Assessing phylogenetic resolution among mitochondrial, nuclear, and morphological datasets in Nothonotus darters (Teleostei: Percidae)

External morphological characters are the basis of our understanding of diversity and species relationships in many darter clades. The past decade has seen the publication of many studies utilizing mtDNA sequence data to investigate darter phylogenetics, but only recently have nuclear genes been used to investigate darter relationships. Despite a long tradition of use in darter systematics few studies have examined the phylogenetic utility of external morphological characters in estimating relationships among species in darter clades. We present DNA sequence data from the mitochondrial cytochrome b (cytb) gene, the nuclear encoded S7 intron 1, and discretely coded external morphological characters for all 20 species in the darter clade Nothonotus. Bayesian phylogenetic analyses result in phylogenies that are in broad agreement with previous studies. The cytb gene tree is well resolved, while the nuclear S7 gene tree lacks phylogenetic resolution, node support, and is characterized by a lack of reciprocal monophyly for many of the Nothonotus species. The phylogenies resulting from analysis of the morphological dataset lack resolution, but nodes present are found in the cytb and S7 gene trees. The highest resolution and node support is found in the Bayesian combined data phylogeny. Based on our results we propose continued exploration of the phylogenetic utility of external morphological characters in other darter clades. Given the extensive lack of reciprocal monophyly of species observed in the S7 gene tree we predict that nuclear gene sequences may have limited utility in intraspecific phylogeographic studies of Nothonotus darters.     

Dispersal, vicariance, and timing of diversification in Nothonotus darters

The species diversity of North American freshwater fishes is unparalleled among temperate regions of the planet. This diversity is concentrated in the Central Highlands of eastern North America and this distribution pattern has inspired different models involving either dispersal or vicariance to explain the high species diversity of North American fishes. The most popular of these models is the Central Highlands vicariance hypothesis (CHVH), which proposes an ancient and diverse widespread fauna that existed across a previously continuous highland landscape that is much different from today. The mechanisms of isolation in the CHVH involve specific instances of vicariance that affected several diverse lineages of Central Highlands fishes. We tested predictions of the CHVH and alternative models using a cytochrome b-inferred phylogeny of the darter clade Nothonotus. A Bayesian mixed-model method was used for phylogenetic analysis. The phylogenetic data set included all 20 recognized Nothonotus species, and most species were represented with multiple sequences. We were able to convert genetic branch lengths to absolute age using external fossil calibrations in the freshwater perciform fish clade Centrarchidae. Using a well-resolved Nothonotus phylogeny and divergence time estimates, we identify equal numbers of instances of both vicariance and dispersal among disjunct regions of the Central Highlands, biogeographic pseudocongruence, rather recent speciation in Nothonotus, and a surprisingly large amount of speciation within highland areas. With regard to Nothonotus, previous Central Highlands biogeographic models offer little in the way of providing possible mechanisms responsible for diversification in the clade. Patterns of speciation in Nothonotus are similar to those discovered in recent efforts that have included speciation as a parameter into classic models of island biogeography.     

Phylogenetic relationships among Boleosoma darter species (Percidae: Etheostoma)

Darters represent a species rich group of North American freshwater fishes studied in the context of their diverse morphology, behavior, and geographic distribution. We report the first molecular phylogenetic analyses of the Boleosoma darter clade that includes complete species sampling. We estimated the relationship among the species of Boleosoma using DNA sequence data from a mitochondrial (cytochrome b) and a nuclear gene (S7 ribosomal protein intron 1). Our analyses discovered that the two Boleosoma species with large geographic distributions (E. nigrum and E. olmstedi) do not form reciprocally monophyletic groups in either gene trees. Etheostoma susanae and E. perlongum were phylogenetically nested in E. nigrum and E. olmstedi, respectively. While analysis of the nuclear gene resulted in a phylogeny where E. longimanum and E. podostemone were sister species, the mitochondrial gene tree did not support this relationship. Etheostoma vitreum was phylogenetically nested within Boleosoma in the mitochondrial DNA and nuclear gene trees. Our analyses suggest that current concepts of species diversity underestimate phylogenetic diversity in Boleosoma and that Boleosoma species likely provide another example of the growing number of discovered instances of mitochondrial genome transfer between darter species.     

