Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Free from mitochondrial DNA: Nuclear genes and the inference of species
trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae)
Thomas J. Near a,⇑, Benjamin P. Keck b
a
b
Department of Ecology and Evolutionary Biology and Peabody Museum of Natural History, Yale University, New Haven, CT 06520, USA
Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e
i n f o
Article history:
Received 2 September 2012
Revised 7 November 2012
Accepted 8 November 2012
Available online xxxx
Keywords:
Molecular clock
Percomorpha
Phylogeny
a b s t r a c t
Investigations into the phylogenetics of closely related animal species are dominated by the use of mitochondrial DNA (mtDNA) sequence data. However, the near-ubiquitous use of mtDNA to infer phylogeny
among closely related animal lineages is tempered by an increasing number of studies that document
high rates of transfer of mtDNA genomes among closely related species through hybridization, leading
to substantial discordance between phylogenies inferred from mtDNA and nuclear gene sequences. In
addition, the recent development of methods that simultaneously infer a species phylogeny and estimate
divergence times, while accounting for incongruence among individual gene trees, has ushered in a new
era in the investigation of phylogeny among closely related species. In this study we assess if DNA
sequence data sampled from a modest number of nuclear genes can resolve relationships of a species-rich
clade of North American freshwater teleost fishes, the darters. We articulate and expand on a recently
introduced method to infer a time-calibrated multi-species coalescent phylogeny using the computer
program BEAST. Our analyses result in well-resolved and strongly supported time-calibrated darter species tree. Contrary to the expectation that mtDNA will provide greater phylogenetic resolution than
nuclear gene data; the darter species tree inferred exclusively from nuclear genes exhibits a higher frequency of strongly supported nodes than the mtDNA time-calibrated gene tree.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Since the early 1980s, molecular phylogenetic studies of closely
related animal species and lineages have relied heavily on the use
of mitochondrial (mtDNA) gene sequences. The reasons for the
utility and frequent use of mtDNA are well known and extensively
reviewed elsewhere (e.g., Avise, 2004). Over the past 10 years a
growing number of studies demonstrated a high prevalence of
mtDNA introgression among closely related animal species (Shaw,
2002; Chan and Levin, 2005), which is often characterized by little
or no introgression of alleles from nuclear genes (Bossu and Near,
2009; Keck and Near, 2010). For example, a recent investigation of
the phylogenetics of darters, a species-rich freshwater fish clade
endemic to North America, discovered that more than 12% of the
248 species contained mtDNA genomes of a heterospecific origin
(Near et al., 2011). This high frequency of mtDNA transfer among
closely related species diminishes the enthusiasm for mtDNA as
a primary source of data to infer the phylogenetics of the most apical branches in the Animal Tree of Life.
Coupled with this reappraisal of the role for mtDNA in the inference of animal phylogeny is the fact that the science of phylogenet⇑ Corresponding author.
E-mail addresses: [email protected] (T.J. Near), [email protected] (B.P. Keck).
ics is in the early stages of a transformation. The standard protocol
that has developed over the past three decades for analysis of
nucleotide data sampled from multiple genes is to concatenate
the data into a single matrix and effectively treat the data as a single gene dataset. Earlier debates in phylogenetics centered on the
issue of whether independent datasets, such as phenotypic and
molecular data, should be combined or analyzed separately (e.g.,
Kluge, 1989; Bull et al., 1993). However, recent skepticism of the
efficacy of data concatenation in producing robust estimates of
phylogeny stems from the realization that individual gene trees
may not be congruent with the underlying species phylogeny
and that gene tree heterogeneity is commonly observed regardless
of the taxonomic scope of the phylogenetic investigation (Edwards,
2009). Processes of lineage sorting and ancestral polymorphism
will result in gene trees that are incongruent among genes and
with the containing species phylogeny, particularly in situations
where lineages have recently diversified or where the lineage’s history is characterized by short time intervals between lineage diversification events (Tajima, 1983; Pamilo and Nei, 1988; Avise, 1989;
Takahata, 1989; Maddison, 1997). In such instances, the standard
practice of data concatenation can result in an erroneous estimate
of the species phylogeny, often with strong node support for incorrectly inferred clades (Gadagkar et al., 2005; Degnan and Rosenberg, 2006; Kubatko and Degnan, 2007). The most likely gene
1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.11.009
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
2
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
tree that evolves in the context of such a species tree will be incongruent with the species phylogeny and combining data to infer the
most commonly observed gene tree ‘‘can be asymptotically guaranteed to produce an incorrect estimate’’ of the species phylogeny
(Degnan and Rosenberg, 2006). Such anomalous gene trees may
not pose a substantial problem to empirical estimation of the species tree, as mutational variance across individual loci lessens the
likelihood that the most frequently observed gene tree is incongruent with the species phylogeny (Huang and Knowles, 2009); however, the concatenation method still fails to adequately convey and
accommodate appreciable heterogeneity among individual gene
trees in phylogeny inference. Fortunately, newly developed ‘‘species tree’’ methods allow the estimation of species phylogenies
from multilocus data that considers the processes of lineage sorting and expected patterns of gene tree heterogeneity and deep coalescence (Maddison and Knowles, 2006; Edwards et al., 2007;
Heled and Drummond, 2010).
