Species tree inference in a recent radiation of orioles (Genus Icterus): multiple markers and methods reveal cytonuclear discordance in the northern oriole group

Recent computational advances provide novel opportunities to infer species trees based on multiple independent loci. Thus, single gene trees no longer need suffice as proxies for species phylogenies. Several methods have been developed to deal with the challenges posed by incomplete and stochastic lineage sorting. In this study, we employed four Bayesian methods to infer the phylogeny of a clade of 11 recently diverged oriole species within the genus Icterus. We obtained well-resolved and mostly congruent phylogenies using a set of seven unlinked nuclear intron loci and sampling multiple individuals per species. Most notably, Bayesian concordance analysis generally agreed well with concatenation; the two methods agreed fully on eight of nine nodes. The coalescent-based method ^∗BEAST further supported six of these eight nodes. The fourth method used, BEST, failed to converge despite exhaustive efforts to optimize the tree search. Overall, the results obtained by new species tree methods and concatenation generally corroborate our findings from previous analyses and data sets. However, we found striking disagreement between mitochondrial and nuclear DNA involving relationships within the northern oriole group. Our results highlight the danger of reliance on mtDNA alone for phylogenetic inference. We demonstrate that in spite of low variability and incomplete lineage sorting, multiple nuclear loci can produce largely congruent phylogenies based on multiple species tree methods, even for very closely-related species.     

A multilocus phylogeny of Asian noodlefishes Salangidae (Teleostei: Osmeriformes) with a revised classification of the family

A group of small and transparent Asian noodlefishes (Osmeriformes: Salangidae) are commercially important fishery species, however, interrelationships among these fishes remain unresolved in previous studies using mitochondrial markers. We re-examine phylogenetic relationships of Salangidae by including complete taxon sampling, based on seven nuclear loci and one mitochondrial gene using a multilocus coalescence-based species-tree method. Our results show a well-resolved phylogeny of Salangidae that does not agree with previous hypotheses. The topology test suggests that our hypothesis represents the most likely phylogeny. Using the inferred species-tree as criterion, we recombine the rank of subfamilies and genera in the Salangidae, and erect a new genus Neosalangichthys. Our revised classification of Salangidae is well supported by reinterpreting previously proposed diagnostic characters. Finally, re-defined synapomorphic characters are used to erect a key to the genera of Salangidae.     

Phylogeny and species delimitation of North European Lumbricillus (Clitellata,Enchytraeidae)

The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of non‐tropical marine and brackish waters world‐wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non‐monophyletic group. In this study, species boundaries and the phylogeny of this genus is re‐assessed using more than 300 DNA‐barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non‐monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister‐group to the genus Grania or as sister‐group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.     

Mitochondrial DNA phylogeny of two morphologically enigmatic fishes in the subfamily Schizothoracinae (Teleostei: Cyprinidae) in the Qinghai-Tibetan Plateau

The complete mitochondrial DNA (mtDNA) cytochrome b gene (1140 bp) was sequenced in Herzenstein macrocephalus and Gymnocypris namensis and in 13 other species and sub-species ( n = 22), representing four closely related genera in the subfamily Schizothoracinae. Conflicting taxonomies of H. macrocephalus and G. namensis have been proposed because of the character instability among individuals. Parsimony, maximum likelihood and Bayesian methods produced phylogenetic trees with the same topology and resolved several distinctive clades. Previous taxonomic treatments, which variously placed these two species of separate genera or as sub-species, are inconsistent with the mtDNA phylogeny. Both H. macrocephalus and G. namensis appear in a well-supported clade, which also includes nine species of Schizopygopsis , and hence should be transferred to the genus Schizopygopsis . Morphological changes are further illustrated, and their adaptive evolution in response to the local habitat shifts during the speciation process appears to be responsible for conflicting views on the systematics of these two species and hence the contrasting taxonomic treatments. These species are endemic to the Qinghai-Tibetan Plateau, a region with a history of geological activity and a rich diversity of habitats that may have result in the parallel and reversal evolution of some morphological characters used in their taxonomies. Our results further suggest that speciation and morphological evolution of fishes in this region may be more complex than those previously expected.     

