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.     

History repeated: recent and historical mitochondrial introgression between the current darter Etheostoma uniporum and rainbow darter Etheostoma caeruleum (Teleostei: Percidae)

Incongruence between recognized taxonomy and phylogenetic relationships between two species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus , Etheostoma uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout their sympatric distribution in the Ozark Highlands of the central United States. Sequences from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum . However, both species were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by those of E. caeruleum . Some E. uniporum haplotypes are shared with geographically proximate E. caeruleum , and this is consistent with recent hybridization, while other E. uniporum haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression is likely asymmetric sneaking behaviour by male E. uniporum , a mating tactic observed in related species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in a historically small female effective population. Additional evidence for darter hybridization will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern North America.     

Molecular evidence for hybridization between two Australian desert skinks, Ctenotus leonhardii and Ctenotus quattuordecimlineatus (Scincidae: Squamata)

Australian scincid lizards in the genus Ctenotus constitute the most diverse vertebrate radiation in Australia. However, the evolutionary processes that have generated this diversity remain elusive, in part because both interspecific phylogenetic relationships and phylogeographic structure within Ctenotus species remain poorly known. Here we use nucleotide sequences from a mitochondrial locus and two nuclear introns to investigate broad-scale phylogeographic patterns within Ctenotus leonhardii and C. quattuordecimlineatus, two geographically widespread species of skinks that were found to have a surprisingly close genetic relationship in a previous molecular phylogenetic study. We demonstrate that the apparent close relationship between these ecologically and phenotypically distinct taxa is attributable to mitochondrial introgression from C. quattuordecimlineatus to C. leonhardii. In the western deserts, Ctenotus leonhardii individuals carry mtDNA lineages that are derived from C. quattuordecimlineatus mtDNA lineages from that geographic region. Coalescent simulations indicate that this pattern is unlikely to have resulted from incomplete lineage sorting, implicating introgressive hybridization as the cause of this regional gene-tree discordance.     

THE INTERFACE BETWEEN PHYLOGENETICS AND POPULATION GENETICS: INVESTIGATING GENE TREES,SPECIES TREES,AND POPULATION DYNAMICS IN THE PHYLLOPHAGA FRATERNA SPECIES GROUP

This study uses traditional and contemporary phylogenetic and population genetic analyses to assess the causes of discordance (i.e., lineage sorting and introgression) among mitochondrial and nuclear gene trees for a clade of eastern North American scarab beetles (fraterna species group, genus Phyllophaga). I estimated gene trees using individual and combined analysis of one mitochondrial and two nuclear loci in MrBayes , and inferred a species tree using a hierarchical coalescent approach based on all loci in the program Best . Because hybridization violates the assumptions of Best , I tested for introgression by comparing species monophyly between the mitochondrial and nuclear gene trees based on the prediction that cytoplasmic genomes introgress more readily than nuclear genomes. Haplotype exclusivity was identified using Bayesian tests of monophyly and the genealogical sorting index. I used the results of the phylogenetic analyses and monophyly tests to develop an explicit hypothesis of introgression that could be tested in the program IMa. Results from these analyses provided evidence for introgression across clades within the fraterna group. The tiered analytical approach used in this study demonstrated how the use of multiple methods can identify when assumptions are violated and methods are prone to yield misleading results.     

Phylogeography and post-glacial colonization patterns of the rainbow darter, Etheostoma caeruleum (Teleostei: Percidae)