Most applications of species tree methods are focused on closely related species radiations (e.g., Kubatko et al., 2011; McCormack et al., 2011), but these methods have also been applied to
deep time phylogenetic investigations of major clades, such as
mammals, turtles, and iguanian lizards (Thomson et al., 2008; Liu
et al., 2009; Barley et al., 2010; Meredith et al., 2011; Townsend
et al., 2011). Still lacking, however, is a study that simultaneously
infers a species tree and estimates divergence times and for a clade
of relatively high diversity that has its origins earlier than the Late
Miocene, and with taxon sampling that allows resolution of nodes
that range from the youngest to the oldest in the clade. Reconstructing phylogenies for such clades is especially challenging if
there is pervasive disagreement between gene trees and underlying species phylogeny.
In this study we apply a species tree inference method to darters, a species rich clade of freshwater fishes endemic to eastern
North America that have been the subject of several molecular
phylogenetic studies using DNA sequences of mitochondrial genes,
or combinations of nuclear and mitochondrial DNA sequence data.
The ages of darter lineages span from approximately 35 million
years (Ma) to less than 1 Ma (Near and Benard, 2004; Hollingsworth and Near, 2009; Near et al., 2011) and the clade as a whole
offers an opportunity to determine if a set of nuclear gene sequences can resolve phylogenetic relationships among closely related animal lineages, and assess the performance of species tree
inference methods for clades with diversification events that span
appreciable breadths of evolutionary time. Our results indicate that
resolution of phylogenetic relationships among closely related animal species and lineages is possible with DNA sequences sampled
from a modest number of nuclear genes, and coalescent based species tree analyses provide robust and well-supported phylogenetic
trees with fairly dense taxon sampling. Relative to inferences from
species tree analyses, phylogenies inferred from data concatenation appear to overestimate support for relationships among darter
lineages, but levels of node support observed in the species tree are
slightly higher than the mtDNA gene tree.
2. Materials and methods
2.1. DNA sequencing, alignment, and model selection
The specimens of the 92 species used in this study are a subset
of the specimens of 245 darter species included in the phylogenetic
analyses presented in Near et al. (2011), except Etheostoma fusiforme collected from Canoochee Creek, Emanuel County, Georgia,
USA (YFTC 6174), Nothonotus maculatus collected from the Elk River, Clay County, West Virginia USA (YFTC 9939), and Percina stictogaster collected from the South Fork of the Kentucky River, Clay
County, Kentucky USA (YFTC 807). Species sampling is detailed in
Supplementary Table S1. Two species, Perca flavescens and Sander
vitreus, were used as outgroups in all phylogenetic analyses. These
two species are classified, along with darters, in Percidae (Collette
and Banarescu, 1977; Wiley, 1992; Song et al., 1998). The darter
species sampled for this study were selected to include all of the
major lineages in the clade (e.g., Near et al., 2011), as well as providing phylogenetic contrasts of closely related species.
Protocols used in DNA extractions and the PCR amplification and
sequencing of the RAG1 exon 3 locus are given in Near et al. (2011).
