An Overview of 24 Years of Molecular Phylogenetic Studies in Phallales (Basidiomycota) With Notes on Systematics, Geographic Distribution, Lifestyle, and Edibility

The order Phallales (Basidiomycota) is represented by gasteroid fungi with expanded and sequestrate basidiomata, known as stinkhorns and false truffles. In phalloids, the first DNA sequence was published in 1997, and after that, some studies aimed to resolve phylogenetic conflicts and propose new species based on DNA markers; however, the number of families and genera in the order still generates controversies among researchers. Thus, this work aims to provide an overview of Phallales diversity represented by selected DNA markers available in public databases. We retrieved Phallales sequences from DNA databases (GenBank and UNITE) of seven markers: ITS (internal transcribed spacer), nuc-LSU (nuclear large subunit rDNA), nuc-SSU (nuclear small subunit rDNA), mt-SSU (mitochondrial small subunit rDNA), ATP6 (ATPase subunit 6), RPB2 (nuclear protein-coding second largest subunit of RNA polymerase), and TEF1-α (translation elongation factor subunit 1α). To compose our final dataset, all ITS sequences retrieved were subjected to BLASTn searches to identify additional ITS sequences not classified as Phallales. Phylogenetic analyses based on Bayesian and maximum likelihood approaches using single and combined markers were conducted. All ITS sequences were clustered with a cutoff of 98% in order to maximize the number of species hypotheses. The geographic origin of sequences was retrieved, as well as additional information on species lifestyle and edibility. We obtained a total of 1,149 sequences, representing 664 individuals. Sequences of 41 individuals were unidentified at genus level and were assigned to five distinct families. We recognize seven families and 22 genera in Phallales, although the delimitation of some genera must be further revisited in order to recognize only monophyletic groups. Many inconsistencies in species identification are discussed, and the positioning of genera in each family is shown. The clustering revealed 118 species hypotheses, meaning that approximately 20% of all described species in Phallales have DNA sequences available. Information related to geographic distribution represents 462 individuals distributed in 46 countries on all continents, except Antarctica. Most genera are saprotrophic with only one putative ectomycorrhizal genus, and 2.1% of the legitimate specific names recognized in Phallales are confirmed edible species. Great progress in the molecular analyses of phalloids has already been made over these years, but it is still necessary to solve some taxonomic inconsistencies, mainly at genus level, and generate new data to expand knowledge of the group.

Parametarhizium (Clavicipitaceae) gen. nov. With Two New Species as a Potential Biocontrol Agent Isolated From Forest Litters in Northeast China

A novel genus Parametarhizium with two new entomopathogenic species, Parametarhizium changbaiense and Parametarhizium hingganense, was introduced based on their morphological characteristics and a multigene phylogenetic analysis, which were isolated from the forest litters collected in Northeast China. To infer their phylogenetic relationships, a six-gene dataset consisting of DNA fragments of [nuclear small subunit rDNA (SSU) + LSU + TUB + TEF + RPB1 + RPB2] was used for phylogenetic analysis, including 105 related fungi. The new genus Parametarhizium formed a monophyletic clade basal to Metarhizium and its related genera (formerly Metarhizium sensu lato). Parametarhizium can be morphologically distinguished from related genera by the combination of the following characteristics: formation of white to yellow colonies on different media, candelabrum-like arrangement of cylindrical or obpyriform phialides, and small subglobose to ellipsoidal conidia. Both P. hingganense and P. changbaiense exhibited anti-insect activities against three farmland pests Monolepta hieroglyphica, Callosobruchus chinensis, and Rhopalosiphum maidis. This is the first report of entomopathogenic fungi exhibiting the anti-insect activity against Mo. hieroglyphica.

New contributions to two ciliate genera (Ciliophora, Heterotrichea) based on morphological and molecular analyses, with description of a new Gruberia species

Background Heterotrichous ciliates are common members of microeukaryote communities which play important roles in both the transfer of material and the flow of energy in aquatic food webs. This group has been known for over two centuries due to their large body size and cosmopolitan distribution. Nevertheless, species identification and phylogenetic relationships of heterotrichs remain challenging due to the lack of accurate morphological information and insufficient molecular data. Results The morphology and phylogeny of two heterotrichous ciliates, namely Gruberia foissneri spec. nov. and Linostomella vorticella (Ehrenberg, 1833) Aescht in Foissner et al., 1999, were studied using rigorous methods (living morphology, stained preparations, and small subunit rDNA sequence data). Gruberia foissneri spec. nov. is morphologically very similar to G. uninucleata Kahl, 1932, however, it can be distinguished from the latter by having more ciliary rows (about 32 vs. about 20) and macronuclear shape (sausage-shaped vs. ellipsoid). Based on a combination of previous and present studies, an improved diagnosis of L. vorticella is supplied and several taxonomic anomalies are clarified. In addition, phylogenetic analyses based on SSU rDNA sequence data support the generic assignment of these two species. Conclusions Modern ciliate taxonomy should be performed by means of detailed living observation, stained preparations and molecular information. For those species that have been reported in previous studies, it is necessary to provide as much useful information as possible using state-of-the-art methods in order to resolve taxonomic anomalies.

