Species-level Lineages Research Articles

Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species

Accurate species delimitations are of great importance for effectively characterizing biological diversity. Our criteria for delimiting species have changed dramatically over the last decades with the increasing availability of molecular data and improvement of analytical methods to evaluate these data. Whereas reciprocal monophyly is often seen as an indicator for the presence of distinct lineages, recently diverged species often fail to form monophyletic groups. At the same time, cryptic species have repeatedly been detected in numerous organismal groups. In this study, we addressed the species delimitation in the crustose lichen-forming fungal genus Diploschistes using multilocus sequence data from specimens representing 16 currently accepted species. Our results indicate the presence of previously undetected, cryptic species-level lineages in the subgenus Limborina. In the subgenus Limborina, samples from different continents currently classified under the same species were shown to be only distantly related. At the same time, in parts of subgen. Diploschistes characterized by short branches, none of the currently accepted species formed monophyletic groups. In spite of the lack of monophyly in phylogenetic reconstructions, a multispecies coalescent method provided support for eight of the nine accepted species in subgen. Diploschistes as distinct lineages. We propose to reduce D. neutrophilus to synonymy with D. diacapsis and point out that additional sampling will be necessary before accepting additional species in subgen. Limborina.

Hidden diversity before our eyes: Delimiting and describing cryptic lichen-forming fungal species in camouflage lichens (Parmeliaceae, Ascomycota)

Molecular data provide unprecedented insight into diversity of lichenized fungi, although morphologically cryptic species-level lineages circumscribed from sequence data often remain undescribed even in well-studies groups. Using diagnostic characters from DNA sequence data and support from the multispecies coalescent model, we formally describe a total of eleven new species and resurrect two others in the hyperdiverse lichen-forming fungal family Parmeliaceae. These include: four in the genus Melanelixia – M. ahtii sp. nov., M. epilosa comb. nov., M. hawksworthii sp. nov., and M. robertsoniorum sp. nov.; six in Melanohalea – M. austroamericana sp. nov., M. beringiana sp. nov., M. clari sp. nov., M. columbiana sp. nov., M. davidii sp. nov., and M. tahltan sp. nov.; and three species in Montanelia – M. occultipanniformis sp. nov., M. saximontana comb. nov., and M. secwepemc sp. nov. Morphological, ecological and geographical features were revised to corroborate species descriptions. These species can consistently be distinguished by differences in nucleotide position characters in the fungal barcoding marker (ITS) and high speciation probabilities. This study helps close the “taxonomic gap” between molecular species delimitation studies and formal taxonomy by incorporating statistical evaluation of lineage independence, diagnostic differences in DNA data, and additional consideration of differences in morphology and species distributions.

Lineage diversification of fringe-toed lizards (Phrynosomatidae: Uma notata complex) in the Colorado Desert: Delimiting species in the presence of gene flow

Multi-locus nuclear DNA data were used to delimit species of fringe-toed lizards of the Uma notata complex, which are specialized for living in wind-blown sand habitats in the deserts of southwestern North America, and to infer whether Quaternary glacial cycles or Tertiary geological events were important in shaping the historical biogeography of this group. We analyzed ten nuclear loci collected using Sanger sequencing and genome-wide sequence/single-nucleotide polymorphism (SNP) data collected using restriction-associated DNA (RAD) sequencing. A combination of species discovery methods (concatenated phylogenies, parametric and non-parametric clustering algorithms) and species validation approaches (coalescent-based species tree/isolation-with-migration models) were used to delimit species, infer phylogenetic relationships, and to estimate effective population sizes, migration rates, and speciation times. Uma notata, U. inornata, U. cowlesi, and an undescribed species from Mohawk Dunes, Arizona (U. sp.) were supported as distinct in the concatenated analyses and by clustering algorithms, and all operational taxonomic units were decisively supported as distinct species by ranking hierarchical nested speciation models with Bayes factors based on coalescent-based species tree methods. However, significant unidirectional gene flow (2NM>1) from U. cowlesi and U. notata into U. rufopunctata was detected under the isolation-with-migration model. Therefore, we conservatively delimit four species-level lineages within this complex (U. inornata, U. notata, U. cowlesi, and U. sp.), treating U. rufopunctata as a hybrid population (U. notata×cowlesi). Both concatenated and coalescent-based estimates of speciation times support the hypotheses that speciation within the complex occurred during the late Pleistocene, and that the geological evolution of the Colorado River delta during this period was an important process shaping the observed phylogeographic patterns.