The polymerase chain reaction was used to amplify 12 additional
nuclear encoded exons using primers and PCR conditions provided
in Li et al. (2007, 2010) for ENC1, Glyt, plagl2, Ptr, SH3PX3, Sidkey,
sreb2, tbr1, UBE3A, UBE-like, zic1, and znf. Double-stranded amplification products were desalted and concentrated using AMPure
beads (Agencourt Biosciences, Beverly, MA). Both strands of the
purified PCR fragments were used as templates and amplified for
sequencing using the original amplification primers and a Prism
Dye Terminator Reaction Kit (Applied Biosystems, Foster City, CA)
with minor modifications to the manufacturer’s protocols. The
nucleotides were sequenced on a 3700 or 3730XL automated DNA
sequencer (Applied Biosystems, Foster City, CA). Contiguous sequences were built using Sequencher (Gene Codes, Ann Arbor, MI)
from DNA sequences of the complementary heavy and light strands.
All of the genes used in our phylogenetic analyses are proteincoding, therefore, the DNA sequence alignments were constructed
from alignments of the translated amino acid sequences constructed using the computer program MUSCLE (Edgar, 2004). The
combined 13-gene dataset contained 10,779 base pairs. Each gene
was treated as a separate data partition and the optimal molecular
evolutionary model for each gene was determined through model
fitting and using the Akaike Information Criterion (AIC) as executed
in the computer program MrModeltest 2.3 (Nylander, 2004).
The presence of recombination was assessed for each of the 12
nuclear loci using the program GARD as implemented at the website www.datamonkey.org (Kosakovsky Pond et al., 2006a,b). This
method uses a genetic algorithm and phylogenetic trees to search
for putative recombination break points, identifies their locations,
and identifies clades involved with the recombination events using
AIC (Kosakovsky Pond et al., 2006a).
The phylogenetic resolution in the nuclear gene inferred species
tree was compared to that from a time-calibrated mtDNA gene
tree. An alignment of the mtDNA gene cytb for 248 darter species
that was used in Near et al. (2011) was subsampled for the 92 species included in the alignments of the 12 nuclear genes. Bayesian
posterior node support was plotted against the estimated age of
the node for both the time-calibrated nuclear species tree and
the cytb time tree.
2.2. Determining the age priors
In our effort to estimate divergence times of darter lineages
using a time calibrated multi-species coalescent species tree analysis, we used age priors for key nodes in the darter phylogeny that
were estimated from a relaxed molecular clock analysis using three
genes sampled for 245 of the 248 known darter species (Near et al.,
2011). The dataset contained a single mtDNA gene (cytb) and two
nuclear loci (S7 intron 1 and RAG1 exon 3), and the cytb sequences
from species with a known history of mtDNA introgression were
treated as missing data (Table 4 in Near et al., 2011). Divergence
times were estimated from the combined three gene dataset using
an uncorrelated lognormal (UCLN) model of molecular evolutionary rate heterogeneity as implemented in the computer program
BEAST version 1.6.1 (Drummond et al., 2006; Drummond and Rambaut, 2007). The posterior molecular evolutionary rate estimates of
each gene were estimated from a set of fossil-calibrated UCLN re-
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
laxed clock analyses of the percomorph lineage Centrarchidae and
the posterior rate estimates were used as rate priors to estimate
divergence times of darters. These rates are presented in Near
et al. (2011: Table 2). The mean and highest posterior density of
the centrarchid molecular evolutionary rates were used to construct a normal prior on the molecular evolutionary rate for each
of the three genes. The molecular evolutionary model used for each
gene is presented in Near et al. (2011). The molecular clock and
molecular evolution models were partitioned by gene, a birth–
death branching prior was used, and the UCLN BEAST analyses
were run three times where each run consisted of 3.0 107 generations. Plotting the marginal posterior probabilities using the computer program Tracer version 1.5 assessed convergence of model
parameter values and estimated node heights to their optimal posterior distributions.
2.3. Bayesian inference of gene trees and concatenated data phylogeny
Estimation of gene trees from each locus and phylogenetic analysis of the concatenated nuclear gene dataset were performed
using a parallel version of the computer program MrBayes version
3.1.2 (Ronquist and Huelsenbeck, 2003; Altekar et al., 2004) on a
Linux cluster. For each analysis MrBayes was run three separate
times for 5.0 107 generations with two simultaneous runs each
with four chains (one cold and three heated chains with a heating
parameter = 0.02 to ensure appropriate mixing). The cold chain
was sampled once every 5000 generations. The ‘‘preset
ratepr = variable’’ command was used to accommodate amongpartition rate variation (Marshall et al., 2006).
