New evolutionary lineages, unexpected diversity, and host specificity in the parabasalid genus Tetratrichomonas

We studied morphological and molecular polymorphism of 53 Tetratrichomonas isolates obtained from amphibian, reptilian, mammalian hosts, and from a slug with the aid of protargol staining and analyses of ITS1-5.8S rRNA-ITS2, SSU rRNA, and alpha-tubulin gene sequences. The phylogenetic tree based on the concatenate of all sequences showed the monophyly of the genus Tetratrichomonas with respect to the genus Trichomonas. Our data suggest that two parabasalid genera, Pentatrichomonoides and Trichomonoides, may belong to the genus Tetratrichomonas. Tetratrichomonas isolates were divided into 16 robust host-specific and monophyletic groups that probably represent separate, mostly new, species. As only five Tetratrichomonas species were described from the examined host taxa so far, our study uncovered considerable species diversity within the genus. The wide host range, high level of species-specific host specificity, and newly revealed biodiversity make the genus Tetratrichomonas a valuable model for studying evolution of parasites.     

Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments

Characterisation of the protists of cold environments provides important background for assessing the effects of climate change on microbial communities. Tetramitus angularis n. sp., from aquatic environments in Iceland and Switzerland, is the first vahlkampfiid recognised to have a characteristic Tetramitus flagellate stage combined with pre-formed excystment pores, which are not typical of this genus. T. angularis amoebae have a typical vahlkampfiid locomotive form and contain prominent lipid inclusions. Flagellates have a collar and cytostome, and can be mono- to multi-nucleate with corresponding change in cell shape from cylindrical to ellipsoidal and variable number of flagella. Cysts are round to semi-angular and have 2-5 pores closed by protruding, translucent plugs. A second organism, T. parangularis n. sp. from Alaska, has similar cysts but a flagellate stage has not been recognised; ITS sequence divergence is consistent with species criteria in the Vahlkampfiidae. Phylogenetic analysis of sequence data for the 5.8S rDNA region clusters the new spp. with T. rostratus, T. entericus and T. waccamawensis.     

Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA

The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.     

Tetramitus thermacidophilus n. sp., an amoeboflagellate from acidic hot springs

ABSTRACT. Tetramitus thermacidophilus n. sp. is a novel thermophilic and acidophilic amoeboflagellate isolated from acidic hot springs in the Caldera Uzon (Kamchatka, Russia) and in Pisciarelli Solfatara (Naples, Italy). We describe it based on physiological, morphological, and sequence data. It was grown in monoxenic culture on the archaeon Acidianus brierleyi as food. Tetramitus thermacidophilus multiplies in a pH range from 1.2 to 5 and in a temperature range from 28 °C to 54 °C. The shortest doubling time was 4.5 h at pH 3 at 45 °C. Its spindle-shaped biflagellated stage was only rarely found in culture. The amoeboid stage shows the typical locomotive form of vahlkampfiid amoebae. Sequence comparisons of the internal transcribed spacer sequences and the small subunit rRNA genes confirm that T. thermacidophilus is a novel species within the genus Tetramitus and that both isolates belong to that species.     

Isolation of a new vahlkampfiid amoeba from soil: Paravahlkampfia lenta n. sp.

When the genus Paravahlkampfia was distinguished from the genus Vahlkampfia on the basis of significant differences in small subunit ribosomal DNA sequences, Paravahlkampfia ustiana was the only described species of this new genus. More recently, a vahlkampfiid strain has been isolated (from soil from an upland farm in Scotland) which has trophozoite and cyst morphology more similar to P. ustiana than to other non-flagellating vahlkampfiid species. Also, it clusters with P. ustiana in phylogenetic trees derived from 5.8S ribosomal DNA sequences. However, it is significantly different from P. ustiana in both phenotype and internal transcribed spacer sequence. Consequently, a new species, Paravahlkampfia lenta, is proposed.     

Species boundaries within Saprolegnia (Saprolegniales, Oomycota) based on morphological and DNA sequence data

Saprolegnia is a common and widespread genus of Oomycetes, however species identifications are difficult and uncertain. To test whether keys based on morphological characters could identify species as determined by molecular characters we determined partial DNA sequences for the 28S rRNA gene and the complete internal transcribed spacer (ITS) region for 55 isolates belonging to Saprolegnia and one isolate of Protoachlya hypogyna that exhibited saprolegnoid zoospore discharge in water culture. Phylogenetic analyses of the combined sequence data yielded 10 robustly supported clades that probably represent separate species. Morphological analyses of all isolates revealed that each DNA-based clade could be delimited from others by autapomorphic or unique combinations of morphological character states but not without employing several features previously not used at the species level. Taxonomic implications of these results are discussed and recommendations for less equivocal characterization of new Saprolegnia species are made.     