Phylogenetic relationships of the New World Troidini swallowtails (Lepidoptera: Papilionidae) based on COI, COII, and EF-1alpha genes

A phylogeny of the Neotropical members of the Tribe Troidini (Lepidoptera: Papilionidae) was obtained with sequences of three protein-coding genes: two mitochondrial (COI and COII), and one nuclear (EF-1alpha). Parsimony and Bayesian analyses of 33 taxa resulted in very similar trees regardless of method used with the 27 troidines always forming a monophyletic clade. Within Troidini, the genus Battus is sister group to the remaining troidines, followed by a clade formed by the Paleotropical taxa (here represented by three exemplars). The genus Euryades is the next branch, and sister group of Parides. The genus Parides is monophyletic, and is divided into four main groups by Maximum Parsimony analysis, with the most basal group composed of tailed species restricted to SE Brazil. Character optimization of ecological and morphological traits over the phylogeny proposed for troidines indicated that the use of several species of Aristolochia is ancestral over the use of few or a single host-plant. For the other three characters, the ancestral states were the absence of long tails, forest as the primary habitat and oviposition solitary or in loose group of several eggs.     

A multigene species tree for Western Mediterranean painted frogs (Discoglossus)

Painted frogs (Discoglossus) are an anuran clade that originated in the Upper Miocene. Extant species are morphologically similar and have a circum-Mediterranean distribution. We assembled a multilocus dataset from seven nuclear and four mitochondrial genes for several individuals of all but one of the extant species and reconstructed a robust phylogeny by applying a coalescent-based species-tree method and a concatenation approach, both of which gave congruent results. The earliest phylogenetic split within Discoglossus separates D. montalentii from a clade comprising all other species. Discoglossus montalentii is monophyletic for haplotype variation at all loci and has distinct morphological, bioacoustic and karyotypic characters. We find moderate support for a sister-group relationship between the Iberian taxa and the Moroccan D. scovazzi, and high support for a D. pictus -D. sardus clade distributed around the Tyrrhenian basin. Topological discordance among gene trees during the speciation of D. galganoi, D. scovazzi, D. pictus and D. sardus is interpreted as the consequence of nearly simultaneous, vicariant diversification. The timing of these events is unclear, but possibly coincided with the final geotectonic rearrangement of the Western Mediterranean in the Middle Miocene or later during the Messinian salinity crisis. The Iberian taxa D. galganoi galganoi and D. g. jeanneae are reciprocally monophyletic in mitochondrial DNA but not in nuclear gene trees, and are therefore treated as subspecies of D. galganoi.     

基于16S rDNA和28S rDNA D2基因序列与形态特征联合分析的中国角顶叶蝉亚科系统发育研究（半翅目： 叶蝉科）

首次在国内利用28S rDNA D2区段和16S rDNA基因序列,结合50个形态特征对角顶叶蝉亚科(Deltocephalinae)［半翅目（Hemiptera）: 叶蝉科（Cicadellidae）］19个属进行系统发育分析研究。从无水乙醇浸泡保存的标本中提取基因组DNA并扩增了19个内群和1种外群Typhlocybinae［半翅目(Hemiptera): 叶蝉科(Cicadellidae)］种类的28S rDNA D2基因片段并测序,同时扩增了16S rDNA基因片段并测序11条,采用了GenBank中1个种类的16S rDNA同源序列。采用PAUP*4.0和MrBayes3.0两个分析软件和3种建树方法,利用同源28S D2 rDNA和16S rDNA两个基因序列与形态特征结合进行系统发育分析研究。分析结果表明,二叉叶蝉族Macrostelini是一个单系,并在角顶叶蝉亚科的系统发育中处于基部的位置,是内群中最原始的族;角顶叶蝉族Deltocephalini中除了纹翅叶蝉属Nakaharanus,其余各属构成单系;殃叶蝉族Euscelini内属的归属比较混乱,可能是一个并系群,属间差异有待进一步研究。隆额叶蝉族Paralimnini与顶带叶蝉族Athysanini是姐妹群。带叶蝉属Scaphoideus与纹翅叶蝉属Nakaharanus是姐妹群,二者与木叶蝉属Phlogotettix的关系最近,三者构成一个单系,建议将三者归为带叶蝉族Scaphoideini。研究结果还表明,小眼叶蝉族Xestocephalini和Balcluthini的系统发育位置不明,有待进一步研究。     

Phylogenetic relationships of North American garter snakes (Thamnophis) based on four mitochondrial genes: how much DNA sequence is enough?