Cryptosporidium ratti n. sp. (Apicomplexa: Cryptosporidiidae) and genetic diversity of Cryptosporidium spp. in brown rats (Rattus norvegicus) in the Czech Republic

The diversity and biology of Cryptosporidium that is specific for rats (Rattus spp.) are not well studied. We examined the occurrence and genetic diversity of Cryptosporidium spp. in wild brown rats (Rattus norvegicus) by microscopy and polymerase chain reaction (PCR)/sequencing targeting the small subunit rDNA (SSU), actin and HSP70 genes. Out of 343 faecal samples tested, none were positive by microscopy and 55 were positive by PCR. Sequence analysis of SSU gene revealed the presence of Cryptosporidium muris (n = 4), C. andersoni (n = 3), C. ryanae (n = 1), C. occultus (n = 3), Cryptosporidium rat genotype I (n = 23), Cryptosporidium rat genotype IV (n = 16) and novel Cryptosporidium rat genotype V (n = 5). Spherical oocysts of Cryptosporidium rat genotype I obtained from naturally-infected rats, measuring 4.4–5.4 μm × 4.3–5.1 μm, were infectious to the laboratory rats, but not to the BALB/c mice (Mus musculus) nor Mongolian gerbils (Meriones unguiculatus). The prepatent period was 3 days post infection and the patent period was longer than 30 days. Naturally- and experimentally-infected rats showed no clinical signs of disease. Percentage of nucleotide similarities at the SSU, actin, HSP70 loci between C. ratti n. sp. and the rat derived C. occultus and Cryptosporidium rat genotype II, III, IV, and V ranged from 91.0 to 98.1%. These genetic variations were similar or greater than that observed between closely related species, i.e. C. parvum and C. erinacei (93.2–99.5%). Our morphological, genetic and biological data support the establishment of Cryptosporidium rat genotype I as a new species, Cryptosporidium ratti n. sp.

The endemic Cladophorales (Ulvophyceae) of ancient Lake Baikal represent a monophyletic group of very closely related but morphologically diverse species

© 2018 Phycological Society of America Lake Baikal, the oldest lake in the world, is home to spectacular biodiversity and extraordinary levels of endemism. While many of the animal species flocks from Lake Baikal are famous examples of evolutionary radiations, the lake also includes a wide diversity of endemic algae that are not well investigated with regards to molecular-biological taxonomy and phylogeny. The endemic taxa of the green algal order Cladophorales show a range of divergent morphologies that led to their classification in four genera in two families. We sequenced partial large- and small-subunit rDNA as well as the internal transcribed spacer region of 14 of the 16 described endemic taxa to clarify their phylogenetic relationships. One endemic morphospecies, Cladophora kusnetzowii, was shown to be conspecific with the widespread Aegagropila linnaei. All other endemic morphospecies formed a monophyletic group nested within the genus Rhizoclonium (Cladophoraceae), a very surprising result, in stark contrast to their morphological affinities. The Baikal clade represents a species flock of closely related taxa with very low genetic differentiation. Some of the morphospecies were congruent with lineages recovered in the phylogenies, but due to the low phylogenetic signal in the rDNA sequences the relationships within the Baikal clade were not all well resolved. The Baikal clade appears to represent a recent radiation, based on the low molecular divergence within the group, and it is hypothesized that the large morphological variation results from diversification in sympatry from a common ancestor in Lake Baikal.

The endemic Cladophorales (Ulvophyceae) of ancient Lake Baikal represent a monophyletic group of very closely related but morphologically diverse species

© 2018 Phycological Society of America Lake Baikal, the oldest lake in the world, is home to spectacular biodiversity and extraordinary levels of endemism. While many of the animal species flocks from Lake Baikal are famous examples of evolutionary radiations, the lake also includes a wide diversity of endemic algae that are not well investigated with regards to molecular-biological taxonomy and phylogeny. The endemic taxa of the green algal order Cladophorales show a range of divergent morphologies that led to their classification in four genera in two families. We sequenced partial large- and small-subunit rDNA as well as the internal transcribed spacer region of 14 of the 16 described endemic taxa to clarify their phylogenetic relationships. One endemic morphospecies, Cladophora kusnetzowii, was shown to be conspecific with the widespread Aegagropila linnaei. All other endemic morphospecies formed a monophyletic group nested within the genus Rhizoclonium (Cladophoraceae), a very surprising result, in stark contrast to their morphological affinities. The Baikal clade represents a species flock of closely related taxa with very low genetic differentiation. Some of the morphospecies were congruent with lineages recovered in the phylogenies, but due to the low phylogenetic signal in the rDNA sequences the relationships within the Baikal clade were not all well resolved. The Baikal clade appears to represent a recent radiation, based on the low molecular divergence within the group, and it is hypothesized that the large morphological variation results from diversification in sympatry from a common ancestor in Lake Baikal.