GEOGRAPHIC AND TEMPORAL ASPECTS OF MITOCHONDRIAL REPLACEMENT IN NOTHONOTUS DARTERS (TELEOSTEI: PERCIDAE: ETHEOSTOMATINAE).

A growing number of molecular studies have identified mitochondrial replacement among closely related animal species, but there has been limited investigation into the phylogenetic, geographic, and temporal patterns, especially in more inclusive clades. We present a phylogenetic analysis of DNA sequences collected from mitochondrial and nuclear genes sampled from all 20 species of the darter clade Nothonotus and reveal extensive mtDNA replacement in N. rufilineatus. Using phylogenetic trees, haplotype networks, analysis of molecular variance (AMOVAs), and distributions of minimum pairwise genetic distances, we discovered that the mtDNA of N. rufilineatus has been replaced by that of different sympatric species of Nothonotus in different river drainages. In the Cumberland River, N. rufilineatus populations were fixed for N. camurus mtDNA. In the upper Tennessee River, N. rufilineatus contained N. chlorobranchius and N. camurus mtDNA. Most surprising, our analyses indicated that N. rufilineatus has acted as a “conduit species,” facilitating the introgression of N. chlorobranchius mtDNA into N. camurus in the upper Tennessee River. We identified several potential mechanisms for the observed pattern of introgression, and suggest experiments to assess their relative contributions. Comparisons among darter subclades indicated that the mitochondrial lineage of the clade is most influential in determining if the lineage is a mitochondrial donor or recipient.     

The influence of life‐history strategy on genetic differentiation and lineage divergence in darters (Percidae: Etheostomatinae)

Recent studies determined that darters with specialized breeding strategies can exhibit deep lineage divergence over fine geographic scales without apparent physical barriers to gene flow. However, the extent to which intrinsic characteristics interact with extrinsic factors to influence population divergence and lineage diversification in darters is not well understood. This study employed comparative phylogeographic and population genetic methods to investigate the influence of life history on gene flow, dispersal ability, and lineage divergence in two sympatric sister darters with differing breeding strategies. Our results revealed highly disparate phylogeographic histories, patterns of genetic structure, and dispersal abilities between the two species suggesting that life history may contribute to lineage diversification in darters, especially by limiting dispersal among large river courses. Both species also showed striking differences in demographic history, indicating that extrinsic factors differentially affected each species during the Pleistocene. Collectively, our results indicate that intrinsic and extrinsic factors have influenced levels of gene flow among populations within both species examined. However, we suggest that life‐history strategy may play a more important role in lineage diversification in darters than previously appreciated, a finding that has potentially important implications for understanding diversification of the rich North American freshwater fish fauna.     

Geomorphic Disturbance and its Impact on Darter (Teleostomi: Percidae) Distribution and Abundance in the Pearl River Drainage, Mississippi

We examined channel and point bar changes over time in the Pearl River, a meandering, Coastal Plain stream in Mississippi and Louisiana. We interpreted extreme changes in bar area as evidence of channel instability and related this to the diversity and abundance of darters in the river. Darters were less diverse and abundant in more disturbed reaches in comparison with relatively undisturbed reaches. Darter abundance was positively correlated with proximity to extreme bar-area changes. The relationship between channel instability and darter abundance observed here points to the importance of landscape level approaches to in-stream habitat management. The results suggest that river management practices that prevent or mitigate extreme changes in channel or sediment dynamics should be adopted for the benefit of benthic communities.     