Aim To examine the effects of historical climate change and drainage isolation on the distribution of mitochondrial DNA cytochrome b genetic variation within the rainbow darter, Etheostoma caeruleum (Percidae: Etheostomatinae). Location Eastern North American streams including tributaries to the Mississippi River, Great Lakes, Potomac River and Hudson Bay drainages. Methods Parsimony analyses, Bayesian analyses and haplotype networks of mitochondrial DNA sequences. Results Four major clades were recovered from sampled populations of E. caeruleum. Three of four clades are distributed in the western portion of the species’ range (primarily west of the Mississippi River). Samples from this region do not form a monophyletic group, and sequences often vary greatly between samples from adjacent stream systems (up to 7.2% divergence). A basal clade includes samples from the White River system in the Ozark Highlands. The northern Ozarks–upper Midwest clade includes samples from Missouri River tributaries and the upper Midwest (Hudson Bay, upper Mississippi River, and western Lake Michigan drainage). The eastern clade is composed of individuals from the Ohio River, Great Lakes and Potomac River. The Mississippi River corridor clade includes samples from middle and lower Mississippi River tributaries. Main conclusions The four major clades of E. caeruleum are deep allopatric lineages with well‐defined boundaries and have additional phylogeographical structure within each clade. The Ozark Highlands have the greatest levels of diversity relative to distributional area, with marked cytochrome b subdivisions between adjacent stream systems. Samples from previously glaciated areas do not have a subset of the cytochrome b diversity found in unglaciated areas, but four separate source areas are identified based on phylogenetic analyses. Dispersal into previously glaciated areas followed several known glacial outlets and, based on sequence divergence between populations, may have occurred during different glacial or interglacial stages. The disjunct distribution and cytochrome b pattern of E. caeruleum in the Mississippi River corridor clade is consistent with late Pleistocene and Recent changes in the course and characteristics of the middle and lower Mississippi River. Phylogeographical boundaries between clades of E. caeruleum correspond to independent sources of biogeographical information and provide insight into historical stream drainage relationships, post‐glacial colonization and drainage isolation patterns.     

Close relatedness between mitochondrial DNA from seven Anser goose species

The phylogenetic relationships of seven goose species and two of the subspecies representing the genus Anser were studied by approximately 1180 bp of mitochondrial DNA tRNAglu, control region and tRNAphe sequences. Despite obvious morphological and behavioural affinities among the species, their evolutionary relationships have not been studied previously. The small amount of genetic differentiation observed in the mitochondrial DNA indicates an extremely close evolutionary relationship between the Anser species. The sequence divergences between the species (0.9–5.5%) are among the lowest reported for avian species with speciation events of Anser geese dating to late Pliocene and Pleistocene. The species grouped into four mtDNA lineages: (1) snow and Ross’ goose, (2) greylag goose, (3) white‐fronted goose, and (4) bean, pink‐footed and lesser white‐fronted goose. The phylogenetic relationships of the most closely related species, bean, pink‐footed and lesser white‐fronted goose, indicate a period of rapid cladogenesis. The poor agreement between morphological relationships and the phylogenetic relationships indicated by mtDNA sequences implies that either ancestral polymorphism and lineage sorting, hybridization and introgression or convergent evolution has been involved.     

MITOCHONDRIAL DNA VARIATION AND EVOLUTION OF THE DEATH VALLEY PUPFISHES (CYPRINODON,CYPRINODONTIDAE)

A phylogenetic analysis of mitochondrial DNA (mtDNA) restriction sites was used to examine the geographic history of the Cyprinodon nevadensis complex of pupfishes, a group of four species (seven extant subspp.) in two endorheic (closed) basins of the Death Valley System in California and Nevada (Owens River Valley and Ash Meadows-Death Valley). The mtDNA results suggest that the group contains mtDNAs from two divergent clades. One such clade is represented by the mtDNAs of the Owens Valley pupfish (C. radiosus) and the existing species in the Colorado River (C. macularius), while the other includes the mtDNAs of the Ash Meadows-Death Valley species (C. nevadensis, C. salinus, and C. diabolis) and a species located much farther to the east (C. fontinalis from the Guzman Basin, Chihuahua, Mexico). These results, together with evidence from other studies, suggest two separate invasions of the Death Valley System by pupfishes carrying phylogenetically divergent mtDNAs. The C. nevadensis complex apparently is either an artificial group or else it is monophyletic and its genetic history includes loss of the original mtDNA in either Owens Valley or Ash Meadows-Death Valley following genetic introgression after an invasion by a pupfish carrying a divergent mtDNA.     

