Molecular phylogeny of asexual entomopathogenic fungi with special reference to Beauveria bassiana and Nomuraea rileyi

The phylogenetic lineage, taxonomic affiliation and interrelationships of important asexual entomopathogenic fungal genera were studied using the sequences of partial regions of β-tubulin and rRNA genes. The species structures of Beauveria bassiana and Nomuraea rileyi were also investigated. A total of 147 fungal entries covering 94 species were analysed. Phylogenetic analysis placed all the asexual entomopathogenic fungal species analysed, in the family Clavicipitaceae of the order Hypocreales of Ascomycota. Deep phylogenetic lineages were observed in B. bassiana iterating the complex nature of this species. Some of the isolates assigned to this species separated out more distinctly than morphologically distinguishable genera. Cryptic speciation was also evident in N. rileyi. It is concluded that the asexual fungi with entomopathogenic habit arose from a single lineage in sexual Clavicipitaceae.     

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.     

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD)

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.     

Extended phylogeny of the Craspedida (Choanomonada)

Currently choanoflagellates are classified into two distinct orders: loricate Acanthoecida and non-loricate Craspedida. The morphologically based taxonomy of the order Craspedida is in need of a revision due to its controversial, paraphyletic and inconsistent systematics and nomenclature. In this study, we add molecular data (SSU and parts of the LSU rDNA) of six new Craspedida species isolated from saline, brackish and freshwater habitats to the existing knowledge. Four of these six organisms could be described as new species: Paramonosiga thecata, “Salpingoeca” euryoecia, “Salpingoeca” ventriosa, “Sphaeroeca” leprechaunica, whereas two are assigned to previous morphologically described species: “Salpingoeca” fusiformis Saville Kent, 1880 and “Salpingoeca” longipes Saville Kent, 1880. Paramonosiga is established as a new genus of the Craspedida based on its phylogenetic position. Extending the dataset by six additional sequences shows that the craspedid taxonomy is still unsolved as the type specimen Salpingoeca gracilis has not yet been sequenced and hence a clear assignment to the genus Salpingoeca is not possible. Trying to assign morphological and ecological data to phylogenetic clades is not successful. We give an improved/emended morphological diagnosis for the two redescribed species and add molecular data for all six species, shedding light on their phylogenetic position.     

Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.     

Genome-based taxonomic classification of the closest-to-Comamonadaceae group supports a new family Sphaerotilaceae fam. nov. and taxonomic revisions

Many genera closest to the family Comamonadaceae have not been classified into any family; moreover, some of them are not monophyletic groups beyond the genus level. To resolve the taxonomic uncertainty of the closest-to-Comamonadaceae (CTC) group, we performed 16S rRNA gene- and genome-based phylogenetic analyses combined with genome relatedness indices and phenotypic traits comparison. Phylogenies based on the 16S rRNA gene and genome sequences demonstrated that the CTC group formed a coherent and robust monophyletic lineage and was sister to the family Comamonadaceae, thereby proposing the CTC group as a novel family, Sphaerotilaceae fam. nov. The resolved genus- and species-level taxonomic relationships of this new family were then validated by the phylogenomic reconstruction and comparisons of genome relatedness indices including digital DNA-DNA hybridization and average nucleotide identity (ANI) as well as comprehensive phenotypic analysis for type strains. Finally, we reclassified all misidentified genera and species, resulting in 19 new combinations, and proposed Sphaerotilaceae-specific thresholds of ANI and average amino acid identity for genus delineation. Collectively, this study has established a sound taxonomic framework of the novel family Sphaerotilaceae and will help guide future taxonomic efforts and prevent the propagation of taxonomic errors.     

Cladistic analysis of Taxaceae s. l.

Taxaceae s. l. is a wider concept of classification treating five genera of Taxaceae s. str. and Cephalotaxus together. Cephalotaxus is morphologically very similar to the five genera of Taxaceae s. str. Various models of classification for six genera have already been published. However, the phylogenetic position and genuine relationships of these genera and species are still confusing. A cladistic analysis of Taxaceae s. l. has been carried out to resolve the problem existing in their phylogeny and to provide a new approach regarding the relationships of these six genera. Parsimony analyses were based on 28 characters and eight genera including two outgroups Agathis and Sciadopitys. The most parsimonious tree retained with branch length 38 and 194 rearrangement trials. Consistency index was 0.68 and retention index was 0.66. Principally, two main clades were found: one represented by Austrotaxus forming the base of the tree and another by the remaining five genera. Taxus + Pseudotaxus clade split after Austrotaxus, and Cephalotaxus was sister to Torreya + Amentotaxus clade. Taxus + Pseudotaxus clade was supported by the highest bootstrap value. Finally, cladistic analysis does not support existence of Cephalotaxaceae. Therefore, it would be better to classify Cephalotaxus within Taxaceae s. l. with the other five genera.     