Phylogeographic analysis of the cornsnake (Elaphe guttata) complex as inferred from maximum likelihood and Bayesian analyses

Most phylogeographic studies have used maximum likelihood or maximum parsimony to infer phylogeny and bootstrap analysis to evaluate support for trees. Recently, Bayesian methods using Marlov chain Monte Carlo to search tree space and simultaneously estimate tree support have become popular due to its fast search speed and ability to create a posterior distribution of parameters of interest. Here, I present a study that utilizes Bayesian methods to infer phylogenetic relationships of the cornsnake (Elaphe guttata) complex using cytochrome b sequences. Examination of the posterior probability distributions confirms the existence of three geographic lineages. Additionally, there is no support for the monophyly of the subspecies of E. guttata. Results suggest the three geographic lineages partially conform to the ranges of previously defined subspecies, although Shimodaira-Hasegawa tests suggest that subspecies-constrained trees produce significantly poorer likelihood estimates than the most likely trees reflecting the evolution of three geographic assemblages. Based on molecular support, these three geographic assemblages are recognized as species using evolutionary species criteria: E. guttata, Elaphe slowinskii, and Elaphe emoryi [phylogeographic, maximum likelihood, maximum parsimony, bootstrap, Bayesian, Markov chain Monte Carlo, cornsnake, Cytochrome b, geographic lineages, E. guttta, E. slowinskii, and E. emoryi].     

Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander,Plethodon kentucki: Incongruence among Data Sets and Methods

Species are a fundamental unit of biodiversity, yet can be challenging to delimit objectively. This is particularly true of species complexes characterized by high levels of population genetic structure, hybridization between genetic groups, isolation by distance, and limited phenotypic variation. Previous work on the Cumberland Plateau Salamander, Plethodon kentucki, suggested that it might constitute a species complex despite occupying a relatively small geographic range. To examine this hypothesis, we sampled 135 individuals from 43 populations, and used four mitochondrial loci and five nuclear loci (5693 base pairs) to quantify phylogeographic structure and probe for cryptic species diversity. Rates of evolution for each locus were inferred using the multidistribute package, and time calibrated gene trees and species trees were inferred using BEAST 2 and *BEAST 2, respectively. Because the parameter space relevant for species delimitation is large and complex, and all methods make simplifying assumptions that may lead them to fail, we conducted an array of analyses. Our assumption was that strongly supported species would be congruent across methods. Putative species were first delimited using a Bayesian implementation of the GMYC model (bGMYC), Geneland, and Brownie. We then validated these species using the genealogical sorting index and BPP. We found substantial phylogeographic diversity using mtDNA, including four divergent clades and an inferred common ancestor at 14.9 myr (95% HPD: 10.8–19.7 myr). By contrast, this diversity was not corroborated by nuclear sequence data, which exhibited low levels of variation and weak phylogeographic structure. Species trees estimated a far younger root than did the mtDNA data, closer to 1.0 myr old. Mutually exclusive putative species were identified by the different approaches. Possible causes of data set discordance, and the problem of species delimitation in complexes with high levels of population structure and introgressive hybridization, are discussed.     

Morphological disparity opposes latitudinal diversity gradient in lacertid lizards

While global variation in taxonomic diversity is strongly linked to latitude, the extent to which morphological disparity follows geographical gradients is less well known. We estimated patterns of lineage diversification, morphological disparity and rates of phenotypic evolution in the Old World lizard family Lacertidae, which displays a nearly inverse latitudinal diversity gradient with decreasing species richness towards the tropics. We found that lacertids exhibit relatively constant rates of lineage accumulation over time, although the majority of morphological variation appears to have originated during recent divergence events, resulting in increased partitioning of disparity within subclades. Among subclades, tropical arboreal taxa exhibited the fastest rates of shape change while temperate European taxa were the slowest, resulting in an inverse relationship between latitudinal diversity and rates of phenotypic evolution. This pattern demonstrates a compelling counterexample to the ecological opportunity theory of diversification, suggesting an uncoupling of the processes generating species diversity and morphological differentiation across spatial scales.     

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.     

