Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America

White-nose syndrome (WNS)  of bats, caused by the fungus previously known as Geomyces destructans, has decimated populations of insectivorous bats in eastern North America. Recent work on fungi associated with bat hibernacula uncovered a large number of species of Geomyces and allies, far exceeding the number of described species. Communication about these species has been hindered by the lack of a modern taxonomic evaluation, and a phylogenetic framework of the group is needed to understand the origin of G. destructans and to target closely related species and their genomes for the purposes of understanding mechanisms of pathogenicity. We addressed these issues by generating DNA sequence data for the internal transcribed spacer (ITS) region, nuclear large subunit (LSU) rDNA, MCM7, RPB2, and TEF1 from a diverse array of Geomyces and allies that included isolates recovered from bat hibernacula as well as those that represent important type species. Phylogenetic analyses indicate Geomyces and allies should be classified in the family Pseudeurotiaceae, and the genera Geomyces, Gymnostellatospora, and Pseudogymnoascus should be recognized as distinct. True Geomyces are restricted to a basal lineage based on phylogenetic placement of the type species, Geomyces auratus. Thus, G. destructans is placed in genus Pseudogymnoascus. The closest relatives of Pseudogymnoascus destructans are members of the Pseudogymnoascus roseus species complex, however, the isolated and long branch of P. destructans indicates that none of the species included in this study are closely related, thus providing further support to the hypothesis that this pathogen is non-native and invasive in eastern North America. Several conidia-producing isolates from bat hibernacula previously identified as members of Pseudeurotium are determined to belong to the genus Leuconeurospora, which is widespread, especially in colder regions. Teberdinia hygrophila is transferred to Pseudeurotium as Pseudeurotium hygrophilum, comb. nov., in accordance with the one name per fungus system of classification, and two additional combinations are made in Pseudogymnoascus including Pseudogymnoascus carnis and Pseudogymnoascus pannorum. Additional sampling from other regions of the world is needed to better understand the evolution and biogeography of this important and diverse group of fungi.     

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren’t. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ² test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ² test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Phylogenetic reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes) based on multigene analyses

Hyaloscyphaceae is the largest family in Helotiales, Leotiomycetes. It is mainly characterized by minute apothecia with well-differentiated hairs, but its taxonomic delimitation and infrafamilial classification remain ambiguous. This study performed molecular phylogenetic analyses using multiple genes including the ITS-5.8S rDNA, the D1–D2 region of large subunit of rDNA, RNA polymerase II subunit 2, and the mitochondrial small subunit. The primary objective was to evaluate the phylogenetic utility of morphological characters traditionally used in the taxonomy of Hyaloscyphaceae through reassessment of the monophyly of this family and its genera. The phylogenetic analyses inferred Hyaloscyphaceae as being a heterogeneous assemblage of a diverse group of fungi and not supported as monophyletic. Among the three tribes of Hyaloscyphaceae only Lachneae formed a monophyletic lineage. The presence of hairs is rejected as a synapomorphy, since morphologically diversified hairs have originated independently during the evolution of Helotiales. The true- and false-subiculum in Arachnopezizeae are hypothesized to have evolved through different evolutionary processes; the true-subiculum is likely the product of a single evolutionary origin, while the false-subiculum is hypothesized to have originated multiple times. Since Hyaloscyphaceae sensu lato was not resolved as monophyletic, Hyaloscyphaceae sensu stricto is redefined and only applied to the genus Hyaloscypha.     

