Phylogenetic analysis of the internal transcribed spacer region of Japanese Lilium species

 The DNA from 16 Lilium species and one variety endemic to or naturalized in Japan were obtained and their internal transcribed spacer regions of nuclear ribosomal DNA (nrDNA) were amplified by PCR and sequenced by cycle sequencing. Phylogenetic analysis of the ITS sequences supported the validity of Comber’s classification system. It has also provided molecular evidence for the transfer of Lilium dauricum to sect. Sinomartagon. The phylogenetic relationships revealed by ITS DNA analysis were supported by previously published crossability data. The molecular phylogeny of Japanese Lilium species was discussed with reference to the putative migration routes of these species. Received: 30 June 1998 / Accepted: 19 October 1998     

Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species

The phylum Nematoda includes the genus Longidorus, a remarkable group of invertebrates that are polyphagous root‐ectoparasites of many plants including various agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Longidorus is complicated by phenotypic plasticity (intraspecific variability and minor interspecific differences) leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in southern Spain that detected 11 species of Longidorus. We developed a comparative study amongst these related species by considering morphological and morphometric features together with molecular data from nuclear ribosomal RNA genes [D2‐D3 expansion segments of large ribosomal subunit (28S), internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of our molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of three new species of the genus, described herein as Longidorus baeticus sp. nov. , Longidorus oleae sp. nov. , and Longidorus andalusicus sp. nov. , and eight known species (Longidorus alvegus, Longidorus crataegi, Longidorus fasciatus, Longidorus intermedius, Longidorus iuglandis, Longidorus magnus, Longidorus rubi, and Longidorus vineacola). Phylogenetic analyses of Longidorus spp. based on the three molecular markers resulted in a general consensus of these species grouping, as lineages were maintained for the majority of species (i.e. species with a conoid‐rounded lip region, amphidial fovea asymmetrically bilobed, female tail bluntly rounded), but not in some others (i.e. positions of L. crataegi, L. intermedius, and L. rubi were quite variable). To date, this is the most complete phylogenetic analysis for Longidorus and Paralongidorus species, with the highest number of species included. No correspondence between phylogenetic trees and morphological characters was found for ribosomal markers, with the exception of amphidial shape. Thus, polyphasic identification, based on integration of molecular analysis with morphology, is a tool beyond doubt in Longidorus identification. © 2013 The Linnean Society of London     

Phylogenetic analyses reveal deeply divergent species lineages in the genus Sphaerobolus (Phallales: Basidiomycota)

Phylogenetic analyses of 27 artillery fungus (Sphaerobolus sp.) isolates were conducted to identify species boundaries in the genus Sphaerobolus. Multiple gene genealogies inferred from maximum likelihood, Bayesian, and maximum-parsimony analyses of sequence data from individual loci (mtSSU, ITS, EF 1-alpha, and LSU) and a combined dataset (mtSSU, ITS, and EF 1-alpha) concordantly indicate the existence of three deeply divergent lineages in the genus Sphaerobolus, each representing a phylogenetic species. These three phylogenetic species correspond to two known species: Sphaerobolus iowensis and Sphaerobolus stellatus, and a newly discovered species. Suprageneric phylogenetic analyses of the mtSSU and LSU datasets containing representatives of related genera of the gomphoid-phalloid clade of Homobasidiomycetes suggested that the undescribed taxon likely is more closely related to S. stellatus than to S. iowensis.     

