Nuclear rDNA Research Articles

Lichenomphalia altoandina, a new species of Hygrophoraceae from the Chilean Altiplano

ABSTRACTLichenomphalia is a lichenized agaric genus characterized by its omphalinoid basidiomes. Lichenomphalia species are associated with unicellular green algae in the genus Coccomyxa and are mainly distributed in polar and alpine habitats. The aim of this work is to describe L. altoandina, a new species from northern Chile that grows among cushion plants over 3000 m above sea level in the Andes Mountains. The species is remarkable for living in highly saline environments, in some cases virtually on salt crusts. Lichenomphalia altoandina differs from other known species and particularly from L. aurantiaca, the most morphologically similar species, in its smooth and broader stipe and its slightly larger spores. Lichenomphalia altoandina is also morphologicaly and ecologically more similar to the core Lichenomphalia clade. Our phylogenetic study based on nuclear rDNA ITS and partial 28S sequences shows that L. altoandina belongs to the Protolichenomphalia clade and is sister to an unknown lineage, L. aff. umbellifera, from New Zealand.

Atractiella rhizophila, sp. nov., an endorrhizal fungus isolated from the Populus root microbiome

ABSTRACTAmong fungi isolated from healthy root mycobiomes of Populus, we discovered a new endorrhizal fungal species belonging to the rust lineage Pucciniomycotina, described here as Atractiella rhizophila. We characterized this species by transmission electron microscopy (TEM), phylogenetic analysis, and plant bioassay experiments. Phylogenetic sequence analysis of isolates and available environmental and reference sequences indicates that this new species, A. rhizophila, has a broad geographic and host range. Atractiella rhizophila appears to be present in North America, Australia, Asia, and Africa and is associated with trees, orchids, and other agriculturally important species, including soybean, corn, and rice. Despite the large geographic and host range of this species sampling, A. rhizophila appears to have exceptionally low sequence variation within nuclear rDNA markers examined. With inoculation studies, we demonstrate that A. rhizophila is nonpathogenic, asymptomatically colonizes plant roots, and appears to foster plant growth and elevated photosynthesis rates.

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

AbstractA protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.

Pseudoheterocaryum, a new genus segregated from Heterocaryum (Boraginaceae) on the basis of molecular data

The phylogeny of Heterocaryum and Suchtelenia was examined using sequence data from the internal transcribed spacer region of the nuclear rDNA (ITS) and plastid trnL intron and trnL–trnF intergenic spacer (trnL–F) regions. Results indicated that Heterocaryum is non-monophyletic because of the inclusion of Suchtelenia calycina (C.A.Mey.) A.DC. Heterocaryum laevigatum (Kar. & Kir.) A.DC. formed a distinct branch sister to S. calycina and remaining Heterocaryum species. Hence, all species of Heterocaryum except H. laevigatum (type species of the genus) are transferred to a new genus, Pseudoheterocaryum. Taxonomic descriptions are presented for Pseudoheterocaryum and Heterocaryum, as well as a diagnostic key to the three genera included in the present study.

Re-description of Chrysaora pacifica (Goette, 1886) (Cnidaria, Scyphozoa) from Korean Coastal Waters: Morphology and Molecular Comparisons

The nomenclature of the sea nettle jellyfish from Korea was initially described as Dactylometra quinquecirrha Agassiz, 1862. However its identity has been questioned on the basis of its local distribution and molecular data. Here, we examined morphology and DNA sequences of nuclear rDNA using specimens collected from southern Korean waters in August 2014. Based on morphological characteristics (bell size, umbrella pattern, number of tentacles and lappets) and distribution locality, we reassign the Korean D. quinquecirrha to Chrysaora pacifica (Goette, 1886), and provide a re-description accordingly. The molecular identity of C. pacifica was further confirmed by comparison of nuclear ribosomal DNA sequences.

A new species of Spirorchis MacCallum, 1918 (Digenea: Schistosomatoidea) and Spirorchis cf. scripta from chicken turtle, Deirochelys reticularia (Emydidae), with an emendation and molecular phylogeny of Spirorchis.