On the identity of Liolaemus nigromaculatus Wiegmann,?1834 (Iguania,Liolaemidae) and?correction?of its type locality

In the current study, we review the taxonomic status of Liolaemus nigromaculatus. Despite being the nominal species of the nigromaculatus group and being the second species of the genus Liolaemus that was described, this species is of uncertain type locality and its true identification is a matter of discussion. After carefully analyzing several digital pictures of the holotype (juvenile male), reviewing all of the literature concerning the issue, examining specimens of nearly all recognized species of the nigromaculatus group, and determining the locations visited by the specimen collector, we are able to point out the following: 1) Liolaemus nigromaculatus was collected between Puerto Viejo and Copiapó of the Atacama region in Chile, and not in Huasco 2) Liolaemus bisignatus is a nomen nudum, and populations attributed to Liolaemus bisignatus should be referred to as Liolaemus nigromaculatus. 3) There is agreement that Liolaemus copiapoensis is indistinguishable from populations currently referred to as Liolaemus bisignatus (= Liolaemus nigromaculatus), 4) Populations found in Huasco (currently considered the type locality of Liolaemus nigromaculatus) are very similar to those found in Caldera (currently considered Liolaemus bisignatus) and should be designated as Liolaemus nigromaculatus, and 5) Liolaemus oxycephalus and Liolaemus inconspicuus are not synonymous with Liolaemus nigromaculatus, although their true identities are difficult to determine. We also detail several characteristic based on the holotype of Liolaemus nigromaculatus, in addition to drawing diagnostic comparisons between this species and others belonging to the nigromaculatus group.     

Isolation and identification of entomopathogenic nematodes and their symbiotic bacteria from Hérault and Gard (Southern France)

Isolation and identification of native nematode-bacterial associations in the field are necessary for successful control of endemic pests in a particular location. No study has yet been undertaken to recover and identify EPN in metropolitan France. In the present paper, we provide results of a survey of EPN and their symbiotic bacteria conducted in Hérault and Gard regions in Southern France. Molecular characterization of isolated nematodes depicted three different Steinernema species and one Heterorhabditis species, H. bacteriophora. Steinernema species recovered were identified as: S. feltiae and S. affine and an undescribed species. Xenorhabdus symbionts were identified as X. bovienii for both S. feltiae and S. affine. Phylogenetic analysis placed the new undescribed Steinernema sp. as closely related to S. arenarium but divergent enough to postulate that it belongs to a new species within the “glaseri-group”. The Xenorhabdus symbiont from this Steinernema sp. was identified as X. kozodoii. All Heterorhabditis isolates recovered were diagnosed as H. bacteriophora and their bacterial symbionts were identified as Photorhabdus luminescens. Molecular characterization of these nematodes enabled the distinction of two different H. bacteriophora strains. Bacterial symbiontic strains of these two H. bacteriophora strains were identified as P. luminescens ssp. kayaii and P. luminescens ssp. laumondii.     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

Ruscus hypophyllum: A New Host for Aphelenchoides fragariae

Aphelenchoides fragariae was isolated from the phylloclades of the ornamental plant Ruscus hypophyllum (Liliaceae). Rotylenchus buxophilus, Scutellonema brachyurum, and Meloidogyne were identified as the most common plant-parasitic nematodes in the soil near the roots. The pathology and life history of A. fragariae were closely related to the climate. To our knowledge, this is the first report of R. hypophyllum as a host of plant-parasitic nematodes.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Morphological and Molecular Characterization of Lymnaeid Snails and Their Potential Role in Transmission of Fasciola spp. in Vietnam

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Uniquely derived upper molar morphology of Eocene Amphipithecidae (Primates: Anthropoidea): Homology and phylogeny