The discovery of Neotropical Lepidosira (Collembola,Entomobryidae) and its systematic position

We herein present the first reliable record of Lepidosira from Neotropical Region. Lepidosira neotropicalis sp. n. from Brazil is described and illustrated in detail, including its complete mitochondrial genome. We perform a Bayesian phylogenetic analysis to place the new species within the Entomobryidae, and at the same time to test previous contrasting hypotheses on Lepidosira position within the Entomobryinae versus Seirinae for the first time. Phylogenetic analyses were based on one mitochondrial and two nuclear genes, Cytochrome Oxidase subunit I, 18S ribosomal RNA and 28S ribosomal RNA, respectively. Lepidosira neotropicalis sp. n. resembles L. sundana Yoshii and Suhardjono and L. nigropunctata (Nguyen) in dorsal chaetotaxy of abdominal segments I and II, but differs from all other species by the combination of head (dorsally and ventrally) and dorsal trunk chaetotaxy, plus empodial complex morphology. Our phylogenetic analyses support the placement of Lepidosira within Entomobryinae, as the sister group of Lepidocyrtoides. Overall, our revision enables a more objective diagnosis to Lepidosira and suggests that the genus is in need of a full revision due to its variable morphology, and lack of data needed to evaluate its monophyly. Finally, we provide an identification key for Neotropical genera of Entomobryinae.     

Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC‐IGS and 16S–23S ITS as markers: investigation of horizontal gene transfer

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S–23S ITS) and phycocyanin intergenic spacer (PC‐IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC‐IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC‐IGS and 16S–23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC‐IGS. Thus, PC‐IGS is a suitable marker, along with 16S–23S ITS for phylogenetic studies of cyanobacteria.     

Phytophthora chrysanthemi sp. nov., a new species causing root rot of chrysanthemum in Japan

A new species of Phytophthora was isolated from stem and root rot of chrysanthemum in the Gifu and Toyama prefectures of Japan. The species differs from other Phytophthora species morphologically, and is characterized by nonpapillate, noncaducous sporangia with internal proliferation, formation of both hyphal swellings and chlamydospores, homothallic nature, distinctive intercalary antheridia, and funnel-shaped oogonia. The new species can grow even at 35°C, with an optimum growth temperature of 30°C in V8 juice agar medium. In phylogenetic analyses based on five nuclear regions (LSU rDNA; genes for translation elongation factor 1α, β-tubulin, 60 S ribosomal protein L10, and heat shock protein 90), the isolates formed a monophyletic clade. Although the rDNA ITS region shows a high resolution and has proven particularly useful for the separation of Phytophthora species, it was difficult to align the sequences for phylogenetic analysis. Therefore, ITS region analysis using related species as defined by the multigene phylogeny was performed, and the topology of the resulting tree also revealed a monophyletic clade formed by the isolates of the species. The morphological characteristics and phylogenetic relationships indicate that the isolates represent a new species, Phytophthora chrysanthemi sp. nov. In pathogenicity tests, chrysanthemum plants inoculated with the isolates developed lesions on stems and roots within 3 days, and the symptoms resembled the ones originally observed. Finally, the pathogen’s identity was confirmed by re-isolation from lesions of infected plants.     

假暗盘菌属一新种——中华假暗盘菌

报道了分离自福建蛇足石杉上的假暗盘菌属Pseudoplectania一新种,中华假暗盘菌Pseudoplectania sinica。基于形态学及分子系统学分析对其进行了鉴定。研究标本保存于齐鲁医药学院实验室和中国普通微生物菌种保藏管理中心（CGMCC）。     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Phylogenetic placement of <Emphasis Type="Italic">Marasmiellus juniperinus</Emphasis>

Recent collections and the type specimen of Marasmiellus juniperinus, the type species of the genus, were examined. Phylogenetic placement, based on ribosomal large subunit (LSU) and internally transcribed spacer (ITS) sequences, is within the lentinuloid clade, nested among Gymnopus taxa. This placement dictates genus name usage and phylogenetic position of other putative species of Marasmiellus. The mating system is tetrapolar.     