Phylogenetic relationships of Amazonetta,Speculanas, Lophonetta,and Tachyeres: four morphologically divergent duck genera endemic to South America

We studied the phylogenetic relationships of four duck genera endemic to South America: Brazilian teal Amazonetta brasiliensis, spectacled duck Speculanas specularis, crested duck Lophonetta specularioides, and four species of steamer ducks Tachyerespatachonicus, T. leucocephalus, T. pteneres, T. brachypterus. Genetic divergence within and among species was compared using population‐level sampling of the mitochondrial DNA (mtDNA) control region, supplemented with three additional mtDNA genes and six independent nuclear loci from one individual of each species and a variety of outgroup taxa. The monophyly of these four morphologically divergent South American genera was strongly supported. Within this clade, Amazonetta and Speculanas were supported as sister species in all analyses, but different gene regions yielded conflicting or ambiguous results for Lophonetta and Tachyeres. This lack of resolution resulted from little informative variation in nuclear loci and high levels of homoplasy in the mtDNA control region. Control region sequences from the four Tachyeres species fell into two distinct clades. In one clade, T. patachonicus and T. leucocephalus share a set of closely related haplotypes (≤0.6% sequence divergence); while no identical haplotypes were shared between species, the control region phylogeny was insufficiently resolved to either support or reject reciprocal monophyly. The second clade, ~1.7% divergent from the first, comprised haplotypes of the Falkland Islands species T. brachypterus and a captive individual of T. pteneres. These distinctive South American ducks likely experienced two bouts of rapid diversification, thus making analysis of their phylogenetic relationships difficult. Incomplete lineage sorting, founder effects, and perhaps introgression likely have contributed to obscuring the relationships among steamer ducks.     

Assessing natural and disturbed population structure in European grayling Thymallus thymallus: melding phylogeographic,population genetic and jurisdictional perspectives for conservation planning

Natural and human‐mediated population structure of European grayling Thymallus thymallus was assessed in primarily Austrian reaches of the Danube River catchment. Data on phylogeographic structure based on mitochondrial DNA (mtDNA) were combined with variation found across 13 microsatellite loci to assess introgression stemming from stocking activities. Populations north of the Alps generally showed no signs of long‐term introgression from allochthonous stocks, although one population comprised two distinct genotypic groups, one of which may stem from recently stocked material. South of the Alps, levels of introgression with stocks from the north were extensive and only one of six populations is believed to represent a reference state. Allelic diversity and expected heterozygosity were positively correlated with mtDNA admixture supporting more recent introgression and that there is little or no reproductive barrier between these two major lineages north and south of the Alps. A third unrelated mtDNA lineage is described from the Lafnitz, a tributary of the Raab drainage. The high lineage diversity in the upper Austrian Danube is not concordant with an existing model of T. thymallus evolutionarily significant units in Europe. Caveats in naming such units or following a strict hierarchical conservation unit structure for broadly distributed species with complex phylogeographic distributions stretching over various jurisdictions are discussed. The necessity of using both phylogeographic and population genetic approaches in evaluating the history and conservation value of populations in a conservation context is additionally highlighted.     

Widespread decoupling of mtDNA variation and species integrity in Grammia tiger moths (Lepidoptera: Noctuidae)

Abstract. We investigate the diversity of the North American tiger moth genus Grammia Rambur (Lepidoptera: Noctuidae) by comparing mitochondrial DNA (mtDNA) ‘barcode’ fragments of cytochrome oxidase I with non‐molecular characters such as morphology, ecology, behaviour and distribution. Mitochondrial DNA genealogy is strikingly at odds with morpho‐species taxonomy for most of the 28 sampled species, as haplotypic polyphyly not only is taxonomically widespread, but involves multiple shared haplotypes among two to four species. Morpho‐ecological traits show that those species sharing haplotypes are often not closely related. Furthermore, high mtDNA divergences occur within species. Haplotypic variation is highly discordant with species taxonomy, but variation at a continental scale reveals significant geographic structuring of haplogroups, transcending morpho‐species boundaries. A nested clade analysis and comparison of non‐molecular with mtDNA data indicate that most discordance between mtDNA and taxonomy in Grammia is explained best by taxonomically and geographically widespread ongoing hybridization events resulting in mtDNA introgression. We hypothesize that broad areas of sympatry, interspecifically compatible genitalic structure, and species overlap in pheromone components facilitate hybridization, with disparate interspecies abundances promoting mitochondrial introgression. The molecular evolution of Grammia challenges the view that interspecific gene exchange occurs rarely and is restricted to recently diverged species. These results show the value of mtDNA in detecting cryptic hybridization, while highlighting the inherent dangers of drawing taxonomic conclusions based solely on mtDNA.     

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.     

Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae)

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.     

Deep sympatric mitochondrial divergence without reproductive isolation in the common redstart Phoenicurus phoenicurus

Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage‐specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation.     

