A mirage of cryptic species: Genomics uncover striking mitonuclear discordance in the butterfly Thymelicus sylvestris

Mitochondrial DNA (mtDNA) sequencing has led to an unprecedented rise in the identification of cryptic species. However, it is widely acknowledged that nuclear DNA (nuDNA) sequence data are also necessary to properly define species boundaries. Next generation sequencing techniques provide a wealth of nuclear genomic data, which can be used to ascertain both the evolutionary history and taxonomic status of putative cryptic species. Here, we focus on the intriguing case of the butterfly Thymelicus sylvestris (Lepidoptera: Hesperiidae). We identified six deeply diverged mitochondrial lineages; three distributed all across Europe and found in sympatry, suggesting a potential case of cryptic species. We then sequenced these six lineages using double‐digest restriction‐site associated DNA sequencing (ddRADseq). Nuclear genomic loci contradicted mtDNA patterns and genotypes generally clustered according to geography, i.e., a pattern expected under the assumption of postglacial recolonization from different refugia. Further analyses indicated that this strong mtDNA/nuDNA discrepancy cannot be explained by incomplete lineage sorting, sex‐biased asymmetries, NUMTs, natural selection, introgression or Wolbachia‐mediated genetic sweeps. We suggest that this mitonuclear discordance was caused by long periods of geographic isolation followed by range expansions, homogenizing the nuclear but not the mitochondrial genome. These results highlight T. sylvestris as a potential case of multiple despeciation and/or lineage fusion events. We finally argue, since mtDNA and nuDNA do not necessarily follow the same mechanisms of evolution, their respective evolutionary history reflects complementary aspects of past demographic and biogeographic events.     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Patterns of chloroplast diversity among western European Dactylorhiza species (Orchidaceae)

In Europe, the genus Dactylorhiza comprises a bewildering variety of forms that are difficult to sort into discrete species. Most Dactylorhiza species are diploid or tetraploid and contrasting hypotheses have been proposed to explain the complex variation within this group. Using PCR-RFLP analysis in eight putative species, we could identify four highly differentiated chloroplast DNA lineages. The first lineage (clade A) included the unique haplotype found in D. sambucina. Clade B grouped four haplotypes belonging mostly to D. incarnata. Clades C and D included 27 haplotypes detected in diploid D. fuchsii and in all tetraploid species investigated. Eighty percent of the chloroplast variation were consistent with currently accepted species boundaries. The imperfect agreement between chloroplast variation and species boundaries may be ascribed to incomplete lineage sorting and/or reticulation. Our cpDNA results provide strong evidence that the allotetrapolyploids have been formed through asymmetric hybridization with a member of the D. fuchsii / maculata group as the maternal parent.     

Nuclear locus divergence at the early stages of speciation in the Orchard Oriole complex

As two lineages diverge from one another, mitochondrial DNA should evolve fixed differences more rapidly than nuclear DNA due to its smaller effective population size and faster mutation rate. As a consequence, molecular systematists have focused on the criteria of reciprocal monophyly in mitochondrial DNA for delimiting species boundaries. However, mitochondrial gene trees do not necessarily reflect the evolutionary history of the taxa in question, and even mitochondrial loci are not expected to be reciprocally monophyletic when the speciation event happened very recently. The goal of this study was to examine mitochondrial paraphyly within the Orchard Oriole complex, which is composed of Icterus spurius (Orchard Oriole) and Icterus fuertesi (Fuertes' Oriole). We increased the geographic sampling, added four nuclear loci, and used a range of population genetic and coalescent methods to examine the divergence between the taxa. With increased taxon sampling, we found evidence of clear structure between the taxa for mitochondrial DNA. However, nuclear loci showed little evidence of population structure, indicating a very recent divergence between I. spurius and I. fuertesi. Another goal was to examine the genetic variation within each taxon to look for evidence of a past founder event within the I. fuertesi lineage. Based on the high amounts of genetic variation for all nuclear loci, we found no evidence of such an event – thus, we found no support for the possible founding of I. fuertesi through a change in migratory behavior, followed by peripheral isolates speciation. Our results demonstrate that these two taxa are in the earliest stages of speciation, at a point when they have fixed differences in plumage color that are not reflected in monophyly of the mitochondrial or nuclear DNA markers in this study. This very recent divergence makes them ideal for continued studies of species boundaries and the earliest stages of speciation.     