Chicken turtles, Deirochelys reticularia (Latreille in Sonnini et Latreille) (Testudines: Emydidae) from Alabama, USA were infected by Spirorchis collinsi Roberts et Bullard sp. n. and Spirorchis cf. scripta. The new species is most easily differentiated from its congeners by the combination of having caeca that extend far beyond the genitalia, intercaecal genitalia positioned in the middle portion of the body, a testicular column that nearly abuts the caecal bifurcation, a cirrus sac positioned between the testes and ovary, a massive Mehlis' gland, an elongate, longitudinal metraterm that extends anteriad beyond the level of the ovary, a pre-ovarian genital pore, and a prominent, intercaecal Manter's organ. The specimens of S. cf. scripta differed from the holotype and published descriptions of Spirorchis scripta Stunkard, 1923 by several subtle morphological features, perhaps comprising intraspecific variation, but collectively warranted a detailed description herein. Based on examinations of the aforementioned specimens plus the holotype, paratypes and vouchers of morphologically-similar congeners, Spirorchis MacCallum, 1918 is emended to include the presence of oral sucker spines, a pharynx, lateral oesophageal diverticula ('plicate organ') and a median oesophageal diverticulum ('oeseophageal pouch'). Phylogenetic analysis of the nuclear large subunit rDNA (28S) recovered S. collinsi sister to Spirorchis picta Stunkard, 1923, > 99% similarity between S. cf. scripta and S. scripta, and a monophyletic Spirorchis MacCallum, 1918. No blood fluke infection has been reported previously from these drainages, Alabama, or this turtle species. This is the first new species of Spirorchis to be described from North America in 26 years.

Novel species of Gliocladiopsis (Nectriaceae, Hypocreales, Ascomycota) from avocado roots (Persea americana) in Australia

Root rot of avocado (Persea americana) is an important disease in seedling nurseries as well as in the field in eastern and southern Australia. During an investigation into the causal organisms of avocado root rot, 19 isolates of Gliocladiopsis were obtained from necrotic lesions on avocado roots and examined by morphology and comparison of DNA sequences from three gene loci (the internal transcribed spacer region of the nuclear rDNA, Histone H3 and β-tubulin). Three new species of Gliocladiopsis are described as a result of phylogenetic analysis of these data. One of the new species, G. peggii, formed a monophyletic group that may represent an unresolved species complex as it contained a polytomy that included a well-supported clade comprising two subclades. Gliocladiopsis peggii is sister to G. mexicana, which is known from soil in Mexico. The remaining two new species, G. whileyi and G. forsbergii, formed a clade sister to G. curvata, which is known from Ecuador, Indonesia and New Zealand.

Antarctomyces pellizariae sp. nov., a new, endemic, blue, snow resident psychrophilic ascomycete fungus from Antarctica.

In the present study, we have identified and characterised a new snow resident ascomycete blue stain fungus from Antarctica named Antarctomyces pellizariae sp. nov. Menezes, Godinho, Porto, Gonçalves and Rosa, using polyphasic taxonomy techniques. This fungal species was recovered from the seasonal snow of the Antarctic Peninsula. Antarctomyces pellizariae displayed different macro- and micromorphology when compared with A. psychrotrophicus Stchigel and Guarro, the only other Antarctomyces species reported until date. Antarctomyces pellizariae showed psychrophilic behavior and very low growth rate at 22-25°C, quite different from A. psychrotrophicus that has a higher growth rate at mesophilic temperatures. In addition, micromorphological characteristics and the analysis of the nuclear rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II regions revealed that A. pellizariae is a new species that is related to A. psychrotrophicus and Thelebolus species. Since the Antarctic Peninsula is reported to be one of the main regions of the earth experiencing the effects of global change in climate, species, such as A. pellizariae, might provide information about these effects on the endemic Antarctic biota. In addition, A. pellizariae displayed psychrophilic behavior and might be a source of interesting anti-freeze compounds that might prove useful in biotechnological processes.

From the Tunnels into the Treetops: New Lineages of Black Yeasts from Biofilm in the Stockholm Metro System and Their Relatives among Ant-Associated Fungi in the Chaetothyriales.