Genome-based taxonomy of genera Halomicrobium and Halosiccatus,and description of Halomicrobium salinisoli sp. nov

The genera Halosiccatus and Halomicrobium are the most closely related genera within the family Haloarculaceae (class Halobacteria). All species of these two genera are closely related to each other in phylogenetic analyses based on their 16S rRNA gene sequences, and also using the sequences of four housekeeping genes. The genus Halosiccatus was proposed based on inferred phylogeny using only one of the three distinct 16S rRNA genes detected in strain DC8^T, while Halomicrobium zhouii, one of three species of Halomicrobium, was omitted from the reference species used in these analyses. The related 16S rRNA gene sequence similarities of type strains of Halomicrobium katesii and Halomicrobium mukohataei were as high as 99.5%–99.7%, much higher than the threshold values proposed as species boundaries. These issues could have resulted in taxonomic inaccuracies in the genera Halosiccatus and Halomicrobium, and a thorough study was undertaken to clarify the status of all species in both genera. Based on phylogenetic and phylogenomic analyses, the current four species of the two genera form a single clade with high bootstrap confidence, indicating that the genus Halosiccatus should be merged with Halomicrobium. Halomicrobium katesii Kharroub et al. 2008 is proposed as a later heterotypic synonym of Halomicrobium mukohataei (Ihara et al. 1997) Oren et al. 2002. An additional species is also described (strains LT50^T and TH30), and was isolated from different Gobi saline soil samples of Tarim Basin, Xinjiang, China. Phenotypic, chemotaxonomic, genomic and phylogenetic properties indicated that strains LT50^T (=CGMCC 1.15187^T = JCM 30837^T) and TH30 (=CGMCC 1.15189 = JCM 30839) represent a novel species of the genus Halomicrobium, for which the name Halomicrobium salinisoli sp. nov. is proposed.     

Description of three new Alteromonas species Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. isolated from marine environments,and proposal for reclassification of the genus Salinimonas as Alteromonas

In the course of a bioprospective study of marine prokaryotes for cosmetic purposes, four strains, MD_567^T, MD_652^T, MD_674 and PS_109^T, were isolated that 16S rRNA gene affiliation indicated could represent three new species within the family Alteromonadaceae. A thorough phylogenetic, genomic and phenotypic taxonomic study confirmed that the isolates could be classified as three new taxa for which we propose the names Alteromonas antoniana sp. nov., Alteromonas lipotrueae sp. nov. and Alteromonas lipotrueiana sp. nov. In addition, the consistent monophyletic nature of the members of the genera Alteromonas and Salinimonas showed that both taxa should be unified, and therefore we also propose the reclassification of the genus Salinimonas within Alteromonas, as well as new combinations for the species of the former. As the specific epithets profundi and sediminis are already used for Alteromonas species, we created the nomina nova “Alteromonas alteriprofundi” nom. nov. and Alteromonas alterisediminis nom. nov. to accommodate the new names for “Salinimonas profundi” and Salinimonas sediminis. Whole genome comparisons also allowed us to detect the unexpected codification of aromatic hydrocarbon biodegradative compounds, such as benzoate and catechol, whose activity was then demonstrated phenotypically. Finally, the high genomic identity between the type strains of Alteromonas stellipolaris and Alteromonas addita indicated that the latter is a junior heterotypic synonym of Alteromonas stellipolaris.     

Nematodes of elasmobranch fishes from the southern coast of Brazil

New records for nematode species recovered from elasmobranch fishes in Brazil are established and new systematical arrangements proposed. Parascarophis sphyrnae Campana-Rouget, 1955 from the spiral valve of Sphyrna zygaena is referred for the first time in South America as a new host record. Procamallanus (S.) pereirai Annereaux, 1946, from the spiral valve of Raja castelnaui is reported parasitizing an elasmobranch host. Nematode larvae of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are listed from the stomach and spiral valves of several hosts. Anisakidae larvae previously referred in Brazil in the genus Phocanema should be reallocated in Pseudoterranova. Nematodes of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are reported for the first time parasitizing elasmobranchs in Brazil.     