Molecular patterns of differentiation in canyon treefrogs (Hyla arenicolor): evidence for introgressive hybridization with the Arizona treefrog (H. wrightorum) and correlations with advertisement call differences

Detection of genetic and behavioural diversity within morphologically similar species has led to the discovery of cryptic species complexes. We tested the hypothesis that US populations of the canyon treefrog (Hyla arenicolor) may consist of cryptic species by examining mate‐attraction signals among three divergent clades defined by mtDNA. Using a multi‐locus approach, we re‐analysed phylogenetic relationships among the three clades and a closely related, but morphologically and behaviourally dissimilar species, the Arizona treefrog (H. wrightorum). We found evidence for introgression of H. wrightorum’s mitochondrial genome into H. arenicolor. Additionally, the two‐clade topology based on nuclear data is more congruent with patterns of call variation than the three‐clade topology from the mitochondrial dataset. The magnitude of the call divergence is probably insufficient to promote isolation of the nuclear DNA‐defined clades should they become sympatric, but further divergence in call properties significant in species identification could promote speciation in the future.     

AFLPs resolve cytonuclear discordance and increase resolution among barcheek darters (Percidae: Etheostoma: Catonotus)

The use of mitochondrial DNA (mtDNA) sequences in phylogenetic analysis has been the subject of increasing scrutiny. A recent phylogenetic study of barcheek darters (Percidae: Etheostoma: Catonotus) revealed cytonuclear discordance, discordance among mtDNA loci, and discordance between mtDNA and morphometric hypotheses. In particular, mtDNA analyses hypothesized a paraphyletic barcheek darter clade, and a combined mtDNA and nuclear sequence topology was not well resolved. Here, we used amplified fragment length polymorphisms to test the monophyly of barcheek darters and to resolve relationships within the group. By including multiple populations of each species, we were able to generate a highly resolved tree that supports both the monophyly of barcheek darters as well as recently elevated species within the group. Analysis of three mtDNA loci indicates that saturation of highly variable sites best explains the discordant topologies among mtDNA partitions.     

Speciation on the coasts of the new world: phylogeography and the evolution of bindin in the sea urchin genus Lytechinus

Abstract Beginning with E. Mayr's study in 1954, tropical sea urchins have played an important role in studies of speciation in the sea, but what are the processes of cladogenesis and divergence that give rise to new species in this group? We attempt to answer this question in the genus Lytechinus. Unlike the majority of other tropical sea urchin genera, which have circumtropical distributions, Lytechinus is mostly confined to the tropics and subtropics of the New World. We sequenced a region of mitochondrial cytochrome oxidase I and the entire molecule of nuclear bindin (a sperm gamete recognition protein) of nearly all species in the genus, and we assayed isozymes of three partially sympatric closely related species and subspecies. We found that in both mitochondrial DNA (mtDNA) and in bindin the genus Lytechinus is paraphyletic, encompassing Sphaerechinus granularis as the sister species of L. euerces. The rest of the species are arranged in an Atlantic clade composed of L. williamsi and L. variegatus, and a Pacific clade containing L. anamesus, L. pictus, L. semituberculatus, and L. panamensis. Divergence between these clades suggests that they were separated no later than the closure of the Isthmus of Panama, and possibly before this time. Our data confirm that L. anamesus and L. pictus from California are a single species, and provide no evidence of differentiation between L. variegatus variegatus from the Caribbean and L. variegatus atlanticus from Bermuda. Lytechinus variegatus variegatus mtDNA is distinct from that of L. variegatus carolinus from the North American seaboard and the Gulf of Mexico, whereas their bindins are very similar. However, there is clear evidence of introgression of mtDNA between the two subspecies and they share alleles in all sampled isozyme loci. Lytechinus williamsi from the Caribbean shares mtDNA haplotypes with L. variegatus variegatus, and they also share isozymes in all assayed loci. Their bindin, however, is distinct and coalesces within each morphospecies. A private clade of mtDNA in L. williamsi may be indicative of former differentiation in the process of being swamped by introgression, or of recent speciation. Recent sudden expansions in effective population size may explain the predominance of a few mitochondrial haplotypes common to the two species. Despite the high divergence of bindin (relative to differentiation of mtDNA) between L. variegatus and L. williamsi, comparison of amino acid replacement to silent substitutions by various methods uncovered no evidence for positive selection on the bindin of any clade of Lytechinus. With the possible exception of L. williamsi and L. variegatus, our results are consistent with a history of allopatric speciation in Lytechinus. The molecular results from Lytechinus, along with those of other similar studies of sea urchins, suggest that the general speciation patterns deduced in the middle of last century by Mayr from morphology and geography have held up, but also have uncovered peculiarities in the evolution of each genus.     