Ultrastructural and molecular phylogenetic delineation of a new order,the Rhizophydiales (Chytridiomycota)

In the order Chytridiales, Rhizophydium is a morphologically defined genus based upon the production of a monocentric, inoperculate, epibiotic sporangium, an endobiotic rhizoidal axis which branches, and an epibiotic resting spore. Despite its simple morphology, over 220 species of Rhizophydium have been described. Recent phylogenetic analyses using nuLSU rRNA (28 S rRNA) gene sequences of a geographically diverse sampling of Rhizophydium cultures revealed that the classical genus Rhizophydium is genetically more variable than previously understood and actually represents multiple genera. In the present study, we use zoospore ultrastructural characters and 28 S rRNA and 5.8 S ribosomal gene sequences of 96 isolates in culture to circumscribe the monophyletic Rhizophydium clade as a new order, Rhizophydiales. Correspondingly, zoospores of members of the Rhizophydiales exhibit a unique suite of ultrastructural character states that further define the order and distinguish it from the order Chytridiales. Molecular analyses reveal several strongly supported clades within the Rhizophydiales. Three of those clades encompass a broad range of isolates and are defined as new families Rhizophydiaceae, Terramycetaceae, and Kappamycetaceae. To resolve close relationships within Terramycetaceae, combined 28 S rRNA and ITS1–5.8 S–ITS2 sequences were analysed and details of zoospore ultrastructural character states determined, with two new genera, Terramyces and Boothiomyces, described. Two species formerly classified in Rhizophydium are transferred to the new genera. This work provides a framework for additional taxonomic revisions within the new order Rhizophydiales and compares genetic variation useful in defining genera, species, and populations within this lineage of chytrids. A broader sampling of representatives is needed before taxonomic decisions can be made for remaining clades within the Rhizophydiales.     

Knowledge of morphology is still required when identifying new amoeba isolates by molecular techniques

We have isolated several free-living amoeba strains from the environment in Ghana, which have internal transcribed spacers, including the 5.8S rDNA, sequences similar to sequences attributed to Vahlkampfiidae (Heterolobosea) in databases. However, morphological examination shows that the isolates belong to the Hartmannellidae (Amoebozoa). We provide evidence that the sequences in the databases are wrongly classified as belonging to a genus or species of the Vahlkampfiidae, but rather belong to strains of the genus Hartmannella.     

AMPLIFICATION OF THE POLYMORPHIC 5.8S rRNA GENE FROM SELECTED AUSTRALIAN GIGARTINALEAN SPECIES (RHODOPHYTA) BY POLYMERASE CHAIN REACTION1

We have initiated comparative studies of ribosomal RNA (rRNA) gene structure to explore its potential to provide taxonomically useful data within the large red algal order Gigartinales. In southern Australia, this group is extremely diverse and includes large numbers of endemic taxa, many of potential economic importance. The 5.8S rRNA gene occurs in the middle region of the ribosomal DNA (rDNA) cistron and is flanked by two internal transcribed spacers (ITSs). These spacers contain regions of DNA, which are highly consented at the generic level and above, interspersed with highly divergent sequences. The 5.8S and associated ITS s of 11 species of Gigartinales (including five species of the largest Australian endemic marine algal genus, Mychodea), plus five taxa belonging to other orders, were amplified by the polymerase chain reaction. The size of the 5.8S rDNA and its flanking ITSs varied not only within and between genera, but also at the species level. However, this rDNA sequence appears to be relatively constant within populations find may be useful as a populational marker.     