Identification and Characterization of Verticillium longisporum Lineage A1/D1 from Brassica Crops in Manitoba, Canada

Verticillium stripe in canola (Brassica napus L.) caused by Verticillium longisporum was first reported in Manitoba in 2014. In this study, Brassica crops including canola, mustard (Brassica juncea) and radish (Raphanus sativus) with visible symptoms of Verticillium stripe were collected from Portage La Prairie, Manitoba, and the pathogens were isolated. Isolates from canola and radish were identified to V. longisporum, which produced longer conidia (7.92–12.00 µm) than Verticillium dahliae (4.32–7.04 µm). An isolate derived from mustard was characterized as V. dahliae. Molecular diagnostics with 18S rDNA, 5.8S rDNA and mating-type marker primers were used to confirm the identification of Verticillium isolates. PCR-RFLP of the mitochondrial small subunit rDNA and the cytochrome b gene were also employed to distinguish V. longisporum isolates from V. dahliae. The multi-gene characterization approach allowed for lineage determination, and V. longisporum isolates from canola and radish were in the A1/D1 group. Isolates of Verticillium longisporum from canola inoculated onto the canola cultivar ‘Westar’ caused symptoms of stem striping, stunting and short plants. Re-isolated fungal strains from infected stems were again inoculated onto canola plants, in order to confirm that V. longisporum was the causal agent of Verticillium stripe disease in the pathogenicity test.

New contributions to two ciliate genera (Ciliophora, Heterotrichea) based on morphological and molecular analyses, with description of a new Gruberia species

Background: Heterotrichous ciliates are common members of microeukaryote communities which play important roles in both the transfer of material and the flow of energy in aquatic food webs. This group has been known for over two centuries due to their large body size and cosmopolitan distribution. Nevertheless, species identification and phylogenetic relationships of heterotrichs remain challenging due to the lack of accurate morphological information and insufficient molecular data.Results: The morphology and phylogeny of two heterotrichous ciliates, namely Gruberia foissneri spec. nov. and Linostomella vorticella (Ehrenberg, 1833) Aescht in Foissner et al., 1999, were studied using rigorous methods (living morphology, stained preparations, and small subunit rDNA sequence data). Gruberia foissneri spec. nov. is morphologically very similar to G. uninucleata Kahl, 1932, however, it can be distinguished from the latter by having more ciliary rows (about 32 vs. about 20) and macronuclear shape (sausage-shaped vs. ellipsoid). Based on a combination of previous and present studies, an improved diagnosis of L. vorticella is supplied and several taxonomic anomalies are clarified. In addition, phylogenetic analyses based on SSU rDNA sequence data support the generic assignment of these two species.Conclusions: Modern ciliate taxonomy should be performed by means of detailed living observation, stained preparations and molecular information. For those species that have been reported in previous studies, it is necessary to provide as much useful information as possible using state-of-the-art methods in order to resolve taxonomic anomalies.

Evaluation of five regions as DNA barcodes for identification of Lepista species (Tricholomataceae, Basidiomycota) from China

Background Distinguishing among species in the genus Lepista is difficult because of their similar morphologies. Methods To identify a suitable DNA barcode for identification of Lepista species, we assessed the following five regions: internal transcribed spacer (ITS), the intergenic spacer (IGS), nuclear ribosomal RNA subunit, mitochondrial small subunit rDNA, and tef1. A total of 134 sequences from 34 samples belong to eight Lepista species were analyzed. The utility of each region as a DNA barcode was assessed based on the success rates of its PCR amplification and sequencing, and on its intra- and inter-specific variations. Results The results indicated that the ITS region could distinguish all species tested. We therefore propose that the ITS region can be used as a DNA barcode for the genus Lepista. In addition, a phylogenetic tree based on the ITS region showed that the tested eight Lepista species, including two unrecognized species, formed eight separate and well-supported clades.

Novel contributions to the peritrich family Vaginicolidae (Protista: Ciliophora), with morphological and phylogenetic analyses of poorly known species of Pyxicola, Cothurnia and Vaginicola

The classification of loricate peritrich ciliates is difficult because of an accumulation of several taxonomic problems. In the present work, three poorly described vaginicolids, Pyxicola pusilla, Cothurnia ceramicola and Vaginicola tincta, were isolated from the surface of two freshwater/marine algae in China. In our study, the ciliature of Pyxicola and Vaginicola is revealed for the first time, demonstrating the taxonomic value of infundibular polykineties. The small subunit rDNA, ITS1-5.8S rDNA-ITS2 region and large subunit rDNA of the above species were sequenced for the first time. Phylogenetic analyses based on these genes indicated that Pyxicola and Cothurnia are closely related. The present study suggested that the loricate species probably represent a distinct lineage in peritrich evolution and both genera Cothurnia and Thuricola are monophyletic. Pyxicola pusilla, Cothurnia ceramicola and Vaginicola tincta are recircumscribed.