The extinct Southeast Asian primate family Amphipithecidae is regularly cited in discussions of anthropoid origins, but its phylogenetic position remains controversial. In part, the lack of consensus regarding amphipithecid relationships can be attributed to uncertainty regarding the homology of upper molar structures in this group. Here, we describe a virtually pristine upper molar of Pondaungia cotteri from the late middle Eocene Pondaung Formation of Myanmar, which is the first example of a relatively unworn and well-preserved amphipithecid upper molar ever recovered. The distolingual upper molar cusp in this new specimen of Pondaungia appears to be a lingually displaced and enlarged metaconule, rather than a hypocone or pseudohypocone as previous workers have thought. Reassessment of the upper molar morphology of other amphipithecids and putative amphipithecids reveals a very similar pattern in Siamopithecus, Myanmarpithecus and Ganlea, all of which are interpreted as having upper molars showing many of the same derived features apparent in Pondaungia. In contrast, the upper molar morphology of Bugtipithecus diverges radically from that of undoubted amphipithecids, and the latter taxon is excluded from Amphipithecidae on this basis. Phylogenetic analyses of several character–taxon matrices culled from the recent literature and updated to reflect the new information on amphipithecid upper molar morphology yield similar results. Consensus tree topologies derived from these analyses support amphipithecid monophyly and stable relationships within Amphipithecidae. Amphipithecids appear to be stem members of the anthropoid clade.     

Semicircular canal system in early primates

Mammals with more rapid and agile locomotion have larger semicircular canals relative to body mass than species that move more slowly. Measurements of semicircular canals in extant mammals with known locomotor behaviours can provide a basis for testing hypotheses about locomotion in fossil primates that is independent of postcranial remains, and a means of reconstructing locomotor behaviour in species known only from cranial material. Semicircular canal radii were measured using ultra high resolution X-ray CT data for 9 stem primates (“plesiadapiforms”; n = 11), 7 adapoids (n = 12), 4 omomyoids (n = 5), and the possible omomyoid Rooneyia viejaensis (n = 1). These were compared with a modern sample (210 species including 91 primates) with known locomotor behaviours. The predicted locomotor agilities for extinct primates generally follow expectations based on known postcrania for those taxa. “Plesiadapiforms” and adapids have relatively small semicircular canals, suggesting they practiced less agile locomotion than other fossil primates in the sample, which is consistent with reconstructions of them as less specialized for leaping. The derived notharctid adapoids (excluding Cantius) and all omomyoids sampled have relatively larger semicircular canals, suggesting that they were more agile, with Microchoerus in particular being reconstructed as having had very jerky locomotion with relatively high magnitude accelerations of the head. Rooneyia viejaensis is reconstructed as having been similarly agile to omomyids and derived notharctid adapoids, which suggests that when postcranial material is found for this species it will exhibit features for some leaping behaviour, or for a locomotor mode requiring a similar degree of agility.     

Intestinal Parasites among Wild Rodents in Northern Gangwon-do,Korea

To determine geographical patterns of natural parasite infections among wild rodents, a total of 46 wild rodents from 3 different localities in northern Gangwon-do (Province), Korea were examined for intestinal parasite infections. Along with nematodes such as hookworms and Syphacia spp., Plagiorchis muris (2 specimens) (Trematoda) were collected from striped field mice, Apodemus agrarius. In a Korean wood mouse, Apodemus peninsulae, the overall nematode infections were similar to A. agrarius, but an adult worm of Echinostoma hortense (Trematoda) was collected. In addition, 2 species of cestodes, i.e., Hymenolepis nana and Hymenolepis diminuta, were collected from A. agrarius. Through this survey, A. agrarius and A. peninsule were confirmed as the natural definite hosts for zoonotic intestinal helminths, i.e., P. muris, E. hortense, H. nana, and H. diminuta, in northern Gangwon-do, Korea. Considering increased leisure activities around these areas, seasonal and further comprehensive surveys on wild rodents seem to be needed to prevent zoonotic parasite infections.     

Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries

Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidic and degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a poor acid savanna soil from Venezuela was characterized, and the members of the collection were compared to other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulating Centrosema in American countries were closely related to different species of the genus Bradyrhizobium. However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that they formed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineages that were divergent among themselves and with respect to those formed by Centrosema strains isolated in other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed for the first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhizobium. The nodC genes of the Centrosema strains were divergent among themselves and with respect to the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume. According to these results, the currently known Centrosema-nodulating strains represent several new putative species and symbiovars of the genus Bradyrhizobium.