Tidying up the genusLetharia: introducingL. lupinasp. nov. and a new circumscription forL. columbiana

AbstractWestern North America is the global centre of diversity forLetharia, a distinctive and cryptically diverse genus of lichenized fungi belonging to theParmeliaceae. The genus is characterized by a shrubby, fruticose habit and presence of vulpinic acid. Previous studies using multiple fungal nuclear loci revealed the existence of two distinct species-level lineages within the traditional concept ofL. vulpinaand four such lineages withinL. columbiana. Here we use molecular sequence data in an attempt to settle long-standing taxonomic issues in the genus. Our results confirm the widespread existence withinL. vulpinas. lat. of two distinct species-level groups, each forming a mutually exclusive partnership with a separate algal clade withinTrebouxia jamesiis. lat. Accordingly, we formally describe the segregate speciesL. lupinasp. nov. Our results also support the evolutionary independence of four candidate species previously circumscribed fromL. columbianas. lat. One of these lineages,L.‘gracilis’, has already received species recognition asL. gracilis, while a second,L.‘lucida’, is epitypified here againstL. columbianas. str. Based on results from species delimitation analyses under the multispecies coalescent model, the two remaining lineages,L.‘barbata’ andL.‘rugosa’, also warrant formal taxonomic recognition; however, we refrain from describing these species pending additional studies of diagnostic characters, ecological preference, and distributions.

Cercospora cf. flagellaris and Cercospora cf. sigesbeckiae Are Associated with Cercospora Leaf Blight and Purple Seed Stain on Soybean in North America.

Cercospora kikuchii has long been considered the causal agent of Cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean, but a recent study found C. cf. flagellaris associated with CLB and PSS in Arkansas (United States) and Argentina. Here, we provide a broader perspective on the distribution of C. cf. flagellaris on soybean and alternate hosts within the United States (Arkansas, Louisiana, Mississippi, Missouri, and Kansas). We used a multilocus phylogenetic approach with data from actin, calmodulin, translation elongation factor 1-α, histone 3, the internal transcribed spacer region of rDNA and the mating-type locus to determine that two species, C. cf. flagellaris (200 of 205 isolates) and C. cf. sigesbeckiae (five of 205 isolates), are associated with CLB and PSS in the United States. In our phylogenetic analyses, species-level lineages were generally well-supported, though deeper-level evolutionary relationships remained unresolved, indicating that these genes do not possess sufficient phylogenetic signal to resolve the evolutionary history of Cercospora. We also investigated the potential for sexual reproduction in C. cf. flagellaris in Louisiana by determining the frequency of MAT1-1/MAT1-2 mating-type idiomorphs within the Louisiana population of C. cf. flagellaris. Though the MAT 1-2 idiomorph was significantly more common in our collection, the presence of both mating types suggests the potential for sexual reproduction exists.

Hidden Genetic Diversity in an Asexually Reproducing Lichen Forming Fungal Group.