Rock-inhabiting fungi harbour species-rich, poorly differentiated, extremophilic taxa of polyphyletic origin. Their closest relatives are often well-known species from various biotopes with significant pathogenic potential. Speleothems represent a unique rock-dwelling habitat, whose mycobiota are largely unexplored. Isolation of fungi from speleothem biofilm covering bare granite walls in the Kungsträdgården metro station in Stockholm yielded axenic cultures of two distinct black yeast morphotypes. Phylogenetic analyses of DNA sequences from six nuclear loci, ITS, nuc18S and nuc28S rDNA, rpb1, rpb2 and β-tubulin, support their placement in the Chaetothyriales (Ascomycota). They are described as a new genus Bacillicladium with the type species B. lobatum, and a new species Bradymyces graniticola. Bacillicladium is distantly related to the known five chaetothyrialean families and is unique in the Chaetothyriales by variable morphology showing hyphal, meristematic and yeast-like growth in vitro. The nearest relatives of Bacillicladium are recruited among fungi isolated from cardboard-like construction material produced by arboricolous non-attine ants. Their sister relationship is weakly supported by the Maximum likelihood analysis, but strongly supported by Bayesian inference. The genus Bradymyces is placed amidst members of the Trichomeriaceae and is ecologically undefined; it includes an opportunistic animal pathogen while two other species inhabit rock surfaces. ITS rDNA sequences of three species accepted in Bradymyces and other undescribed species and environmental samples were subjected to phylogenetic analysis and in-depth comparative analysis of ITS1 and ITS2 secondary structures in order to study their intraspecific variability. Compensatory base change criterion in the ITS2 secondary structure supported delimitation of species in Bradymyces, which manifest a limited number of phenotypic features useful for species recognition. The role of fungi in the speleothem biofilm and relationships of Bacillicladium and Bradymyces with other members of the Chaetothyriales are discussed.

Molecular Identification of Sclerotinia trifoliorum and Sclerotinia sclerotiorum Isolates from the United States and Poland.

Symptoms of clover rot caused by Sclerotinia trifoliorum or S. sclerotiorum are identical, making differentiation and identification of the causal species difficult and time consuming. Polymerase chain reaction (PCR) amplification and nucleotide sequencing were used to examine 40 isolates of S. trifoliorum (29 from Poland, 11 from the United States) and 55 isolates of S. sclerotiorum (26 from Poland, 29 from the United States). We determined that amplification of the β-tubulin and calmodulin genes with TU1/TU2/TU3 and SscadF1/SscadR1 PCR primers and the presence of introns and single-nucleotide polymorphisms (SNP) within the ribosomal DNA (rDNA) as detected with NS1/NS8 and internal transcribed spacer (ITS)1/ITS4 PCR primers are effective for rapidly and accurately differentiating between the two species of Sclerotinia. In addition, our research revealed a lack of intraspecies variation within S. sclerotiorum isolates from the United States and Poland using these same molecular markers. We detected a relatively high degree of intraspecies variability among isolates of S. trifoliorum from the United States and Poland using the presence of introns and SNP within the rDNA. SNP and nuclear small-subunit rDNA analyses revealed distinct groups of S. trifoliorum among the isolates used in this study. The results of this study provide useful information for clover breeders and pathologists looking to develop clover varieties with durable resistance.

Sporoschisma from submerged wood in Yunnan, China

We are studying the lignicolous freshwater fungi along a north–south latitudinal gradient in Asia. In this paper, fresh collections of Sporoschisma from submerged wood in Yunnan Province, China, are characterized by morphology and ITS and nuclear large subunit 28S rDNA sequences. The phylogenetic analysis of combined ITS and LSU sequence data placed the isolates within the family Chaetosphaeriaceae, order Chaetosphaeriales. The collections were identified as Sporoschisma hemipsilum, S. mirabile, S. nigroseptatum, S. phaeocentron, and S. taitense, while S. mirabile, S. nigroseptatum, S. phaeocentron, and S. taitense are the first records in China. Sporoschisma aquaticum is described as new species. The conidial characters of Sporoschisma species obtained in this study are summarized. Descriptions and illustrations are provided for the six species of Sporoschisma with notes on their taxonomy.

Morphology and Molecular Phylogeny of the Soil Ciliate Anteholosticha rectangula sp. nov. from King George Island, Maritime Antarctica

The soil ciliate Anteholosticha rectangula nov. spec. was discovered on King George Island in maritime Antarctica. Morphology and the nuclear SSU rDNA sequence were used to describe and infer the phylogenetic position of the new species. Anteholosticha rectangula is morphologically similar to A. bergeri and A. verrucosa , differing primarily by the morphology of the nuclear apparatus and dorsal kineties, respectively. The morphological features of related species are compared and discussed to confirm the validity of the new species. Molecular phylogenetic tree supports the previously reported polyphyly of the genus Anteholosticha .

Monophyly of Anthozoa (Cnidaria): why do nuclear and mitochondrial phylogenies disagree?