Phylogenetic Relationships Among Colletotrichum Pathogens of Strawberry and Design of PCR Primers for their Identification

Isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides pathogenic to strawberry plants were examined by sequence analysis of the 5.8S‐ITS region. Phylogenetic relationships among isolates of Colletotrichum are, for the most part, congruent with the molecular groups established in earlier works. 5.8S‐ITS sequence analysis showed a high level of genetic divergence within C. acutatum. Isolates of this species clustered into two very distinct clusters with further subdivision. The divergences between C. fragariae and C. gloeosporioides were too low to distinguish them as separate species. On the basis of the sequence data, specific primers were designed both to identify isolates belonging to the genus Colletotrichum, and to distinguish isolates of the species C. acutatum. The specificity of these primers was validated by testing a wide range of strawberry isolates of Colletotrichum, non‐strawberry isolates of Colletotrichum and other fungi used as controls. Although the 5.8S‐ITS sequences were not polymorphic enough to allow the construction of C. gloeosporioides‐specific primers, specific PCR amplification followed by an MvnI digestion provides a tool to specifically identify strawberry isolates of C. gloeosporioides.     

Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species

The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~ 83%. There was > 40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.     

Re-evaluation of the phylogenetic relationship among species of the genus Tricholoma

The internal transcribed spacer sequences spanning the regions between the 17S and 25S rRNAs (ITS1 and ITS2) and including the complete sequence of the 5.8S rRNA were used for phylogenetic analyses. This approach to define phylogenetic relationships within the genus Tricholoma was tested using different isolates of T. terreum. Fruitbodies identified in nature were analysed in order to allow use of morphology for taxonomy. The isolates from different locations were closely related as could be expected for one species. Thus, the method could be applied to different Tricholoma species. Three clusters within the genus Tricholoma can be distinguished with four additional species not included in any of these clusters. Molecular analyses of two Cortinarius species confirm a phylogenetically distinct genus.     

Molecular systematics of Helicoma, Helicomyces and Helicosporium and their teleomorphs inferred from rDNA sequences

Three genera of asexual, helical-spored fungi, Helicoma, Helicomyces and Helicosporium traditionally have been differentiated by the morphology of their conidia and conidiophores. In this paper we assessed their phylogenetic relationships from ribosomal sequences from ITS, 5.8S and partial LSU regions using maximum parsimony, maximum likelihood and Bayesian analysis. Forty-five isolates from the three genera were closely related and were within the teleomorphic genus Tubeufia sensu Barr (Tubeufiaceae, Ascomycota). Most of the species could be placed in one of the seven clades that each received 78% or greater bootstrap support. However none of the anamorphic genera were monophyletic and all but one of the clades contained species from more than one genus. The 15 isolates of Helicoma were scattered through the phylogeny and appeared in five of the clades. None of the four sections within the genus were monophyletic, although species from Helicoma sect. helicoma were concentrated in Clade A. The Helicosporium species also appeared in five clades. The four Helicomyces species were distributed among three clades. Most of the clades supported by sequence data lacked unifying morphological characters. Traditional characters such as the thickness of the conidial filament and whether conidiophores were conspicuous or reduced proved to be poor predictors of phylogenetic relationships. However some combinations of characters including conidium colour and the presence of lateral, tooth-like conidiogenous cells did appear to be predictive of genetic relationships.     

Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA)

Sediment samples from rivers, canals and lakes in Arizona (USA) were cultured for free-living amoebae at three different incubation temperatures (22, 37 and 40 degrees C). Isolates belonging to the Vahlkampfiidae were identified by sequencing the PCR-amplified ITS1, 5.8S and ITS2 rDNA. With this molecular method three Naegleria spp. were identified, N. gruberi sensu stricto, N. australiensis and N. tihangensis. Also a strain each of Willaertia magna and Vahlkampfia avara were identified. Three samples yielded two new Tetramitus spp. of which the closest relative is T. ovis. Many Acanthamoeba strains were also isolated. The genotype of these strains was identified using Acanthamoeba-specific primers (JDP1 and JDP2) amplifying a part of the SSUrDNA and sequencing with an internal primer (892c). Five of the Acanthamoeba isolates belong to genotype T5 (A. lenticulata), while five are genotype T4.     

Length Variation of the Nuclear Ribosomal DNA Internal Transcribed Spacer in the Genus Abies,with Reference to Its Systematic Utility in Pinaceae