Stationarity of the chains and convergence of the Metropoliscoupled Markov chain Monte Carlo algorithm were assessed by
plotting the likelihood score and all other parameter values against
the generation number to determine when there was no increase
relative to the generation number using the computer program Tracer version 1.5 (Drummond and Rambaut, 2007). Measuring the
average standard deviation of the split frequencies between those
runs also assessed convergence; it was assumed that the chains
had reached stationarity when this value was less than 0.005. The
first 30% of the sampled generations were discarded as burn in
and the set of posterior phylogenies were summarized in 50%
majority-rule consensus trees. The posterior probability for a given
clade was the frequency that the clade was present among the posterior trees, which translates to the probability that the lineage is
monophyletic given the model and the data (Larget and Simon,
1999; Huelsenbeck and Rannala, 2004). We follow the standard
practice in Bayesian phylogenetics in interpreting a given node in
the summarized posterior phylogeny as strongly supported if the
clade is present in 95% or greater in the posterior distribution of
trees (e.g., Alfaro et al., 2003; Huelsenbeck and Rannala, 2004).
2.4. Estimation of a time-calibrated species tree
The computer program BEAST version 1.6.1 was used to perform a set of multi-species coalescent analyses to estimate a
time-calibrated species tree for the 92 sampled darter species
(Heled and Drummond, 2010). The species trees were inferred
using the 12 nuclear genes that were sequenced for all of the sampled species. The sreb2 locus was not included in the BEAST species tree analyses because five darter species were not sequenced
for the gene (Supplementary Table S1). The computer program
BEAUTI 1.6.1 was used, along with the individual gene alignments
in nexus format, to construct the initial XML files for the BEAST
species tree analyses. The molecular clock and molecular evolutionary models were unlinked across the 12 loci, the UCLN model
of molecular evolutionary rate heterogeneity was used for all loci,
the nucleotide substitution models for each gene were the same as
3
those used in the MrBayes gene tree analyses, and the molecular
evolutionary rate for each locus was estimated. The gene trees inferred from each locus were unlinked, and a birth–death speciation
prior was used (Drummond et al., 2006). A previous study used
external age information to time calibrate a species tree (McCormack et al., 2011), but we outline, in detail, the three steps involved with editing the XML file after an initial set up using
BEAUTI for a time-calibrated multi-species coalescent analysis in BEAST. First, we included a tree in newick format with branch
lengths that were not in conflict with the age priors in the species
tree command with the syntax:
<speciesTree id=‘‘sptree’’ constantRoot=‘‘true’’>
<species idref=‘‘species’’/>
<sppSplitPopulations value=‘‘0.045’’>
<parameter id=‘‘speciesTree.splitPopSize’’/>
</sppSplitPopulations>
<newick>
NEWICK TREE WITH BRANCH LENGTHS GOES HERE;
</newick>
</speciesTree>
Second, a definition for the time to the most recent common
ancestor (tmrca) statistic was constructed for any nodes in the species tree that are calibrated following the syntax:
<tmrcaStatistic id=‘‘Clade_A’’ includeStem=‘‘false’’>
<speciesTree idref=‘‘sptree’’/>
<mrca>
<taxa>
<sp idref=‘‘Species_A’’/>
<sp idref=‘‘Species_B ’’/>
<sp idref=‘‘Species_C ’’/>
<sp idref=‘‘Species_D ’’/>
</taxa>
</mrca>
</tmrcaStatistic>
Third, the external age information is included in the prior commands. In this case an age prior with a normal distribution was used
because the age was taken from the mean and highest posterior
density age estimate from another BEAST analysis:
hnormalPrior mean ¼ \X" stdev ¼ \Y"i
hstatistic idref ¼ \Clade A"=i
h=normalPriori
where the mean age of the prior, ‘‘X’’ matches the mean age from
the posterior distribution of ages from the three gene analysis and
the standard deviation ‘‘Y’’ is set to reflect the 95% HPD of the posterior age estimate from the three gene analysis in the width of the
normal distributed age prior in the BEAST species tree analysis. The
age prior can be modified as lognormal or exponential distributions
if prior age information is obtained from the fossil record (e.g., Ho
and Phillips, 2009; Dornburg et al., 2011).
