Tallapoosa River
MU are evolutionarily about equidistant from the Enitachopco Creek MU.
However, multigenic analysis shows that the Tallapoosa River MU and the
Enitachopco Creek MU are more closely related to each other than either is
to the Little Tallapoosa MU (Fig. 2, Table 4). This discrepancy is likely due
to faster lineage sorting of the haploid unisexually transmitted mitochondrial
genome, which would be more susceptible to genetic drift than the nuclear
genome. Furthermore, the multigenic analysis is based on a larger number
of presumably independently assorting genes, whereas mitochondrial haplotypes
represent essentially a single locus. Thus, it is likely that multigenic
analysis may provide a better understanding of the evolutionary relatedness
of populations than mitochondrial sequence analysis in situations where
populations are divergent at nuclear loci.
The major problem with reconstruction of phylogenies with nuclear
genes is that sorting of gene lineages may be independent of speciation
events (Maddison 1997, Moore 1995, Nichols 2001). That is, alleles of genes
that have arisen prior to any speciation events may be stochastically sorted
during subsequent speciation events. Under these conditions, phylogeny
reconstruction using single genes may be incongruent. An example of this is
seen in the phylogeny of the Tallapoosa Darter based only on the uORF gene,
where Tallapoosa Darters and the Holiday Darter are not resolved as separate
lineages (data not shown). Utilization of multiple genes increases accuracy.
Utilizing a dataset of 106 orthologous genes in eight species of yeast, Rokas
et al. (2003) found that concatenations of 20 or more genes provided accurate
trees with >95% bootstrap values at each branch. Bootstrap support
of >70% was obtained with as few as three concatenated genes. Using computer
simulations, Gadagkar et al. (2005) have found that concatenations of
10 genes provide >95% accuracy. The phylogenetic trees were generated in
this study from concatenated gene sequences. It should be noted that under
some conditions concatenated data do not produce accurate species trees
(Kubatko and Degnan 2007). A number of statistical methodologies are being
developed to estimate species trees from multiple nuclear gene and allele
DNA sequence data (Brumfield et al. 2008, Edwards et al. 2007, Liu et al.
2008). Regardless of methodology, it will probably be necessary to develop
primers for more single-copy nuclear genes than the six genes utilized in this
study to increase accuracy of estimated darter species relationships.
2010 L.G. Kral 383
I thank Ian Stansbury, Catherine Singleton, Levy Trusty, Gregory Ayuk, and Ivey
Holland, students in the department of biology at the University of West Georgia, for
laboratory assistance during various phases of this project. I also thank Thomas Near
for DNA samples of E. coosae and E. brevirostrum. I further thank all the reviewers
of this manuscript for many helpful comments. This study was supported by Faculty
Research Grant and Faculty Research Enhancement Award funds obtained from the
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