A novel clade of sporocarp-forming species of glomeromycotan fungi in the <Emphasis Type="Italic">Diversisporales</Emphasis> lineage

In the early times of taxonomy of arbuscular mycorrhizal fungi (Glomeromycota), exclusively sporocarpic species were described. Since then the focus has mainly shifted to species forming spores singly. For many of the sporocarpic species, no molecular data have been made available, and their phylogenetic position has remained unclear. We obtained small subunit ribosomal rDNA and internal transcribed spacer data from specimens of glomeromycotan sporocarps from tropical areas that were assigned to three morphospecies. The complete sequence of the 18S small rDNA subunit sequence, internal transcribed spacers (ITS) 1 and 2 and 5.8S rDNA subunit, was determined from a sporocarp of Glomus fulvum. Partial sequences of the small subunit and the other regions were obtained from Glomus pulvinatum and the newly described species Glomus megalocarpum. Molecular phylogenetic analyses placed all species analyzed as a monophyletic sister group to the Diversispora spurca/Glomus versiforme clade group (“Glomus group C”) within the Diversisporales. The phylogenetic divergence from other known species suggests that this clade may constitute a new genus. These findings will have important consequences for taxon definition in the Diversisporales. They will facilitate identification of these fungi using rDNA sequences within colonized roots or the environment. Taxonomic novelties: Glomus megalocarpum D. Redecker     

Phylogeny of Allium L. subg. Melanocrommyum (Webb et Berth.) Rouy based on DNA sequence analysis of the internal transcribed spacer region of nrDNA

 Sequence analysis of the ITS region of nuclear ribosomal DNA from subgeneric representatives of Allium L. produced phylogenetic trees which concurred with previous conclusions based on classical taxonomy. Phylogenetic analysis revealed a closer relationship between Nectaroscordum siculum and Allium cernuum (representing Amerallium) than between A. cernuum and the rest of the Allium species employed in this study. The phylogeny of subg. Melanocrommyum based on ITS sequences largely agreed with inferences made by previous researchers based on morphology or a restriction analysis of chloroplast DNA. However, the phylogenetic positions of Allium protensum and Allium macleanii based on ITS sequences did not correspond to their morphological similarity with Allium schubertii and Allium giganteum, respectively. Received: 15 February 1998 / Accepted: 12 March 1998     

Phylogenetic relationship of China Dendrobium species based on the sequence of the internal transcribed spacer of ribosomal DNA

The genetic relationship of 36 Dendrobium species in China was determined based on sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA. Aligned sequences of the complete ITS region obtained from the 36 Dendrobium species and 2 outgroup species (Epigeneium amplum and Epigeneium nakaharaei) by using PCR amplification and direct DNA sequencing. The nrDNA ITS1 of Dendrobium was 225–234 bp and ITS2 was 239–248 bp. Phylogenetic tree was constructed, and seven main clusters were generated among the 36 Dendrobium species. From the results, D. moulmeinense was not grouped in the classification of Dendrobium and E. amplum and E. nakaharaei were shown to be divergent from Dendrobium species. The phylogenetic relationships revealed by ITS DNA analysis partially supported previously published morphological data.     

Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework

In order to investigate the genetic relationships between Dermanyssus gallinae (Metastigmata: Dermanyssidae) (de Geer) isolates from poultry farms in Italy and other European countries, phylogenetic analysis was performed using a portion of the cytochrome c oxidase subunit 1 (cox1) gene of the mitochondrial DNA and the internal transcribed spacers (ITS1+5.8S+ITS2) of the ribosomal DNA. A total of 360 cox1 sequences and 360 ITS+ sequences were obtained from mites collected on 24 different poultry farms in 10 different regions of Northern and Southern Italy. Phylogenetic analysis of the cox1 sequences resulted in the clustering of two groups (A and B), whereas phylogenetic analysis of the ITS+ resulted in largely unresolved clusters. Knowledge of the genetic make‐up of mite populations within countries, together with comparative analyses of D. gallinae isolates from different countries, will provide better understanding of the population dynamics of D. gallinae. This will also allow the identification of genetic markers of emerging acaricide resistance and the development of alternative strategies for the prevention and treatment of infestations.     

Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.     