Recovering cryptic diversity and ancient drainage patterns in eastern North America: historical biogeography of the Notropis rubellus species group (Teleostei: Cypriniformes)

The Central Highlands of North America contain a strikingly diverse assemblage of temperate freshwater fishes and have long been a focus of biogeographic studies. The rosyface shiner complex, Notropis rubellus and related species, is a member of this fauna exhibiting a disjunct highlands distribution occurring in the unglaciated regions of the Central Highlands and glaciated regions of the Central Lowlands. Until recently, N. rubellus was considered a single, widespread species exhibiting geographic variation in morphological characters. However, several studies have revealed that N. rubellus is a multi-species complex with closely related species endemic to drainages within each highland region. We examined genetic variation of the N. rubellus complex using a complete mtDNA cytochrome b gene sequence data set and combined mtDNA and published allozyme data sets. Parsimony and Bayesian analyses of the mitochondrial data set and parsimony analyses of a combined mitochondrial and allozyme data sets were largely consistent. Results of these analyses revealed ancient cryptic diversity within the N. rubellus complex that existed prior to the onset of Pleistocene glaciations. We identified seven strongly supported clades within the N. rubellus complex. Four clades are diagnosed as separate species (N. percobromus, N. rubellus, N. micropteryx and N. suttkusi) and three clades may represent undescribed forms. Relationships among these groups and their biogeographical patterns provided significant inferences on ichthyofaunal distributions in southeastern North America. These include the timing of the origin of the diversity, ancient drainage patterns and barriers to dispersal in the Central Highlands. The observation of increased diversity in N. rubellus suggests there may be greater diversity within other taxa with a similar distribution.     

Molecular systematics,biogeography and taxonomy of forest‐falcons in the Micrastur ruficollis species complex (Aves: Falconidae)

We gathered molecular data to assess phylogenetic and phylogeographic patterns for widespread lineages of Neotropical forest falcons in the genus Micrastur to: 1) investigate the comparative phylogeography of four species from the M. ruficollis complex (M. ruficollis, M. gilvicollis, M. plumbeus and M. mintoni), to identify the temporal and spatial context of the group's diversification; and 2) to reevaluate, based on molecular characters, the taxonomic status and interspecific boundaries within this complex. Molecular phylogenies were based on sequences of the mitochondrial genes ND2 and Cyt b and the nuclear genes FIB5 and MUSK from 119 specimens, including M. mirandollei and M. semitorquatus as outgroups. The phylogenetic trees obtained by BI and a Species Tree analysis recovered the monophyly of currently accepted species belonging to the M. ruficollis complex. The dates in our tree indicate that the separation of species within the complex occurred 2–4 million yr ago, initiating during the Neogene (Pliocene). However, when compared to most such widely distributed Neotropical lineages, the diversification within the M. ruficollis complex appears more recent (i.e. centered in the Late Pleistocene). Our results demonstrate the existence of eleven geographic lineages (subclades) in M. ruficollis, M. gilvicollis and M. mintoni, which differ genetically from each other and therefore can be interpreted as distinct evolutionary lineages and possibly separate species under lineage‐based species concepts. However, BPP results failed to recognize with strong statistical support any of these subclades as distinct species. Distinct subclades in the M. ruficollis complex are limited by the principal tributaries of the Amazon River and the Andes, suggesting that these modern barriers limit gene flow and thereby could have promoted differentiation mostly during the Pleistocene. However, our results indicate widely disparate responses to individual barriers across subclades, supporting lineage‐specific histories throughout the Neotropics.     

Phylogeography and speciation in the <Emphasis Type="Italic">Pseudocrenilabrus philander</Emphasis> species complex in Zambian Rivers