Five nodes were calibrated with age priors: the most recent
common ancestor (MRCA) of all darters, the MRCA of Nothonotus
and Etheostoma, the MRCA of Percina, the MRCA of Nothonotus,
and the MRCA of Carnipellucida. The monophyly of all these nodes
was enforced using ‘‘booleanLikelihood’’ priors on the ‘‘monophylyStatistic’’ commands in BEAST. The MRCA of Etheostoma
was not calibrated because we were interested in assessing the
monophyly of this lineage in the multi-species coalescent species
tree analyses. Details on the age priors derived from the three gene
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
4
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
BEAST UCLN analyses are given in Table 1. The chain lengths were
3 108 generations with parameters sampled every 103 generations. Convergence of parameters values in the BEAST Markov
chain Monte Carlo were assessed by the effective sample sizes that
were calculated using Tracer version 1.5 and visualizing the cumulative split frequencies in the set of posterior trees using AWTY
(Nylander et al., 2008). Generations sampled before convergence
was attained were discarded as burn-in. For comparisons of clade
support (outlined below), a time-calibrated gene tree was inferred
using mtDNA cytb gene sequences from the same set of 92 darter
species sampled for the nuclear genes and calibration settings outlined above. Details on the collection of the cytb data are provided
in Near et al. (2011). The effect of multiple calibration densities on
the marginal prior distribution of the calibrated nodes was assessed by running an empty alignment in BEAST (e.g., Heled and
Drummond, 2012; Warnock et al., 2012).
2.5. Comparison of phylogenetic resolution in the nuclear gene species
tree and the mtDNA gene tree
We assessed if the time-calibrated mtDNA gene tree provided
greater phylogenetic resolution than the nuclear gene-inferred species tree by plotting the posterior probability clade support values
versus the estimated age of the node for the BEAST mtDNA gene
tree and the BEAST nuclear gene species tree. In addition, the proportion of nodes supported with strong Bayesian posterior values
(P0.95) were recorded in each of the two phylogenies for three
age intervals (0–10 Ma, 10–20 Ma, and 20 Ma to the root node).
3. Results
3.1. DNA sequence variation, gene trees, and concatenated data
analyses
The 92 sampled darter species were sequenced for each of the
13 nuclear genes, except Etheostoma artesiae, E. caeruleum, E. fusiforme, E. gracile, and E. olivaceum were not sequenced for sreb2
Table 1
Calibration priors used in BEAST analyses. All ages are given in millions of years (Ma).
Calibrated node
Mean of age
prior (Ma)
Standard
deviation
Credible interval on age
prior (Ma)
Etheostomatinae
NothonotusEtheostoma
Percina
Nothonotus
Carnipellucida
36.0
32.8
3.65
3.40
30.0, 42.0
27.2, 38.4
20.1
22.9
25.4
3.10
3.60
4.17
15.0, 25.2
17.0, 28.8
18.5, 33.6
Table 2
Characteristics of each sampled nuclear gene.
(Supplementary Table S1). The size of the sequenced fragment,
the number of variable sites, and the optimal molecular evolutionary models for each gene are given in Table 2. There was no detectable evidence of recombination in the 13 nuclear loci using GARD
as all genes showed no significant support for internal recombinational breakpoints in the aligned sequences (Kosakovsky Pond
et al., 2006a). All new sequences were deposited in GenBank
(accession numbers KC134418-KC135495).
The gene trees that exhibited the highest resolution and greatest
number of nodes with strong Bayesian posterior support were inferred from Glyt and RAG1, and the genes that provided the lowest
phylogenetic resolution in the inferred gene trees were tbr1 and
zic1 (Table 2). The maximum clade credibility tree summarized from
the posterior set of trees inferred from a Bayesian analysis of the
concatenated 13 nuclear gene dataset was highly resolved, and 85
of the 91 nodes in the phylogeny were supported with Bayesian posterior values greater or equal to 0.95 (Fig. 1). Species sampled from
the clades Carnipellucida (Ammocrypta and Crystallaria), Percina, Nothonotus, and Etheostoma were each monophyletic and supported
with strong (P0.95) Bayesian posterior probabilities (Fig. 1). In this
tree, the clade Percina was resolved as the sister lineage of all other
darters and the MRCA of Carnipellucida, Nothonotus, and Etheostoma
was supported with a strong Bayesian posterior (Fig. 1).