Asexual species with vegetative propagation of both symbiont partners (soredia) in lichens may harbor lower species diversity because they may indeed represent evolutionary dead ends or clones. In this study we aim to critically examine species boundaries in the sorediate lichen forming fungi Parmotrema reticulatum–Parmotrema pseudoreticulatum complex applying coalescent-based approaches and other recently developed DNA-based methods. To this end, we gathered 180 samples from Africa, Asia, Australasia, Europe, North and South America and generated sequences of internal transcribed spacer of nuclear ribosomal DNA (ITS) and DNA replication licensing factor MCM7 (MCM7). The dataset was analysed using different approaches such as traditional phylogeny–maximum likelihood and Bayesian–genetic distances, automatic barcode gap discovery and coalescent-based methods–PTP, GMYC, spedeSTEM and *Beast–in order to test congruence among results. Additionally, the divergence times were also estimated to elucidate diversification events. Delimitations inferred from the different analyses are comparable with only minor differences, and following a conservative approach we propose that the sampled specimens of the P. reticulatum–P. pseudoreticulatum complex belong to at least eight distinct species-level lineages. Seven are currently classified under P. reticulatum and one as P. pseudoreticulatum. In this work we discuss one of only few examples of cryptic species that have so far been found in sorediate reproducing lichen forming fungi. Additionally our estimates suggest a recent origin of the species complex–during the Miocene. Consequently, the wide distribution of several of the cryptic species has to be explained by intercontinental long-distance dispersal events.

Spongiibacter taiwanensis sp. nov., a marine bacterium isolated from aged seawater.

A Gram-reaction-negative, heterotrophic, marine bacterium, designated strain SPT1T, was isolated from an aged seawater sample which was collected from the shallow coastal region of Nanya, Keelung, Taiwan and stored at room temperature for more than 7 years. Strain SPT1T was a motile rod which exhibited monotrichous flagellation. It required NaCl for growth and exhibited optimal growth at 30-35 °C, 1-3 % NaCl and pH 7-8. The strain was a strictly aerobic bacterium, incapable of anaerobic growth by nitrate reduction or denitrification, or by fermenting glucose or other carbohydrates. Cellular fatty acids were dominated by C16 : 1ω7c and/or C16 : 1ω6c (23.4 %), C17 : 1ω8c (18.1 %), C16 : 0 (8.5 %), C18 : 1ω7c (8.4 %) and C10 : 0 3-OH (6.3 %). The predominant isoprenoid quinone was Q-8. Polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid. The DNA G+C content was 57.9 mol%. Phylogeny based on 16S rRNA gene sequences showed that strain SPT1T formed a distinct species-level lineage within the genus Spongiibacter of the class Gammaproteobacteria and shared sequence similarities of 94.4-96.2 % with Spongiibacter marinusand Spongiibacter tropicus, the only two species of the genus Spongiibacterwith validly published names. The 16S rRNA gene sequence similarities between strain SPT1T and other species were less than 93.1 %. Polyphasic taxonomic data obtained in this study indicated that strain SPT1T could be classified as a novel species of the genus Spongiibacter, for which the name Spongiibacter taiwanensis sp. nov. is proposed. The type strain is SPT1T (=JCM 31012T=BCRC 80916T).

Molecular data show thatHypotrachyna sorocheila(Parmeliaceae) is not monophyletic

Abstract Lichens that reproduce by means of vegetative propagules (soredia or isidia) are generally thought to have broad geographic distributions. However, recent studies have shown that some asexually reproducing lichens with broad distribution may be comprised of multiple, independent species-level lineages. Our understanding of species diversity in asexually reproducing lichenized fungal species may be further confounded by the fact that otherwise morphologically similar taxa separated based on the presence or absence of vegetative reproductive structures may in fact be conspecific. In this study, we investigate genetic diversity of the pantropical sorediate species Hypotrachyna sorocheila using molecular sequence data. Specifically, we generated a three-marker dataset for Hypotrachyna subgen. Everniastrum specimens, and reconstructed a multilocus, molecular phylogeny. Our results show that sorediate samples phenotypically identifiable as H. sorocheila do not form a monophyletic group, but form two di...