Haplochromine cichlids form the most species-rich lineage of cichlid fishes that both colonized almost all river systems in Africa and radiated to species flocks in several East African lakes. The enormous diversity of lakes is contrasted by a relatively poor albeit biogeographically clearly structured species diversity in rivers. The present study analyzed the genetic structure and phylogeographic history of species and populations of the genus Pseudocrenilabrus in Zambian rivers that span two major African drainage systems, the Congo- and the Zambezi-system. The mtDNA phylogeny identifies four major lineages, three of which occur in the Congo-system and one in the Zambezi system. Two of the Congo-clades (Lake Mweru and Lunzua River) comprise distinct albeit yet undescribed species, while the fish of the third Congo-drainage clade (Chambeshi River and Bangweulu swamps), together with the fish of the Zambezi clade (Zambezi and Kafue River) are assigned to Pseudocrenilabrus philander. Concerning the intraspecific genetic diversity observed in the sampled rivers, most populations are highly uniform in comparison to lacustrine haplochromines, suggesting severe founder effects and/or bottlenecking during their history. Two bursts of diversification are reflected in the structure of the linearized tree. The first locates at about 3.9% mean sequence divergence and points to an almost simultaneous colonization of the sampled river systems. Subsequent regional diversification (with about 1% mean sequence divergence) occurred contemporaneously within the Kafue River and the Zambezi River. The clear-cut genetic biogeographic structure points to the dominance of geographic speciation in this lineage of riverine cichlid fishes, contrasting the importance of in situ diversification observed in lake cichlids.     

Identifying glacial refugia in a geographic parthenogen using palaeoclimate modelling and phylogeography: the New Zealand stick insect Argosarchus horridus (White)

We have used phylogeographic analysis of mitochondrial DNA (COI and COII genes) and ecological niche modelling (ENM) to reconstruct the population history of Argosarchus horridus (White), a widespread species of New Zealand stick insect. These data were used to address outstanding questions on the role of glacial refugia in determining the distribution and genetic structure of New Zealand species. Phylogeographic analysis shows a general pattern of high diversity in upper North Island and reduced diversity in lower North Island and South Island. The ENM indicates that during the last glacial maximum, A. horridus was largely restricted to refugia around coastal areas of North Island. The ENM also suggests refugia on the northeast coast of South Island and southeast coast of North Island and this prediction is verified by phylogeographic analysis, which shows a clade restricted to this region. Argosarchus horridus is also most likely a geographic parthenogen where males are much rarer at higher latitudes. The higher levels of genetic variation in northern, bisexual populations suggest southern and largely unisexual populations originated from southwardly expanding parthenogenetic lineages. Bayesian skyline analysis also provides support for a recent population size increase consistent with a large increase in geographic distribution in the late Pleistocene. These results exemplify the utility of integrating ENM and phylogeographic analysis in testing hypotheses on the origin of geographic parthenogenesis and effects of Pleistocene environmental change on biodiversity.     

Out of the forest: past and present range expansion of a parthenogenetic weevil pest,or how to colonize the world successfully

Previous research revealed complex diversification patterns in the parthenogenetic weevil Naupactus cervinus. To understand the origin of clonal diversity and successful spreading of this weevil, we investigated its geographic origin and possible dispersal routes and whether parthenogens can persist in habitats under unsuitable environmental conditions. This study is based on samples taken throughout a broad area of the species’ range. We used both mitochondrial and nuclear markers and applied phylogenetic and network analyses to infer possible relationships between haplotypes. Bayesian phylogeographic analyses and ecological niche modeling were used to investigate the processes that shaped genetic diversity and enabled the colonization of new geographic areas. Southeastern Brazil emerges as the original distribution area of N. cervinus. We detected two range expansions, one along natural corridors during the Pleistocene and the other in countries outside South America during recent times. Isolation due to climate shifts during the early Pleistocene led to diversification in two divergent clades, which probably survived in different refugia of the Paranaense Forest and the Paraná River delta. The origin of the clonal diversity was probably a complex process including mutational diversification, hybridization, and secondary colonization. The establishment of N. cervinus in areas outside its native range may indicate adaptation to drier and cooler conditions. Parthenogenesis would be advantageous for the colonization of new environments by preventing the breakup of successful gene combinations. As in other insect pests, the present distribution of N. cervinus results from both its evolutionary history and its recent history related to human activities.     

Hybrid sterility increases with genetic distance in snubnose darters (Percidae: <Emphasis Type="Italic">Etheostoma</Emphasis>)

The process of speciation is often synonymous with the evolution of reproductive isolation, and the tempo at which reproductive barriers evolve can provide insight into the underlying mechanisms of divergence. Darters are a diverse group of North American freshwater fish in which behavioral isolation appears to evolve rapidly; however, little is known about hybrid sterility among darter species. Here, six allopatric pairs of darter species were hybridized via in vitro fertilization and offspring were reared to reproductive age. Hybrid sterility appears more pronounced in male hybrids. Additionally, levels of hybrid sterility increase with genetic distance across species pairs. Understanding sex determination and sex-specific gene expression in darters will provide further insight into the evolution of hybrid sterility.     