The clade of garter snakes (Thamnophis) includes some of the most abundant and well-studied snakes in North America. However, phylogenetic relationships within this group have been little studied. We used DNA sequences of four mitochondrial genes (cytochrome b and NADH dehydrogenase subunits 1, 2, and 4) to estimate relationships among 29 of the 31 recognized species of Thamnophis plus the related species Adelophis foxi. Both maximum parsimony (MP) and maximum-likelihood (ML) analyses of all these genes combined produced well-resolved trees with moderate (70-89%) to strong (90-100%) bootstrap support for most clades. MP and ML trees were very similar, with no strongly supported conflict between the two analyses. These analyses identify a clade of 12 species largely restricted to México (the "Mexican clade"), and a clade containing 15 species that collectively range from Central America to southern Canada (the "widespread clade"). These two groups are identified as sister taxa in both MP and ML analyses. A clade consisting of the ribbon snakes (T. sauritus and T. proximus) and the common garter snake (T. sirtalis) is placed as the sister group to all other Thamnophis (i.e., the Mexican + widespread clades) in our analyses. High bootstrap proportions at several levels in the tree support the inclusion of both Thamnophis validus, which has traditionally been placed in the genus Nerodia, and the poorly known species Adelophis foxi within Thamnophis. We used randomly sampled characters (i.e., standard bootstrapping) and randomly sampled contiguous blocks of characters to examine the effect of number of characters on resolution of and support for relationships within Thamnophis using MP. In general, these analyses indicate that we have reached a point of strongly diminishing returns with respect to the effect of adding mtDNA sequence characters for the current set of taxa; our sample of 3809 mtDNA characters is apparently "enough." The next steps to improve the phylogenetic estimate may be to add nuclear DNA sequences, morphology, or behavior, or to sequence additional mtDNA lineages within species.     

Phylogeny estimation of the radiation of western North American chipmunks (Tamias) in the face of introgression using reproductive protein genes

The causes and consequences of rapid radiations are major unresolved issues in evolutionary biology. This is in part because phylogeny estimation is confounded by processes such as stochastic lineage sorting and hybridization. Because these processes are expected to be heterogeneous across the genome, comparison among marker classes may provide a means of disentangling these elements. Here we use introns from nuclear-encoded reproductive protein genes expected to be resistant to introgression to estimate the phylogeny of the western chipmunks (Tamias: subgenus: Neotamias), a rapid radiation that has experienced introgressive hybridization of mitochondrial DNA (mtDNA). We analyze the nuclear loci using coalescent-based species-tree estimation methods and concatenation to estimate a species tree and we use parametric bootstraps and coalescent simulations to differentiate between phylogenetic error, coalescent stochasticity and introgressive hybridization. Results indicate that the mtDNA gene tree reflects several introgression events that have occurred between taxa of varying levels of divergence and at different time points in the tree. T. panamintinus and T. speciosus appear to be fixed for ancient mitochondrial introgressions from T. minimus. A southern Rocky Mountains clade appears well sorted (i.e., species are largely monophyletic) at multiple nuclear loci, while five of six taxa are nonmonophyletic based on cytochrome b. Our simulations reject phylogenetic error and coalescent stochasticity as causes. The results represent an advance in our understanding of the processes at work during the radiation of Tamias and suggest that sampling reproductive-protein genes may be a viable strategy for phylogeny estimation of rapid radiations in which reproductive isolation is incomplete. However, a genome-scale survey that can statistically compare heterogeneity of genealogical process at many more loci will be necessary to test this conclusion.     

Multilocus analysis of a taxonomically densely sampled dataset reveal extensive non-monophyly in the avian family Locustellidae

The phylogeny of most of the species in the avian passerine family Locustellidae is inferred using a Bayesian species tree approach (Bayesian Estimation of Species Trees, BEST), as well as a traditional Bayesian gene tree method (MrBayes), based on a dataset comprising one mitochondrial and four nuclear loci. The trees inferred by the different methods agree fairly well in topology, although in a few cases there are marked differences. Some of these discrepancies might be due to convergence problems for BEST (despite up to 1×10(9) iterations). The phylogeny strongly disagrees with the current taxonomy at the generic level, and we propose a revised classification that recognizes four instead of seven genera. These results emphasize the well known but still often neglected problem of basing classifications on non-cladistic evaluations of morphological characters. An analysis of an extended mitochondrial dataset with multiple individuals from most species, including many subspecies, suggest that several taxa presently treated as subspecies or as monotypic species as well as a few taxa recognized as separate species are in need of further taxonomic work.     