3.2. Resolution of the time-calibrated species tree
The posterior parameter value estimates from the BEAST multi-coalescent species tree analyses using 12 of the 13 sampled nuclear genes were characterized by high (>200) effective sample
sizes and convergence of the individual runs was confirmed from
assessments using both Tracer and AWTY. The maximum clade
credibility tree and divergence time estimates from the pooled
posterior set of species trees is presented in Fig. 2 where 62 of
the 91 nodes in the species tree were supported with strong
(P0.95) Bayesian posterior probabilities. The Etheostomatinae,
Carnipellucida, Percina, Nothonotus, and the MRCA of Nothonotus
and Etheostoma were constrained as monophyletic because these
nodes were calibrated with external age priors, but Etheostoma
was not constrained and resolved as monophyletic with a Bayesian
posterior support equal to 1.00 (Fig. 2).
Most of the inferred relationships are similar to the phylogenies
presented in Near et al. (2011) that used two nuclear genes and a
single mtDNA gene for a much larger taxon sampling of darters.
Important differences include the non-monophyly of the Percina
subclade Imostoma in the species tree and the resolution of a clade
containing the Etheostoma subclades Richiella, Oopareia, and Stigmacerca, which supports the recognition of the traditional arrangement of Catonotus (e.g., Page, 1975; Page et al., 1992; Porterfield
et al., 1999).
3.3. Phylogenetic resolution in the nuclear gene species tree as
compared to the mtDNA gene tree
Gene
Size in
base pairs
Number of
variable sites
Optimal
model
Nodes in gene tree with
Bayesian posterior P0.95
ENC1
Glyt
plagl2
Ptr
RAG1
SH3PX3
Sidkey
sreb2
tbr1
UBE3A
UBE-like
zic1
znf
810
813
672
705
1314
705
1107
891
579
615
594
729
1245
175
286
157
150
335
136
267
165
92
132
153
103
208
GTG+G
GTG+G
HKY+G
HKY+G
HKY+G
HKY+G
GTG+G
HKY+G
HKY+G
HKY+G
HKY+G
HKY
GTR+G
21
42
24
27
49
25
37
30
13
24
29
13
23
Plotting the clade posterior probability versus the estimated age
of the lineage showed no qualitative differences between the relationship of node support and clade age in the nuclear gene inferred
species tree and the mtDNA gene tree (Fig. 3). However, the proportion of clades with strong (P0.95) support were slightly higher
in the nuclear gene species tree than the mtDNA gene tree for the
most recent age interval, but were much higher for the two oldest
age intervals (Fig. 3).