Phylogeography of the Northern Alligator Lizard (Squamata,Anguidae): Hidden diversity in a western endemic

Western North America includes the California Floristic Province and the Pacific Northwest, biologically diverse regions highlighted by a complex topography, geology, climate and history. A number of animals span these regions and show distinctive patterns of dispersal, vicariance and lineage diversification. Examining phylogeographic patterns in the fauna of this area aids in our understanding of the forces that have contributed to the generation and maintenance of regional biodiversity. Here, we investigate the biogeography and population structure of the Northern Alligator Lizard (Elgaria coerulea), a wide‐ranging anguid endemic to western North America. We sequenced two mtDNA fragments (ND2 and ND4) for 181 individuals across the range of the species and analysed these data with phylogenetic approaches to infer population and biogeographic history, and date major divergences within the taxon. We further used Bayesian clustering methods to assess major patterns of population structure and performed ecological niche modelling (ENM) to aid in our interpretation of geographic structure and diversification of E. coerulea lineages. Our phylogeographic examination of E. coerulea uncovered surprising diversity and structure, recovering 10 major lineages, each with substantial geographic substructure. While some divergences within the species are relatively old (Pliocene, 5.3–2.6 mya), most intraspecific variation appears to be of more recent origin (Pleistocene, 2.6 mya‐11,700 ya). Current diversity appears to have arisen in the Sierra Nevada Mountains and spread west and north since the Pliocene. Finally, our ENMs suggest that much of the Coast Ranges in California provided ideal habitat during the Last Glacial Maxima (LGM) that has since contracted dramatically and shifted northwards, whereas significant portions of the Sierra Nevada were unsuitable during the LGM and have since become more suitable. Interestingly, E. coerulea shares a number of genetic boundaries with other sympatric taxa, suggesting common historical events and geomorphological features have shaped the biota of this region.     

Limited Phylogeographic Signal in Sex-Linked and Autosomal Loci Despite Geographically,Ecologically, and Phenotypically Concordant Structure of mtDNA Variation in the Holarctic Avian Genus Eremophila

Phylogeographic studies of Holarctic birds are challenging because they involve vast geographic scale, complex glacial history, extensive phenotypic variation, and heterogeneous taxonomic treatment across countries, all of which require large sample sizes. Knowledge about the quality of phylogeographic information provided by different loci is crucial for study design. We use sequences of one mtDNA gene, one sex-linked intron, and one autosomal intron to elucidate large scale phylogeographic patterns in the Holarctic lark genus Eremophila. The mtDNA ND2 gene identified six geographically, ecologically, and phenotypically concordant clades in the Palearctic that diverged in the Early - Middle Pleistocene and suggested paraphyly of the horned lark (E. alpestris) with respect to the Temminck''s lark (E. bilopha). In the Nearctic, ND2 identified five subclades which diverged in the Late Pleistocene. They overlapped geographically and were not concordant phenotypically or ecologically. Nuclear alleles provided little information on geographic structuring of genetic variation in horned larks beyond supporting the monophyly of Eremophila and paraphyly of the horned lark. Multilocus species trees based on two nuclear or all three loci provided poor support for haplogroups identified by mtDNA. The node ages calculated using mtDNA were consistent with the available paleontological data, whereas individual nuclear loci and multilocus species trees appeared to underestimate node ages. We argue that mtDNA is capable of discovering independent evolutionary units within avian taxa and can provide a reasonable phylogeographic hypothesis when geographic scale, geologic history, and phenotypic variation in the study system are too complex for proposing reasonable a priori hypotheses required for multilocus methods. Finally, we suggest splitting the currently recognized horned lark into five Palearctic and one Nearctic species.