Phylogenetic relationships of the New World Troidini swallowtails (Lepidoptera: Papilionidae) based on COI, COII, and EF-1α genes

A phylogeny of the Neotropical members of the Tribe Troidini (Lepidoptera: Papilionidae) was obtained with sequences of three protein-coding genes: two mitochondrial (COI and COII), and one nuclear (EF-1α). Parsimony and Bayesian analyses of 33 taxa resulted in very similar trees regardless of method used with the 27 troidines always forming a monophyletic clade. Within Troidini, the genus Battus is sister group to the remaining troidines, followed by a clade formed by the Paleotropical taxa (here represented by three exemplars). The genus Euryades is the next branch, and sister group of Parides. The genus Parides is monophyletic, and is divided into four main groups by Maximum Parsimony analysis, with the most basal group composed of tailed species restricted to SE Brazil. Character optimization of ecological and morphological traits over the phylogeny proposed for troidines indicated that the use of several species of Aristolochia is ancestral over the use of few or a single host-plant. For the other three characters, the ancestral states were the absence of long tails, forest as the primary habitat and oviposition solitary or in loose group of several eggs.     

Multilocus phylogenetics of a rapid radiation in the genus Thomomys (Rodentia: Geomyidae)

Species complexes undergoing rapid radiation present a challenge in molecular systematics because of the possibility that ancestral polymorphism is retained in component gene trees. Coalescent theory has demonstrated that gene trees often fail to match lineage trees when taxon divergence times are less than the ancestral effective population sizes. Suggestions to increase the number of loci and the number of individuals per taxon have been proposed; however, phylogenetic methods to adequately analyze these data in a coalescent framework are scarce. We compare two approaches to estimating lineage (species) trees using multiple individuals and multiple loci: the commonly used partitioned Bayesian analysis of concatenated sequences and a modification of a newly developed hierarchical Bayesian method (BEST) that simultaneously estimates gene trees and species trees from multilocus data. We test these approaches on a phylogeny of rapidly radiating species wherein divergence times are likely to be smaller than effective population sizes, and incomplete lineage sorting is known, in the rodent genus, Thomomys. We use seven independent noncoding nuclear sequence loci (total approximately 4300 bp) and between 1 and 12 individuals per taxon to construct a phylogenetic hypothesis for eight Thomomys species. The majority-rule consensus tree from the partitioned concatenated analysis included 14 strongly supported bipartitions, corroborating monophyletic species status of five of the eight named species. The BEST tree strongly supported only the split between the two subgenera and showed very low support for any other clade. Comparison of both lineage trees to individual gene trees revealed that the concatenation method appears to ignore conflicting signals among gene trees, whereas the BEST tree considers conflicting signals and downweights support for those nodes. Bayes factor analysis of posterior tree distributions from both analyses strongly favor the model underlying the BEST analysis. This comparison underscores the risks of overreliance on results from concatenation, and ignoring the properties of coalescence, especially in cases of recent, rapid radiations.     

Unraveling evolutionary lineages in the limbless fossorial skink genus Acontias (Sauria: Scincidae): are subspecies equivalent systematic units?

Subspecies in the limbless, endemic African fossorial skink genus Acontias constitute ill-defined operational taxonomic units, consequently considerable systematic debate has lingered on the systematic diversity within Acontias. In the present study, the systematic affinities among acontine taxa are explored with the utility of partial sequence data from two mitochondrial gene loci (16S rRNA and cytochrome oxidase subunit 1 (COI)) for all taxa, while two additional loci (12S rRNA, cytochrome b) were used to investigate relationships within the Acontias meleagris complex. Phylogenetic results, derived from the combined analysis, revealed two monophyletic clades. Clade 1 is comprised of small-bodied skinks while clade 2 comprised the medium bodied skinks. Within clade 2 none of the traditionally recognized subspecies formed reciprocally monophyletic groups. Furthermore, constraining the topology and enforcing sister taxa relationships between the assumed subspecies, consistently recovered a topology that was statistically significant worse, indicating that the traditionally designated subspecies groupings probably represent invalid taxonomic units, thus clearly reflecting considerable discord with current taxonomy. The burrowing life style of these lizards has probably led to marked convergent evolution and constrained the development of diagnostic morphological characters among these species. Morphological similarities in color as well as scale architecture within Acontias are labile and highly homoplaseous and do not reflect the evolutionary history of the group. Taxonomic implications of these results are discussed.     