Genome-wide data help identify an avian species-level lineage that is morphologically and vocally cryptic

Species identification has traditionally relied on morphology. However, morphological conservatism can lead to a high incidence of cryptic species, as characters other than morphological ones can be biologically important. In birds, the combined application of bioacoustic and molecular criteria has led to an avalanche of cryptic species discoveries over the last two decades in which findings of deep vocal differentiation have usually been corroborated by molecular data or vice versa. In this study, we use genome-wide DNA data to uncover an unusual case of cryptic speciation in two species within the South-east Asian Streak-eared Bulbul Pycnonotus blanfordi complex, in which both morphology and vocalizations have remained extremely similar. Despite a considerable pre-Pleistocene divergence of these two bulbul species, bioacoustic analysis failed to uncover differences in their main vocalization, but examination of live birds revealed important differences in eye color that had been overlooked in museum material. Our study demonstrates that genome-wide DNA data can be helpful in the detection of cryptic speciation, especially in species that have evolved limited morphological and behavioral differences.

Have we so far only seen the tip of the iceberg? Exploring species diversity and distribution of the giant amphipod Eurythenes

Additional material of the iconic giant amphipod Eurythenes was investigated. Recently, the species E. gryllus has been separated into 12 distinct species-level lineages of which several have been described as distinct species, based on both morphology and genetics. This study revealed three additional species-level lineages from unique sampling localities, showing that with minimal sampling effort, species diversity within Eurythenes can still increase. One species-level lineage was found in the Indian Ocean and another one in the Pacific, which was subsequently identified as E. thurstoni. In addition to the three species already reported from the Southern Ocean (E. maldoror, E. gryllus s.s. and E. andhakarae), a supplementary bathyal species was found in the Weddell Sea. E. gryllus was confirmed to be amphitropical including newly sampled localities around the Kerguelen Islands and additional samples from the Svalbard Archipelago. Building on new and earlier data, geographic and bathymetric distributions of the different species that have been discovered so far are presented here and several factors are evaluated for their likelihood of having triggered past speciation events in this scavenger. Topographic and hydrographical features are discussed but rejected as sufficient reasons for the distributional patterns observed. Bathymetric segregation is interpreted with regard to what is known about the ecology of the species. The previously reported genetic break around 3000 m persists in this new data-set for all species but one. This study underlines the need of processing all individuals sampled, since two or more sympatric species are found in different proportions, and that conclusions regarding diversity and distribution may drastically change when increasing sampling intensity and coverage. Finally, I suggest here that only a mere fraction of all Eurythenes species has yet been discovered and that a more complete knowledge of the ecology of the species is of paramount importance for interpreting their evolution.

Cryptic diversity and symbiont interactions in rock-posy lichens

Identifying factors that influence species interactions is central to research in symbiotic systems. While lichens represent iconic models of symbiosis and play important roles in understanding the biology of symbiotic interactions, patterns of interactions in lichen symbionts and mechanisms governing these relationships are not well characterized. This is due, in part to the fact that current taxonomic approaches for recognizing diversity in lichen symbionts commonly fail to accurately reflect actual species diversity. In this study, we employed DNA-based approaches to circumscribed candidate species-level lineages in rock-posy lichen symbionts (mycobiont=Rhizoplaca s. lat. species; photobiont=Trebouxia species). Our results revealed a high degree of cryptic diversity in both the myco- and photobionts in these lichens. Using the candidate species circumscribed here, we investigated the specificity of the symbionts toward their partners and inferred the relative importance of various factors influencing symbiont interactions. Distinct mycobiont species complexes, ecozones, and biomes are significantly correlated with the occurrence of photobiont OTUs, indicating that complex interactions among mycobiont lineages, ecogeography, and microhabitat determine interactions between photobionts and their mycobionts in lichen symbiosis. One-to-one specificity between mycobiont and photobiont species was not found, with the exception of R. maheui that associated with a single Trebouxia OTU that was not found with other Rhizoplaca s. lat. species. We estimated the most recent common ancestor of the core Rhizoplaca group at c. 62.5Ma, similar in age to the diverse parmelioid core group in the well-studied family Parmeliaceae. However, in contrast to Parmeliaceae, species in Rhizoplaca were found to associate with a narrow range of photobionts. Our study provides important perspectives into species diversity and interactions in iconic lichen symbiotic systems and establishes a valuable framework for continuing research into rock-posy lichens.

Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae) reveals a new genus Turmalinea and highlights the utility of ITS minisatellite-like insertions for molecular identification.

The sequestrate (truffle-like) basidiomycete genera Rossbeevera, Chamonixia, and Octaviania are closely related to the epigeous mushroom genera Leccinum and Leccinellum. In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of Rossbeevera and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, EF-1α) and two mitochondrial DNA loci (ATP6 and mtSSU) as well as precise morphological observations. Phylogenetic analyses of three nuclear loci suggest a complex evolutionary history with sequestrate fruiting bodies present in several clades, including a previously unrecognized sister clade to Rossbeevera. Here we propose a new sequestrate genus, Turmalinea, with four new species and one new subspecies as well as two new species of Rossbeevera. The three-locus nuclear phylogeny resolves species-level divergence within the Rossbeevera-Turmalinea lineage, whereas a separate phylogeny based on two mitochondrial genes corresponds to geographic distance within each species-level lineage and suggests incomplete lineage sorting (ILS) and gene introgression within several intraspecific lineages of Rossbeevera. Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within Rossbeevera probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all Rossbeevera and Turmalinea species. A barcode gap analysis demonstrates that the insertion is more informative for discrimination at various taxonomic levels than the rest of the ITS region and could therefore serve as a unique molecular barcode for these genera.

Integrating phylogenomic and morphological data to assess candidate species-delimitation models in brown and red-bellied snakes (Storeria)

Systematics at the species level is still marked by theoretical and empirical tensions amongst the desires to identify geographical lineages, delimit species, and estimate their relationships. These goals are often confounded because each relies, at least to some extent, on the others being known. However, recently developed methods can simultaneously address all three. Furthermore, next-generation genomic sequencing allows us to generate large-scale molecular data sets to examine variation within species at a fine scale. Finally, a renaissance in morphological species validation allows us to integrate historical species definitions with coalescent models for species delimitation. Here, we investigate the applicability of these methods in an empirical case, in the Nearctic snake genus Storeria. Integrating trait data into species delimitation reduces the number of species delimited from molecular data alone. Whereas molecular data support eight distinct species-level lineages, including morphological data reduces this to four. The taxa Storeria dekayi, Storeria occipitomaculata, Storeria storerioides, and Storeria victa are considered distinct, monotypic species, with no subspecies recognized. We highlight the need for careful assessment of species delimitation, combining both computational genetic methods as well as traditional character-based descriptions. It is now possible to identify phylogeographical lineages, delimit species using molecular and morphological data, and estimate their relationships in a single coherent set of analyses. Moving forward, this will allow for more rapid and objective assessments of cryptic diversity at the species level.

A DNA barcoding approach for identification of hidden diversity in Parmeliaceae (Ascomycota):Parmelia sensu strictoas a case study

Accurate specimen identification is challenging in groups with subtle or scarce taxonomically diagnostic characters, and the use of DNA barcodes can provide an effective means for consistent identification. Here, we investigate the utility of DNA barcode identification of species in a cosmopolitan genus of lichen-forming fungi, Parmelia (Parmeliaceae). Two hundred and two internal transcribed spacer (ITS) sequences generated from specimens collected from all continents, including Antarctica, were analysed, and DNA barcodes of 14 species of Parmelia s.s. are reported. Almost all species show a barcode gap. Overall, intraspecific divergence values were lower than the threshold previously established for Parmeliaceae. However, the mean and range were elevated by deep barcode divergences in three species, indicating the likely occurrence of overlooked species-level lineages. Here, we provide a DNA barcode reference library with well-identified specimens sampled worldwide and sequences from most of the type material to enable easy and fast accurate sample identification and to assist in uncovering overlooked species in Parmelia s.s. Further, our results confirm the efficiency of the ITS region in the identification of species of Parmelia s.s. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ●●, ●●–●●.