4. Discussion
Historically, there has been reluctance on the part of animal
systematists to use sequence data from nuclear genes to investi-
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
5
Fig. 1. Phylogeny of 92 species of darters inferred from a partitioned Bayesian analysis of a combined nucleotide dataset consisting of 13 protein coding nuclear genes. Filled
black circles identify clades with Bayesian posterior support of 1.00 and unfilled circles identify clades with Bayesian support that ranges between 0.99 and 0.95. Branches in
the phylogeny are colored based on the major clade of the lineage: Percina is orange, Crystallaria is purple, Ammocrypta is blue, Nothonotus is green, and Etheostoma is red. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
gate phylogenetic relationships among closely related species because the low number of variable sites, relative to mtDNA genes,
would result in phylogenies with low resolution. The dependency
on the high mutation rate of mtDNA genes has led to strategies
in resolving the phylogeny of species-rich animal clades where
there is a dense taxon sampling of mtDNA genes that target the
most apical nodes, and a sub-sample of taxa for nuclear encoded
genes to resolve the deepest nodes in the tree (e.g., Wiens et al.,
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
6
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
Fig. 2. Maximum clade credibility time-calibrated species tree estimated from 12 protein coding nuclear genes using the multi-species coalescent in BEAST. Absolute time
scale is given in millions of years. The 95% highest posterior density of node age estimates are shown with bars with black nodes identifying clades with a Bayesian posterior
probability of 1.00, gray bars for clades with Bayesian support ranging between 0.99 and 0.95, and open bars labeling lineages with Bayesian posterior probability support less
than 0.95. Posterior probabilities are shown for nodes with less than 0.50 Bayesian support. Branches in the phylogeny are colored based on the major clade of the lineage:
Percina is orange, Crystallaria is purple, Ammocrypta is blue, Nothonotus is green, and Etheostoma is red. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
2005). The use of mtDNA to resolve the most closely related lineages in the Animal Tree of Life is plagued with the reliance on a
single locus to infer a species phylogeny, but also the presence of
mtDNA introgression will hamper attempts to exclusively use
mtDNA gene sequences for phylogeny inference (Shaw, 2002; Near
et al., 2011). Despite commonly expressed expectations, our analyses demonstrate that nuclear gene sequence data in phylogenetic
analyses of closely related animal species results in substantial
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
phylogenetic resolution, and strong support values for resolved
nodes in both data concatenated analyses and inferences of the
species tree (Figs. 1 and 2). Perhaps most surprising is that the nuclear gene species tree exhibited a slightly higher proportion of
nodes with strong Bayesian posterior support when compared
with the mtDNA gene tree, but as expected there were a much
higher number of nodes with strong support in the nuclear gene
species tree at the deepest contrasts in the darter phylogeny
(Fig. 3). The results from our analyses are important because they
demonstrate that nuclear DNA gene sequences can result in resolved phylogenetic hypotheses for closely related animal lineages.
In addition, we provided an articulation of a protocol to use external age information to time-calibrate species trees.
The time-calibrated multi-species coalescent phylogeny of darters results in age estimates that are younger for most nodes when
compared to the time-calibrated tree from analysis of individual
genes or a concatenated three-gene dataset (Near et al., 2011). This
difference in age estimates is expected because divergences among
gene copies are presumed to predate the inferred speciation
events; thus, divergence times estimated from analyses of gene
trees, or concatenated datasets, will tend to overestimate the ages
of diversification events on resolved phylogenies (Edwards and
Beerli, 2000). What is not known is how disparate these age estimates are in practice, and if posteriors from Bayesian age estimates
of concatenated and multi-species coalescent analyses will differ
substantially. Our time-calibrated trees are consistent with the
expectation of older age estimates in the concatenated relaxed
clock analysis and this disparity appears more pronounced for
younger clades. However, there was broad overlap in the posterior
age estimates when age estimates from the time-calibrated multispecies coalescent tree (Fig. 2) were compared with those from a
concatenated three-gene analysis (Near et al., 2011). For example
the age of the MRCA of Etheostoma from the three-gene concatenated relaxed clock analysis was 29.4 Ma, 95% highest posterior
density (HPD): [24.6, 34.7] and older than the estimated age for
this lineage in the time-calibrated species tree analysis 26.2 Ma,
95% HPD: [22.7, 29.7], while the differences for the estimated age
7
of the younger clade Atlantis was greater between the concatenated and species tree analyses, 12.7 Ma 95% HPD: [8.3, 17.3]
and 7.7 Ma 95% HPD: [5.3, 10.1]. The 95% HPD overlapped substantially in the estimates from the two different methods and magnitude in the width of the credible intervals did not appear to differ
between the two different relaxed clock analyses.
The phylogenies inferred from both the concatenated dataset
and the multi-species coalescent provides validation and insight
into relationships among darters presented in a recent phylogenetic investigation based on a single mtDNA gene and two nuclear
genes that sampled all but three of the 248 darter species (Near
et al., 2011). The dense taxon sampling and resolved phylogenetic
hypotheses were used to construct a rank-free, phylogeny-based
classification of darters. The phylogeny-based classification reflects
findings from analysis of molecular phylogenetic datasets that are
different from traditional darter classifications, namely the recognition of Nothonotus as a clade, or genus, distinct from Etheostoma
and phylogenetic resolution of Etheostoma cinereum as closely related to snubnose and greenside darters, all classified in a newly
named clade Simoperca (Near et al., 2011).