Gene trees,species and species trees in the <Emphasis Type="Italic">Ctenosaura palearis</Emphasis> clade

The growing use of molecular systematics in conservation has increased the importance of accurate resolution of taxonomic units and relationships. DNA data relate most directly to genealogies, which need not have perfect relationships with species limits and phylogenies. We used a multilocus gene tree approach to elucidate the relationships between four endangered Central American iguanas. We found support for the proposition that the described species taxa correspond to distinct evolutionary lineages warranting individual protection. We combined gene trees to estimate a phylogeny using Bayesian Estimation of Species Trees (BEST), minimizing deep coalescence, Species Trees from Average Ranks (STAR), and traditional concatenation. The estimate from concatenation conflicted with the other methods, likely owing to the disproportionate effect of mtDNA on concatenated analyses. This illustrates the importance of appropriate treatment of multilocus sequence data in phylogenetics. Our results indicate that these species have gone through recent and rapid speciation, resulting in four closely related narrow-range endemics.     

Molecular phylogeny and evolutionary history of the tit-tyrants (Aves: Tyrannidae)

Tit-tyrants of the genus Anairetes presently consist of six species; five inhabit various regions along the Andean cordillera of South America and one is endemic to the Juan Fernandez Islands off the coast of Chile. Data from mtDNA ND2 and Cyt b sequences were used to construct a phylogeny for all Anairetes species as well as Uromyias agilis, a closely related genus, and Stigmatura as an outgroup, to determine their relationships and history of radiation in South America. Results strongly supported the following paired relationships: A. nigrocristatus-A. reguloides, A. flavirostris-A. alpinus, and A. parulus-A. fernandezianus. This dataset, however, could not resolve basal nodes; therefore relationships among these pairs remains obscure. Moreover the genus Uromyias, controversially separated on morphological criteria from Anairetes, fell within the Anairetes clade, although its exact position could not be ascertained with confidence. The molecular data indicate that this group probably radiated within the past 2 million years, concomitant with highly accentuated cycles of global climatic change. Certain high altitude areas within the Andes may have been stable during global climatic changes and may have served as refugia during the Plio-Pleistocene.     

Reconstructing the phylogeny of "Buarremon" brush-finches and near relatives (Aves, Emberizidae) from individual gene trees

Gene trees are often assumed to be equivalent to species trees, but processes such as incomplete lineage sorting can generate incongruence among gene topologies and analyzing multilocus data in concatenated matrices can be prone to systematic errors. Accordingly, a variety of new methods have been developed to estimate species trees using multilocus data sets. Here, we apply some of these methods to reconstruct the phylogeny of Buarremon and near relatives, a group in which phylogenetic analyses of mitochondrial DNA sequences produced results that were inconsistent with relationships implied by a taxonomy based on variation in external phenotype. Gene genealogies obtained for seven loci (one mitochondrial, six nuclear) were varied, with some supporting and some rejecting the monophyly of Buarremon. Overall, our species-tree analyses tended to support a monophyletic Buarremon, but due to lack of congruence between methodologies, resolution of the phylogeny of this group remains uncertain. More generally, our study indicates that the number of individuals sampled can have an important effect on phylogenetic reconstruction, that the use of seven markers does not guarantee obtaining a strongly-supported species tree, and that methods for species-tree reconstruction can produce different results using the same data; these are important considerations for researchers using these new phylogenetic approaches in other systems.     

A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.     

Species-level phylogeny of 'Satan's perches' based on discordant gene trees (Teleostei: Cichlidae: Satanoperca Günther 1862)

Neotropical rivers are home to the largest assemblage of freshwater fishes, but little is known about the phylogeny of these fishes at the species level using multi-locus molecular markers. Here, we present a phylogeny for all known species of the genus Satanoperca, a widespread group of Neotropical cichlid fishes, based on analysis of six unlinked genetic loci. To test nominal and proposed species limits for this group, we surveyed mtDNA sequence variation among 320 individuals representing all know species. Most nominal species were supported by this approach but we determined that populations in the Xingu, Tapajós, and Araguaia+Paraná Rivers are likely undescribed species, while S. jurupari and S. mapiritensis did not show clear genetic distinction. To infer a phylogeny of these putative species, we conducted maximum likelihood and Bayesian non-clock and relaxed clock analyses of concatenated data from three genes (one mitochondrial, two nuclear). We also used a multi-species coalescent model to estimate a species tree from six unlinked loci (one mitochondrial, five nuclear). The topologies obtained were congruent with other results, but showed only minimal to moderate support for some nodes, suggesting that more loci will be needed to satisfactorily estimate the distribution of coalescent histories within Satanoperca. We determined that this variation results from topological discordance among separate gene trees, likely due to differential sorting of ancestral polymorphisms.     

Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.