Delimiting species in the genus Otospermophilus (Rodentia: Sciuridae), using genetics,ecology, and morphology

We apply an integrative taxonomy approach to delimit species of ground squirrels in the genus Otospermophilus because the diverse evolutionary histories of organisms shape the existence of taxonomic characters. Previous studies of mitochondrial DNA from this group recovered three divergent lineages within Otospermophilus beecheyi separated into northern, central, and southern geographical populations, with Otospermophilus atricapillus nested within the southern lineage of O. beecheyi. To further evaluate species boundaries within this complex, we collected additional genetic data (one mitochondrial locus, 11 microsatellite markers, and 11 nuclear loci), environmental data (eight bioclimatic variables), and morphological data (23 skull measurements). We used the maximum number of possible taxa (O. atricapillus, Northern O. beecheyi, Central O. beecheyi, and Southern O. beecheyi) as our operational taxonomic units (OTUs) and examined patterns of divergence between these OTUs. Phenotypic measures (both environmental and morphological) showed little differentiation among OTUs. By contrast, all genetic datasets supported the evolutionary independence of Northern O. beecheyi, although they were less consistent in their support for other OTUs as distinct species. Based on these data, we support the conclusions from a previous study that synonymized O. atricapillus with O. beecheyi, and we elevate the northern lineage of O. beecheyi to a separate species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 1136–1151.     

Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae)

Vallo, P., Benda, P., Červený, J. & Koubek, P. (2012). Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae). —Zoologica Scripta, 42, 1–12. Hybridization between species may result in introgression of mitochondrial DNA from one species to another. Phylogenetic inference, therefore, may not recover true evolutionary relationships. In bats, there are only a few reported cases of introgressive hybridization. House bats are a genus with obscure phylogeny and taxonomy, caused mainly by morphological similarity. We undertook a detailed analysis of small‐sized West African house bats (Scotophilus), tentatively identified as S. nigritellus, to clarify relationships between two sympatric colour forms. These forms were recovered in paraphyletic position to each other in both mitochondrial and nuclear phylogenies, signifying that they are two distinct species. While the yellow‐bellied form could be assigned beyond doubt to S. nigritellus s. str., the white‐bellied form may be an as yet undescribed species. Moreover, the white‐bellied form clustered as a sister mitochondrial lineage to another species, Scotophilus leucogaster. These sister lineages differed by only 2.6–2.8% sequence divergence, which lies within the intraspecific range for this genus. Two nuclear markers, however, contradicted the sister relationship, showing them instead to be distantly related. The apparent conflict between the mitochondrial and nuclear signals suggests that past hybridization may have occurred between these morphologically distinct species.     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales,Phaeophyceae)

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).     

Using multilocus approach to uncover cryptic diversity within Pseudotrapelus lizards from Saudi Arabia

The agamid Pseudotrapelus lizards inhabit the mountainous areas of the Arabian Peninsula and eastern North Africa. Currently six Pseudotrapelus species are recognised, though diagnostic morphological characters are still lacking, creating great difficulty in describing new species. Recently, two specimens of Pseudotrapelus were collected from the vicinity of Riyadh in central Saudi Arabia, an area that was not sampled in previous phylogenetic studies. In here we used both mitochondrial and nuclear data to investigate the phylogenetic position of the new samples, and assess their phylogenetic relationships with the other recognised species of Pseudotrapelus from across the distribution range of the genus. We used a multilocus approach of haplotype networks, concatenated datasets and species trees, performed mitochondrial and nuclear species delimitation analyses, and estimated divergence times. In general, our results support previous molecular studies and uncover the presence of cryptic diversity within Pseudotrapelus. The phylogenetic structure of the genus is of two major clades and within them seven distinct, delimited phylogenetic groups belonging to the six recognised species and the seventh to the individuals from Riyadh. The Riyadh specimens were distinct in all analyses performed. We suggest that the new specimens from the Riyadh area are a distinct lineage, forming a clade with their phylogenetic relatives, P. sinaitus and P. chlodnickii. The clade formed by these three species diverged during the Late Miocene around 6.4 Ma, with cladogenesis possibly facilitated by vicariance and isolation caused due to climatic fluctuations and the progression of sandy areas. Our results suggest further morphological research is necessary to revise the taxonomic status of this lineage and of the entire genus.     