Biogeography of ‘Cyprinella lutrensis’: intensive genetic sampling from the Pecos River ‘melting pot’ reveals a dynamic history and phylogenetic complexity

Thorough sampling is necessary to delineate lineage diversity for polytypic ‘species’ such as Cyprinella lutrensis. We conducted extensive mtDNA sampling (cytochrome b and ND4) from the Pecos River, Rio Grande, and South Canadian River, New Mexico. Our study focussed on the Pecos River due to its complex geological history and potential to harbour multiple lineages. We used geometric–morphometric, morphometric, and meristic analyses to test for phenotypic divergence and combined nucDNA with mtDNA to test for cytonuclear disequilibrium and combined our sequences with published data to conduct a phylogenetic re‐assessment of the entire C. lutrensis clade. We detected five co‐occurring mtDNA lineages in the Pecos River, but no evidence for cytonuclear disequilibrium or phenotypic divergence. Recognized species were interspersed amongst divergent lineages of ‘C. lutrensis’. Allopatric divergence among drainages isolated in the Late Miocene and Pliocene apparently produced several recognized species and major divisions within ‘C. lutrensis’. Pleistocene re‐expansion and subsequent re‐fragmentation of a centralized lineage founded younger, divergent lineages throughout the Rio Grande basin and Edwards Plateau. There is also evidence of recent introductions to the Rio Grande, Pecos and South Canadian Rivers. Nonetheless, deeply divergent lineages have coexisted since the Pleistocene.     

Chlorophyll-binding proteins revisited - a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Background

Mitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.

Results

DNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.

Conclusions

The majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.     

Molecular phylogenetic status of the Bulgarian marbled polecat (Vormela peregusna,Mustelidae, Carnivora), revealed by Y chromosomal genes and mitochondrial DNA sequences

In this study, we investigated molecular phylogenetic status of the marbled polecat (Vormela peregusna) from Bulgaria, using sequences of two Y-chromosomal genes (SRY and ZFY). The phylogenetic tree inferred using combined sequences of both genes indicated that the marbled polecat was split from genera Lutra, Neovison and Mustela after genus Martes was diverged in family Mustelidae. In addition, we analyzed molecular phylogeography of the Bulgarian population of the marbled polecat, using cytochrome b and control region sequences of mitochondrial DNA (mtDNA). The phylogenetic tree of cytochrome b indicated that the haplotypes of the Bulgarian population comprised two haplogroups, which were the most ancestral clades. Additionally, the control region phylogeny showed that the haplotypes of Bulgaria formed two haplogroups: one was the most ancestral clade, and the other was the derivative clade. One individual with the most ancestral cytochrome b clade had a control region haplotype of the derivative clade. Thus, this study revealed that the most ancestral lineages of the marbled polecat are included in the population of Bulgaria. The Bulgarian population could be a remnant lineage from a basal for the species, which in Pleistocene occupied a relatively large area related to the Balkan-Caucasian.     

Systematics, biogeography, and evolution of the Neotropical peacock basses Cichla (Perciformes: Cichlidae)

To investigate forces influencing diversification in Neotropical fishes, the phylogenetic relationships among species and populations of the cichlid genus Cichla were examined. Mitochondrial DNA was sequenced for 454 individuals of the 5 nominal Cichla species and several putative undescribed species. Phylogenetic analyses support the distinction of two major clades of Cichla. Clade A includes C. temensis and two undescribed species from the lower Amazonas and Xingu Rivers. Clade B includes C. orinocensis, C. monoculus, C. ocellaris. C, intermedia, and an undescribed species from the upper Madeira River. Species boundaries were relatively well-circumscribed for clade B, while incomplete lineage sorting was inferred for clade A. Three probable instances of introgression were observed, including a regional population of C. orinocensis from the Negro River that shows a history of introgression. Biogeographic patterns from Cichla are partially congruent with those seen in several other Neotropical fish clades, and the diversification of Cichla species is inferred to result from both vicariance and sympatric divergence.