Revision of the Western Australian pebble-mimic dragon species-group (Tympanocryptis cephalus: Reptilia: Agamidae).

Recent work on species complexes of the pebble-mimic dragons of the Australian genus Tympanocryptis has greatly clarified evolutionary relationships among taxa and also indicated that species diversity has been severely underestimated. Here we provide a morphological and molecular appraisal of variation in the T. cephalus species-group and find evidence for recognizing five species-level lineages from Western Australia. Four species-level lineages are strongly supported with a combined mitochondrial and nuclear DNA Bayesian analysis (a fifth population from the Gascoyne region lacked tissue samples). Morphologically, we found subtle, yet consistent, differences among the populations in scalation, color and pattern. True T. cephalus Günther is restricted to the coastal Pilbara region and characterized by five dark blotches on the dorsum, keeled ventrals, and other characters. Two other lineages within the Pilbara, from the Hamersley range and Fortescue/northern Pilbara region, differed from T. cephalus senso stricto by possessing a more elongate body and a plain dorsum. Furthermore, the Hamersley lineage differed from the Fortescue lineage by possessing slightly more reddish coloration and feeble keeling on the snout. Although there are few specimens and no tissue samples available for the Gascoyne population, these individuals are larger, have rugose scales on the snout, and possess scattered enlarged tubercles with three large blotches on the dorsum. The name T. cephalus gigas Mitchell is available for this population. The most widespread lineage, and the one best represented in collections and in field guides, occurs throughout central Western Australia. These Goldfield populations are characterized by a protruding snout, narrow rostral, and uniform reddish-brown coloration, often with a dark wash. Based on the genetic and morphological differences, we redescribe T. cephalus, resurrect and elevate T. gigas to a full species and designate a neotype for this taxon, and describe three lineages as new species (T. diabolicus sp. nov., T. fortescuensis sp. nov., T. pseudopsephos sp. nov.).

Molecular systematics of South American marsh rats of the genusHolochilus(Muroidea, Cricetidae, Sigmodontinae)

We present a comprehensive systematic study of Holochilus, a sigmodontine genus of large, herbivorous, and semiaquatic rodents widely distributed in South America. Remarkably, given its complex taxonomic history and large economic as well as epidemiological importance, the alpha taxonomy of Holochilus has not benefited from a molecular-based approach. The study is based on sequences of 1 mitochondrial and 3 nuclear loci that were analyzed by maximum likelihood and Bayesian inference. Analyses include sequences of specimens from localities from Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, Suriname, and Uruguay, representing all but 2 of the species currently recognized in the genus. Of the 4 data matrices, the mitochondrial data set contains the largest geographic coverage and recovered 6 species-level lineages that form 2 well-supported species groups: the brasiliensis species group formed by H. brasiliensis and H. vulpinus and the sciureus species group composed by H. chacarius, H. sciureus, and 2 currently unnamed forms. Surprisingly, in the cytochrome b gene analyses, the 2 species groups are not sister to each other; i.e., Holochilus is not monophyletic, although these topologies lack significant support. However, the monophyly of Holochilus was supported by the 3 nuclear loci as well as by the combined analysis of all 4 loci. These genealogical results are the basis of taxonomic and biogeographic considerations. Presentamos un estudio sistemático comprensivo sobre Holochilus, un género sigmodontino de grandes roedores herbívoros y semi-acuáticos ampliamente distribuido en América del Sur. Llamativamente, dada su compleja historia taxonómica y el gran impacto económico y epidemiológico, la taxonomía alfa de Holochilus no se ha beneficiado de un abordaje basado en evidencia molecular. El estudio se basa en secuencias de 1 gen mitocondrial y de 3 nucleares que fueron analizadas con máxima verosimilitud e inferencia Bayesiana. Los análisis incluyen secuencias de especímenes colectados en localidades de Argentina, Bolivia, Brasil, Colombia, Paraguay, Perú, Surinam y Uruguay, representando todas, con excepción de 2, las especies actualmente reconocidas en el género. La genealogía mitocondrial, que es la que tiene la mayor cobertura geográfica de Holochilus, recobra 6 linajes de nivel de especie que forman 2 grupos de especies bien apoyados: el grupo de especies brasiliensis integrado por H. brasiliensis y H. vulpinus y el grupo de especies sciureus que está compuesto por H. chacarius, H. sciureus y 2 formas aparentemente sin nominar. Llamativamente, en los análisis basados en el gen mitocondrial los 2 grupos de especies no son hermanos; i.e., Holochilus no es monofilético, aunque esta topología no tiene apoyo significativo. Sin embargo la monofilia de Holochilus es apoyada por los análisis de los 3 genes nucleares y por el análisis combinado de los 4 genes. Estos resultados genealógicos son la base de consideraciones taxonómicas y biogeográficas.