The elevation of Nothonotus as a genus by Near and Keck (2005)
based on molecular phylogenetic inferences has generated substantial critique (e.g., Mayden et al., 2006; Smith et al., 2011); however, phylogenetic analyses of AFLP markers, cannot reject the
hypothesis that Etheostoma forms a clade exclusive of Nothonotus
(Smith et al., 2011). On the other hand, the phylogenies resulting
from Bayesian analysis of the concatenated 13 nuclear gene dataset and the species tree multi-species coalescent analysis both
unequivocally support the distinction of Nothonotus relative to
Etheostoma, and there was not a single tree in the set of posterior
trees from these analyses that resolved any species of Nothonotus
as nested within Etheostoma (Fig. 1). While it is unfortunate that
long-standing generic classifications face revision in the context
of objective data-driven phylogenetic analyses, it is worth mentioning that systematists strive to produce and recognize classifications of organisms that reflect phylogenetic relationships. The
concept of Etheostoma that contained Nothonotus as a subgenus
Fig. 3. Plots showing the relationship between clade posterior probability and estimated clade age and proportion of strongly supported clades (Bayesian posterior P0.95)
among three age intervals for the nuclear gene species tree and a mtDNA gene tree (cytochrome b).
Please cite this article in press as: Near, T.J., Keck, B.P. Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related
darter lineages (Teleostei: Percidae: Etheostomatinae). Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.11.009
8
T.J. Near, B.P. Keck / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
was a hypothesis developed well before the introduction of phylogenetic methods, and was simply the group of darters for which no
morphological synapomorphies could be identified (Bailey et al.,
1954; Bailey and Gosline, 1955). Moreover, no published phylogenetic analysis of morphological or molecular data has resulted in a
phylogeny that is incongruent with our elevation of Nothonotus as
a clade distinct from Etheostoma (e.g., Ayache and Near, 2009; Near
et al., 2011; Smith et al., 2011). Analysis of the 13 nuclear genes has
validated our previous phylogenetic results and attempts to produce a phylogeny-based classification of darters, and specifically
our effort to recognize Nothonotus as a distinct clade and not
nested in Etheostoma.
Our inferences of darter phylogeny based on analyses of DNA
sequences sampled 13 exon regions serves as a prospectus for future studies aimed at resolving the most apical nodes in the Animal
Tree of Life that are free from mtDNA sequence data. As the limitations and pitfalls of mtDNA as a source of data for phylogenetic
studies become well known (e.g., Shaw, 2002; Funk and Omland,
2003), the obvious strategy is to explore the phylogenetic utility
of nuclear gene DNA sequences in resolving phylogenetic relationships among closely related species. An increasing skepticism towards the role of mtDNA in these efforts and the adoption of a
strategy that samples multiple nuclear genes is more amenable
to the changing field of molecular phylogenetics, which broadly
recognizes and accommodates the expected incongruence of gene
trees and the underlying species tree due to processes of lineage
sorting (e.g., Edwards, 2009). Phylogenetic trees are the basis for
scores of different comparative methods used to test fundamental
concepts in evolutionary biology that focus on the origin and diversification of biodiversity. As the field of phylogenetics moves forward with more sophisticated approaches finding optimal
phylogenetic trees, the scale of datasets collected from the nuclear
genome to resolve relationships among the most closely related
species will continue to increase dramatically (e.g., Hittinger
et al., 2010; Alföldi et al., 2011; Lemmon et al., 2012; Lemmon
and Lemmon, 2012).
Acknowledgements
B.H. Bauer, C.M. Bossu, G.R. Dinkins, D.A. Etnier, R.C. Harrington,
P.R. Hollingsworth, and C.D. Hulsey provided assistance in the field
and provided discussions on our approaches to resolving darter
phylogeny. K.L. Kuhn provided assistance in the laboratory and
J.P. Joice and G.J. Watkins-Colwell provided collections support.
We thank M.E. Alfaro and M.L. Niemiller for comments on earlier
versions of our manuscript. The Peabody Museum of Natural History and the National Science Foundation (DEB-0716155 and
DEB-1011328) provided support for this work.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.ympev.2012.11.009.
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