Reptodigitus Chapmanii (Nostocales,Hapalosiphonaceae) Gen. Nov.: A Unique Nostocalean (Cyanobacteria) Genus Based on a Polyphasic Approach1

The Nostocales is a monophyletic, heterocytous lineage of cyanobacteria capable of akinete production and division in multiple planes, depending upon family-level clade. While present in a variety of ecosystems, the diversity of the Nostocales has been poorly elucidated. Due to environmentally -induced phenotypic plasticity, morphology alone is often insufficient to determine the true phylogenetic placement of these taxa. In order to bridge this gap, taxonomists now employ the polyphasic approach, combining methods such as morphological analysis, phylogenetic analysis based on DNA sequence and genetic identity based on ribosomal genes, and secondary structure of the 16S-23S ITS and 16S rRNA gene sequences, as well as ecological characterization. Using this combined approach, a new genus and species (Reptodigitus chapmanii gen. et sp. nov.) isolated from the St. Johns River (Jacksonville, Florida, USA) within the Nostocales is herein described. Phylogenetic analyses place this taxon within the Hapalosiphonaceae, sister to the clade containing Fischerella, Hapalosiphon, and Westiellopsis. The 16S-23S ITS secondary folding structure analysis also supports the erection of this new genus.     

Cryptic species in the <Emphasis Type="Italic">Terfezia boudieri</Emphasis> complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5′ end of the ribosomal large subunit gene, a chitin synthase partial sequence, β-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.     

Systematics and evolution of ruminal species of the genus Prevotella

Bacterial species of the genusPrevotella represent a numerically dominant microbial population in the rumen of cattle. They belong to the phylogenetic divisionCytophaga-Flexibacter-Bacteroides (CFB) which is a large group of ecologically diverse bacteria with only a few shared traits. The phylogenetic descent from a common ancestor seems to be unquestionable, however, as judged from the small subunit ribosomal RNA analysis. Only 4 ruminalPrevotella species have been described to date, even though the sequence analysis of directly retrieved 16S rRNA genes indicates a large genetic diversity within this group of rumen bacteria. The closest relatives of ruminalPrevotella spp. are not surprisingly other species of the genusPrevotella, typically inhabiting the gastrointestinal tract, oral cavity and genital areas of other animals and man. The previous phylogenetic analysis showed that species of the genusPrevotella can be split into two groups or superclusters, the “ruminal” and the “non-ruminal prevotellas”. One of 4 currently described ruminalPrevotella spp.,i.e. P. albensis, has been placed outside the supercluster containing ruminalPrevotella spp. and within the supercluster containing the non-ruminalPrevotella spp. However, the number of available small subunit rRNA sequences from this species represents only a fraction of all known ruminalPrevotella sequences.     

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Phylogenetic relationships of coffee-tree species (Coffea L.) as inferred from ITS sequences of nuclear ribosomal DNA

 Phylogenetic relationships of Coffea species were estimated from the sequences of the internal transcribed spacer (ITS 2) region of nuclear ribosomal DNA. The ITS 2 region of 37 accessions belonging to 26 Coffea taxa and to three Psilanthus species was directly sequenced from polymerase chain reaction (PCR)-amplified DNA fragments. The level of variation was high enough to make the ITS 2 a useful tool for phylogenetic reconstruction. However, an unusual level of intraspecific variation was observed leading to some difficulty in interpreting rDNA sequence divergences. Sequences were analysed using Wagner parsimony as well as the neighbour-joining distance method. Coffea taxa were divided into several major groups which present a strong geographical correspondence (i.e. Madagascar, East Africa, Central Africa and West Africa). This organisation is well supported by cytogenetic evidence. On the other hand, the results were in contradiction with the present classification of coffee-tree taxa into two genera, namely Coffea and Psilanthus. Furthermore, additivity of parental rDNA types was not observed in the allotetraploid species C. arabica. Received: 25 July 1996 / Accepted: 18 October 1996