The phylum Cnidaria is usually divided into five classes: Anthozoa, Cubozoa, Hydrozoa, Scyphozoa and Staurozoa. The class Anthozoa is subdivided into two subclasses: Hexacorallia and Octocorallia. Morphological and molecular studies based on nuclear rDNA and recent phylogenomic studies support the monophyly of Anthozoa. On the other hand, molecular studies based on mitochondrial markers, including two recent studies based on mitogenomic data, supported the paraphyly of Anthozoa, and positioned Octocorallia as sister group to Medusozoa (the monophyletic group of Cubozoa, Hydrozoa and Scyphozoa). On the basis of 51 nuclear orthologs from four hexacorallians, four octocorallians, two hydrozoans and one scyphozoan (with poriferans and Homo sapiens as out‐groups), we built a multilocus alignment of 9 873 amino acids, which aimed at minimizing missing data and hidden paralogy, in order to understand the discrepancy between nuclear and mitochondrial phylogenies. Our phylogenetic analyses strongly supported the monophyly of Anthozoa. We compared the level of substitution saturation between our data set, the data sets of two recent phylogenomic studies and one of a mitogenomic study. We found that mitochondrial DNA is more saturated than nuclear DNA at all the phylogenetic levels studied. Our results emphasize the need for a good evaluation of phylogenetic signal.

Assessing Azorella (Apiaceae) and its allies: Phylogenetics and a new classification

Phylogenetic analyses of nuclear rDNA spacers (ITS and ETS) from Azorella and five closely related genera confirm earlier plastid results indicating that Azorella, Huanaca, Mulinum, and Schizeilema are all polyphyletic, and that the monotypic genus Laretia is nested within one of the six subclades of Azorella. Only Stilbocarpa is monophyletic, but that genus is embedded within a larger clade that includes representatives of three other genera (Azorella, Huanaca, and Schizeilema). Both nuclear and plastid datasets identify the same 10 clades, but the placement of these clades remains unstable. A new classification is presented in which these six genera are reduced to a single genus (Azorella) comprising 58 species arranged in 10 sections, one of which is newly described here (Azorella sect. Ranunculus), and one lectotypified (Azorella sect. Glabratae). A total of 13 new combinations are made (A. albovaginata, A. allanii, A. boelckei, A. burkartii, A. colensoi, A. echegarayi, A. echinus, A. hallei, A. lyallii, A. polaris, A. prolifera, A. robusta, A. ulicina), along with three replacement names (A. atacamensis, A. ruizii, A. schizeilema). For each of the 58 accepted species, a full synonymy is provided along with geographic ranges (and nomenclatural notes, where useful).

Cladosiphon takenoensis sp. nov. (Ectocarpales s.l., Phaeophyceae) from Japan

SUMMARYThe new brown algal species Cladosiphon takenoensis H. Kawai (Chordariaceae, Ectocarpales s.l.) is described from Takeno, Hyogo, Japan based on morphology and DNA sequences. The species is a spring annual, growing on subtidal rocks at more or less exposed sites. It resembles C. umezakii in its gross morphology, and the two often grow together, but is distinguishable from C. umezakii in having a more hairy appearance. Cladosiphon takenoensis has a slimy, cylindrical, multiaxial and sympodial erect thallus, branching once to twice, and is provided with long assimilatory filaments (up to 1.8 mm long, composed of up to 100 cells). Unilocular zoidangia are formed on the basal part of assimilatory filaments. The species is genetically most related to C. umezakii and has the same basic thallus structures, but differs from C. umezakii and other Cladosiphon species in lacking phaeophycean hairs and plurilocular zoidangia of the assimilatory filaments. DNA sequences of the mitochondrial cox1 and cox3, chloroplast atpB, psaA, psbA and rbcL genes and the nuclear rDNA ITS2 region support the distinctness of the species. The genus Cladosiphon was paraphyletic in our analyses because the clades of C. okamuranus/C. zosterae and C. takenoensis/C. umezakii were split by Mesogloia vermiculata. However, since the genus‐level taxonomy of Chordariaceae needs considerable revision, we suspend the genus‐level taxonomy of the new species, and tentatively describe it as C. takenoensis.