Finding complexity in complexes: Assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads

Mitonuclear discordance is a frequently encountered pattern in phylogeographic studies and occurs when mitochondrial and nuclear DNA display conflicting signals. Discordance among these genetic markers can be caused by several factors including confounded taxonomies, gene flow, and incomplete lineage sorting. In this study, we present a strong case of mitonuclear discordance in a species complex of toads (Bufonidae: Incilius coccifer complex) found in the Chortís Block of Central America. To determine the cause of mitonuclear discordance in this complex, we used spatially explicit genetic data to test species limits and relationships, characterize demographic history, and quantify gene flow. We found extensive mitonuclear discordance among the three recognized species within this group, especially in populations within the Chortís Highlands of Honduras. Our data reveal nuclear introgression within the Chortís Highlands populations that was most probably driven by cyclical range expansions due to climatic fluctuations. Though we determined introgression occurred within the nuclear genome, our data suggest that it is not the key factor in driving mitonuclear discordance in the entire species complex. Rather, due to a lack of discernible geographic pattern between mitochondrial and nuclear DNA, as well as a relatively recent divergence time of this complex, we concluded that mitonuclear discordance has been caused by incomplete lineage sorting. Our study provides a framework to test sources of mitonuclear discordance and highlights the importance of using multiple marker types to test species boundaries in cryptic species.     

A phylogenomic perspective on diversity,hybridization and evolutionary affinities in the stickleback genus Pungitius

Hybridization and convergent evolution are phenomena of broad interest in evolutionary biology, but their occurrence poses challenges for reconstructing evolutionary affinities among affected taxa. Sticklebacks in the genus Pungitius are a case in point: evolutionary relationships and taxonomic validity of different species and populations in this circumpolarly distributed species complex remain contentious due to convergent evolution of traits regarded as diagnostic in their taxonomy, and possibly also due to frequent hybridization among taxa. To clarify the evolutionary relationships among different Pungitius species and populations globally, as well as to study the prevalence and extent of introgression among recognized species, genomic data sets of both reference genome‐anchored single nucleotide polymorphisms and de novo assembled RAD‐tag loci were constructed with RAD‐seq data. Both data sets yielded topologically identical and well‐supported species trees. Incongruence between nuclear and mitochondrial DNA‐based trees was found and suggested possibly frequent hybridization and mitogenome capture during the evolution of Pungitius sticklebacks. Further analyses revealed evidence for frequent nuclear genetic introgression among Pungitius species, although the estimated proportions of autosomal introgression were low. Apart from providing evidence for frequent hybridization, the results challenge earlier mitochondrial and morphology‐based hypotheses regarding the number of species and their affinities in this genus: at least seven extant species can be recognized on the basis of genetic data. The results also shed new light on the biogeographical history of the Pungitius‐complex, including suggestion of several trans‐Arctic invasions of Europe from the Northern Pacific. The well‐resolved phylogeny should facilitate the utility of this genus as a model system for future comparative evolutionary studies.     