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales,Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400–600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy—in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria—and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup “Pleiochloris,” was included in the genus Ixipapillifera as I. deasonii comb. nov.     

Phylogenetic relationships among four species of Mullidae (Perciformes) inferred from DNA sequences of mitochondrial cytochrome b and 16S rRNA genes

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among four species of mullids. Approximately 238 bp of the mitochondrial 16S ribosomal RNA (rRNA) and 261 bp of the cytochrome b (cytb) genes were sequenced from representatives of three mullid genera (Mullus, Upeneus, Pseudopeneus), present in the Mediterranean Sea. Trees were constructed using three methods: maximum likelihood (ML), neighbor joining (NJ) and parsimony (MP). The results of the analyses of these data together with published data of the same mtDNA segments of two other perciform species (Sparus aurata, Perca fluviatilis), support the previous taxonomic classification of the three genera examined, as well as the classification of the two red mullet species in the same genus.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

New dicrocoeliid digeneans from mammals in Ecuador including a highly genetically divergent new genus from an ancient marsupial lineage

The Dicrocoeliidae is a highly diverse and broadly distributed family of digeneans typically parasitic in the gall bladder and liver of their tetrapod hosts. So far, no study has reported dicrocoeliids, or any digeneans, from the ancient marsupial family Caenolestidae. Herein, we describe a new genus of dicrocoeliids (Otongatrema n. gen.) from Tate's shrew opossum Caenolestes fuliginosus and a new species of Metadelphis (Metadelphis cesartapiai n. sp.) from a phyllostomid bat Anoura peruana collected in Ecuador. Otongatrema can be readily distinguished from the morphologically closest dicrocoeliid genera Concinnum, Conspicuum and Canaania based on the position of the genital pore, distribution/position of the uterus and extent of vitellarium. Metadelphis cesartapiai can be easily differentiated from other Metadelphis spp. based on a combination of morphological characters including body shape and size, distribution of vitellarium, shape of the gonads as well as size of suckers and cirrus sac. In addition, we used newly generated partial sequences of the nuclear 28S rRNA gene and mitochondrial cox1 genes to examine phylogenetic affinities of the new taxa within the Dicrocoeliidae. Both the 28S and cox1 phylogenies confidently positioned Otongatrema as a sister/basal group to all other dicrocoeliids sequenced so far. The phylogenetic position of Otongatrema may be explained by a close co-evolutionary relationship with Caenolestidae, one of the most basal and most ancient groups of marsupials. In addition, our 28S phylogeny provides evidence that the complete or partial loss of intestinal structures has likely occurred independently at least 3 times in the evolutionary history of the Dicrocoeliidae.     

Molecular phylogeny of baculiform desmid taxa (Zygnematophyceae)

The baculiform, rod-like morphotypes belong to several phylogenetic lineages within Desmidiaceae (Zygnematophyceae, Viridiplantae). Some, for example the genus Pleurotaenium, form independent lineages, but reductive evolution of complicated desmid cells toward baculiform morphology also occurred in individual lineages, for example Micrasterias. In this genus, the rod like Triploceras forms evolved from more complex ancestors. In this study, we tested for an independent position of the subtropical and tropical genus Triplastrum, previously separated from Triploceras on the basis of morphological data. In addition, monophyly of Pleurotaenium was also investigated with multiple isolates corresponding to seven species of this genus, including the morphologically dissimilar P. nodosum and P. ovatum. Finally, two isolates of Docidium baculum were also investigated. Molecular phylogenetic analysis of concatenated rbcL?+?coxIII sequence data implied that the baculiform taxa investigated were in three distantly related positions within Desmidiaceae. The genus Triplastrum proved to be unrelated to Triploceras, because it clustered in the “omniradiate” lineage of Desmidiaceae among morphologically dissimilar taxa. The genus Pleurotaenium was monophyletic, but P. ovatum was recovered in a weakly supported sister position to all the other members of the genus. The trnG^ucc phylogeny of Pleurotaenium taxa concurred with the rbcL?+?coxIII phylogram, and generally revealed the poor morphological concepts of some species in this genus. The most common taxa P. ehrenbergii and P. trabecula were resolved as polyphyletic because their strains were distributed among several strongly supported clades. However, strains of P. nodosum and P. archeri formed separate, well supported lineages within the genus.