Long-distance dispersal and speciation of Australasian and American species of Cortinarius sect. Cortinarius

We present a multigene phylogeny (partial nuc rDNA and RPB2) of Cortinarius sect. Cortinarius (i.e. the C. violaceus group), which reveals eight species distributed in Europe, Australasia, South America, Central America and North America. Relaxed molecular clock analyses suggested that diversification began during the Miocene, thus rejecting more ancient Gondwanan origin scenarios among the taxa currently occurring in the northern and southern hemispheres. There was strong support for an Australasian origin of the C. violaceus group with initial dispersal to the Neotropics, followed by migration into North America and Europe. A dispersal-extinction cladogenesis model that includes a parameter for founder effects was the most highly supported biogeographic model in the program BioGeoBEARS. A maximum likelihood analysis showed the most recent common ancestor of sect. Cortinarius was an angiosperm ectomycorrhizal associate. Ancestral associations at the plant family level, however, were ambiguous. Of eight recovered species-level lineages, C. violaceus is the only one that associates with Pinaceae and the only species to associate with both Pinaceae and angiosperms. This analysis showed that long-distance dispersal and founder event speciation have been important factors during evolution of the C. violaceus group.

Integrative taxonomic approach to the cryptic diversity of Diplostomum spp. in lymnaeid snails from Europe with a focus on the 'Diplostomum mergi' species complex.

BackgroundRecent molecular studies have discovered substantial unrecognised diversity within the genus Diplostomum in fish populations in Europe and North America including three species complexes. However, data from the first intermediate host populations are virtually lacking. This study addresses the application of an integrative taxonomic approach to the cryptic species diversity of Diplostomum spp. in natural lymnaeid snail populations in Europe with a focus on the ‘D. mergi’ species complex.MethodsTotals of 1,909 Radix auricularia, 349 Radix peregra, 668 Stagnicola palustris and 245 Lymnaea stagnalis were sampled at five reservoirs of the Ruhr river system in Germany and screened for infections with Diplostomum spp. Cercariae were examined and identified alive, fixed and under scanning electron microscopy. Sequences from the barcode region of the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene and from the internal transcribed spacer cluster (ITS1-5.8S-ITS2) of the rRNA gene were amplified for 51 and 13 isolates, respectively.ResultsDetailed morphological and molecular analyses provided evidence for three named species (Diplostomum spathaceum, D. pseudospathaceum and D. parviventosum), and a further four species-level lineages (‘D. mergi Lineages 2–4’ and ‘Diplostomum sp. Clade Q’ in the lymnaeid snail populations from the Ruhr river basin. The paper provides the first descriptions of molecularly identified cercariae of D. spathaceum and of the cercariae of D. parviventosum, three lineages of the ‘D. mergi’ species complex and of ‘Diplostomum sp. Clade Q’.ConclusionThe integration of molecular and morphological evidence for Diplostomum spp. achieved in this study will serve as a baseline for species identification of these important parasites of snail and fish populations and thus advance further studies on the distribution of Diplostomum spp. in Europe.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0904-4) contains supplementary material, which is available to authorized users.