Use of mitochondrial and nuclear genes to infer the origin of two endemic pigeons from the Canary Islands

DNA nucleotide sequences from two mitochondrial genes (cytochrome b and NADH dehydrogenase subunit 2) and the nuclear intron 7 of β-fibrinogen were obtained to infer the phylogenetic origin of the two endemic Canarian pigeons: Bolle’s Pigeon (Columba bollii) and Laurel Pigeon (C. junoniae). Phylogenetic analyses of mitochondrial and nuclear genes based on maximum parsimony, maximum likelihood and Bayesian inference all converged into a congruent topology: C. bollii clusters together with the Wood Pigeon (C. palumbus) which is common in Europe and Asia, while C. junoniae was found near the base of the clade that includes other species of the genus Columba from the Old World. Laurel Pigeon probably represents an old lineage that might have colonized the Canary Islands a long time ago (20 My) while Bolle’s Pigeon might have arrived on the archipelago much later during the Upper Miocene (5 My). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.     

Recognition of a new generic‐level swallow taxon from central Africa

The forest swallow Petrochelidon fuliginosa is a little‐known species endemic to lowland forests in central Africa; for lack of access to high‐quality genetic material, the species has been omitted from all previous molecular phylogenetic studies of the swallows. The species is currently placed in the genus Petrochelidon, within the ‘mud‐nester’ clade of swallows, yet its plumage, morphology, and nesting behavior do not align well with those of other major swallow lineages. As a consequence, upon securing recent specimens and high‐quality tissue samples, we sequenced DNA from two mitochondrial genes and one nuclear marker to place this species in the swallow phylogenetic tree. Our results placed the forest swallow firmly within the ‘mud nester’ clade, but outside of the clade corresponding to Petrochelidon. This outcome led us to document and describe formally a distinct, generic‐level lineage of swallow endemic to the Lower Guinean forest region of central Africa.     

TESTS OF PHYLOGEOGRAPHIC MODELS WITH NUCLEAR AND MITOCHONDRIAL DNA SEQUENCE VARIATION IN THE STONE CRABS,MENIPPE ADINA AND MENIPPE MERCENARIA

Evolutionary relationships among stone crabs (Menippe) from the Gulf of Mexico and western Atlantic were investigated by comparisons of restriction sites within anonymous nuclear DNA sequences and nucleotide sequences of both mitochondrial and a duplicated nuclear form of the mitochondrial large subunit ribosomal RNA (LSrDNA) gene. A survey of over 100 restriction sites by Southern blot analysis with 10 anonymous nuclear DNA sequence probes failed to reveal any differences between Menippe adina and M. mercenaria. Sequence comparisons of both mitochondrial and nuclear forms of the LSrDNA gene also did not distinguish these species. Although both LSrDNA gene sequences were variable, some haplotypes were shared by the two species, implying either incomplete gene lineage sorting or introgressive hybridization. Based on molecular clock calibrations, we estimate that all of the observed mitochondrial LSrDNA sequences share a common ancestor between 1.5 and 2.7 million years before present (M.Y.B.P.). However, because identical sequences are shared by the two species, these data are also compatible with a more recent common ancestry. These findings conflict with a previously proposed biogeographic scenario for North American Menippe, which featured a relict hybrid zone on the Atlantic Coast. We suggest an alternative scenario based on relatively recent events and ongoing, rather than historical, gene flow.     

Phylogenetic relationships in Nuphar (Nymphaeaceae): evidence from morphology,chloroplast DNA,and nuclear ribosomal DNA

The genus Nuphar consists of yellow-flowered waterlilies and is widely distributed in north-temperate bodies of water. Despite regular taxonomic evaluation of these plants, no explicit phylogenetic hypotheses have been proposed for the genus. We investigated phylogenetic relationships in Nuphar using morphology and sequences of the chloroplast gene matK and of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Two major lineages within Nuphar are consistently resolved with the morphological and molecular data sets. One lineage comprises New World taxa and the other represents a primarily Old World lineage. Relationships within the major lineages were poorly resolved by morphology and ITS, yet certain relationships were elucidated by all analyses. Most notable is the strong support for a monophyletic lineage of dwarf taxa and the alliance of the North American N. microphylla with the Eurasian taxa. Minor discordance between the independent cladograms is accounted for by hybridization. The common taxonomic practice of uniting all North American and Eurasian taxa under one species is not supported phylogenetically.     

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.