Phylogenetic Diversity of Chytridiomycetes in a Temporary Forest Pond Surveyed Using Culture-Based Methods

Temporary forest ponds are depressions that annually cycle between non-inundated and inundated stages and are recognized as biodiversity hotspots for amphibians and other taxa. However, little is known about the microbial communities of temporary forest ponds and less is known about chytrids, an inconspicuous, early diverging lineage of fungi, present in such ponds. We sampled soil and water from a temporary forest pond in the Oakmulgee District of the Talladega National Forest. Chytrids were isolated from the samples using baits and standard isolating techniques. Isolated strains were identified using the monophyletic-species concept based on a molecular phylogeny inferred from an alignment of the nuclear large subunit rDNA. A total of 30 strains were isolated, which represented 7 species. Due to the limitations of a culture-based method in being able to culture all chytrids that are present, we have described only a portion of the chytrid community. This study, however, is foundational to future inventories that apply a multiphasic approach to further reveal chytrid diversity in this understudied and variable habitat.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 μm in length and 6.4 ± 0.52 μm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

Licmophora colosalis sp. nov. (Licmophoraceae, Bacillariophyta), a large epiphytic diatom from coastal waters

:The characteristic wedge-shape and large size of some diatom species in the genus Licmophora can make them seem relatively easy to identify. However, this is not always the case, as many specific diagnoses are based solely on light microscopy, type material is not available and species may be difficult to distinguish from each other. This study provides the description and phylogenetic position of Licmophora colosalis sp. nov., which has large cells and extremely long dichotomously branched stalks that form macroscopic arborescent colonies. Material for this study was collected from a hypersaline Mediterranean lagoon but this species has also been reported from Florida and the Red Sea. It was studied using scanning electron microscopy and sequencing of the nuclear small subunit rDNA (SSU) and the chloroplast marker rbcL. Its colonies and cell morphometry are compared with three morphologically similar taxa: Licmophora remulus, Licmophora gigantea and Licmophora grandis.

Gymnopanella nothofagi, a new genus and species of gymnopoid fungi (Omphalotaceae) from Chilean Nothofagus forest

A novel, lignicolous agaric from Nothofagus forests of southern Chile is described as a new genus and species, Gymnopanella nothofagi. This taxon falls within the family Omphalotaceae as a sister group to Gymnopus in phylogenetic analyses based on sequences spanning the internal transcribed spacer region and D1/D2 region of nuclear 28S rDNA. Morphologically it is characterized by convex to flabellate basidiomata with distinctly gelatinized trama, pileipellis in the form of a cutis with erect fascicles of cylindrical, spirally incrusted hyphae and nonamyloid, broadly ellipsoid basidiospores. This combination of features, in particular the lack of a rameales structure, serve to distinguish Gymnopanella from Gymnopus, Marasmiellus and other similar genera of the Omphalotaceae or Marasmiaceae. The new taxon is known only from Chilean Nothofagus forests at approximately 45–46° south latitude, but concerted searching in similar habitats in surrounding areas or in New Zealand may extend the known range considerably.

Molecular analysis of six Rhynchospio Hartman, 1936 species (Annelida: Spionidae) with comments on the evolution of brooding within the group

Rhynchospio Hartman, 1936 is a small group of spionid polychaetes currently comprising ten described species distributed mainly in the Pacific. Five species examined to date are hermaphrodites producing spermatozoa with long nuclei, oocytes with thin and smooth envelopes, and dorsally brooding their offspring. Since our first molecular analysis of four Rhynchospio species, we have collected additional material from Northern Territory, Australia, and Oregon, USA. Herein, we describe the gamete and adult morphology of the newly collected material and use molecular analyses to provide new insight on the phylogenetic relationships of six Rhynchospio species. Adults of R. cf. foliosa from Oregon are hermaphrodites, but in contrast to other Rhynchospio, they produce spermatozoa with short nuclei (ect-aquasperm), oocytes with thick vesiculate envelopes, and likely have a holopelagic larval development. Analysis of four gene fragments, comprising mitochondrial 16S rDNA, and nuclear 18S, 28S rDNA, and Histone 3 (2516 bp in total) showed Rhynchospio to be a monophyletic group, with R. cf. foliosa being a distant sister to the five other species. Rhynchospio cf. foliosa was closer to M. arctia having ect-aquasperm and vesiculate thick-envelop oocytes (p = 14.40%) than to Spioninae members B. proboscidea and P. elegans, having introsperm and oocytes with thin and smooth envelopes (p = 15.39 and 16.54%, respectively). We hypothesize that brooding might have evolved from free-spawning inside the Rhynchospio clade, but this hypothesis should be tested in a future analysis.