Who’s getting around? Assessing species diversity and phylogeography in the widely distributed lichen-forming fungal genus Montanelia (Parmeliaceae, Ascomycota)

Brown parmelioid lichens comprise a number of distinct genera in one of the most species-rich families of lichen-forming fungi, Parmeliaceae (Ascomycota). In spite of their superficial similarity, a number of studies of brown parmelioids have provided important insight into diversification in lichen-forming fungi with cosmopolitan distributions. In this study we assess species diversity, biogeography and diversification of the genus Montanelia, which includes alpine to temperate saxicolous species. We sampled each of the five known species, four of which are known from broad, intercontinental distributions. In order to identify potential biogeographical patterns, each broadly distributed species was represented by individuals collected across their intercontinental distributions. Molecular sequence data were generated for six loci, including three nuclear protein-coding markers (MCM7, RPB1, and RPB2), two nuclear ribosomal markers (ITS and nrLSU), and a fragment of the mitochondrial small subunit. We used three sequence-based species delimitations methods to validate traditional, phenotype-based species and circumscribe previously unrecognized species-level lineages in Montanelia. Relationships among putative lineages and divergence times were estimated within a coalescent-based multi-locus species tree framework. Based on the results of the species delimitation analyses, we propose that the genus Montanelia is likely comprised of six to nine species-level lineages, including previously unrecognized species-level diversity in the nominal taxa M. panniformis and M. tominii. In contrast, molecular sequence data suggest that M. predisjuncta may be conspecific with the widespread taxon M. disjuncta in spite of distinct morphological differences. The rate-based age estimation of the most recent common ancestor of Montanelia (ca. 23.1Ma) was similar to previous estimates based on the fossil record. Furthermore, our data suggest that diversification in Montanelia occurred largely during the Neogene. At least three Montanelia species are broadly distributed throughout Asia, Europe, and North America with no evidence of phylogeographic substructure. In contrast to broadly distributed Montanelia species, our study suggests Pleistocene-dominated diversification and complex biogeographic history in the M. tominii group. Our analyses provide additional insight for understanding diversification and uncovering cryptic diversity in cosmopolitan species of lichen-forming fungi.

Coalescent-based species delimitation approach uncovers high cryptic diversity in the cosmopolitan lichen-forming fungal genus Protoparmelia (Lecanorales, Ascomycota).

Species recognition in lichen-forming fungi has been a challenge because of unsettled species concepts, few taxonomically relevant traits, and limitations of traditionally used morphological and chemical characters for identifying closely related species. Here we analyze species diversity in the cosmopolitan genus Protoparmelia s.l. The ~25 described species in this group occur across diverse habitats from the boreal -arctic/alpine to the tropics, but their relationship to each other remains unexplored. In this study, we inferred the phylogeny of 18 species currently assigned to this genus based on 160 specimens and six markers: mtSSU, nuLSU, ITS, RPB1, MCM7, and TSR1. We assessed the circumscription of species-level lineages in Protoparmelia s. str. using two coalescent-based species delimitation methods – BP&P and spedeSTEM. Our results suggest the presence of a tropical and an extra-tropical lineage, and eleven previously unrecognized distinct species-level lineages in Protoparmelia s. str. Several cryptic lineages were discovered as compared to phenotype-based species delimitation. Many of the putative species are supported by geographic evidence.