The internal transcribed spacer (1TS) region (1TS1, ITS2 and 5.8S rDNA) of the nuclear ribosomal DNA (nrDNA) was amplified via PCR in 28 taxa of Abies Mill. The amplified fragments showed length polymorphism among species, with species from Central America and two species from North America having a length of approximately 2 500 base pairs (bp) and the remaining taxa having a length of approximately 1 700 bp based on 100 bp and 1 kb ladder standard markers. The complete sequencing of ITS of Abies bracteata showed that the shorter type is 1 697 bp (1TS1 is 1 296 bp, 5.8S + 1TS2 is 401 bp). For the longer one, the partial rrs1 and complete 5.8S + ITS2 sequencing revealed that thelength of 5.8S + ITS2 is the same as that of the shorter type. The length difference of ITS in Abies is mainly due to the length difference in the ITS1 region, a result similar to the previous findings in other genera of Pinaceae. Variation in ITS length seems well correlated with morphological and geographic characters in Abies, suggesting that the length variation may be a phylogenetically informative character within the genus, long ITS was also found in other genera of Pinaceae in the previous studies. The long length of ITS in the family makes the sequencing of the region and subsequent alignment of sequences among species or genera more difficult than in taxa with short ITS, such as angiosperms. Although the length variation of ITS in the genus Abies is significant, the homogenous of ITS sequence between the longer one and the shorter one is obvious if the insertion in the longer ITS is ignored.     

基于rDNAITS序列分析Alternaria与相似属的关系及A.leucanthemi的分类地位

对Alternaria,Stemphylium,Ulocladium,Nimbya,Embellisia等五个形态相似属的代表性种进行了5.8S/ITS区段序列测定,连同从GenBank下载的相关有性型Pleospora,Lewia的同项资料,由Neighbor-joining方法构建系统发育树。序列对比结果显示,所有参试的菌株/种的5.8SrDNA序列保守程度较高,ITS序列变化较大,ITS1比ITS2变化更大。由5.8S/ITS构建的系统发育树可以足够将供试菌区分到属;系统发育树显示Alternaria,Ulocladium,Nimbya和Embellisia系统学关系较近,又存在一定的分化;Stemphylium聚在相对较远的分支上,与上述四属关系较远,相对独立进化;有性型的Pleospora与无性型的Stemphylium被聚到一个分支,这种不同阶段菌株在遗传基础上的一致,充分证明所用方法及所选测序的rDNA区段对本类群真菌的分类很有价值。Alternarialeucanthemi位于系统发育树的外侧,较为独立进化,与Alternaria的其它种亲缘关系较远,却与Stemphyliumspp.和Bipolarisspp.关系较近,对其目前的分类地位提出质疑。     

Phylogeny and taxonomy of the genus Cylindrocladiella

The genus Cylindrocladiella was established to accommodate Cylindrocladium-like fungi that have small, cylindrical conidia and aseptate stipe extensions. Contemporary taxonomic studies of these fungi have relied on morphology and to a lesser extent on DNA sequence comparisons of the internal transcribed spacer regions (ITS 1, 2 and 5.8S gene) of the ribosomal RNA and the ??-tubulin gene regions. In the present study, the identity of several Cylindrocladiella isolates collected over two decades was determined using morphology and phylogenetic inference. A phylogeny constructed for these isolates employing the ??-tubulin, histone H3, ITS, 28S large subunit and translation elongation factor 1-alpha gene regions resulted in the identification of several cryptic species in the genus. In spite of the 18 new Cylindrocladiella species described in this study based on morphological and sequence data, several species complexes remain unresolved.     

Sequence Variation of Ribosomal Internal Transcribed Spacers (ITS) in Commercially Important Phytoseiidae Mites

Preliminary work is needed to assess the usefulness of different markers at different taxonomic scales when a new group is analyzed, such as the commercially important Phytoseiidae mites. We investigate here the level of sequence variation of the nuclear ribosomal spacers ITS 1 and 2 and the 5.8S gene in six species of Phytoseiidae: Neoseiulus californicus, N. fallacis, Euseius concordis, Metaseiulus occidentalis, Typhlodromus pyri and Phytoseiulus persimilis. As expected, the 5.8S gene (148 base pairs) is markedly conserved and displays little variation in between genera comparisons. ITS1 and ITS2 show contrasting patterns: while the ITS2 is short (80–89 bp) and shows little variation, the ITS1 is longer (303–404 bp) and is very variable in sequence. This fact compromises reliable nucleotide homologies when comparing the genera. The comparison of ITS1 sequence similarity at the species level might be useful for species identification, however, the value of ITS in taxonomic studies does not extend to the level of the family. The intraspecific variations of ITS were investigated in three species: N. californicus, N. fallacis and E. concordis. The first species has identical ITS1 sequences and the last two display low polymorphism (2 nucleotide substitutions). The ITS2 and 5.8S sequences were identical in all three subspecies comparisons.