Phylogeny of the Neotropical sages (<Emphasis Type="Italic">Salvia</Emphasis> subg. <Emphasis Type="Italic">Calosphace</Emphasis>; Lamiaceae) and insights into pollinator and area shifts

Salvia subg. Calosphace (Lamiaceae, Lamiales) is a highly diverse clade endemic to the New World. The phylogenetic relationships of Calosphace have been previously investigated using DNA sequences of nuclear ITS region and plastid psbA–trnH intergenic spacer, but the resulting trees lack resolution and support for many clades. The present paper reassesses the phylogenetic relationships of subgenus Calosphace, including a broader taxon sampling, with a special focus on representing previously unsampled sections, and using an additional plastid marker (trnL–trnF region). Our results show increased resolution and overall patterns of support, recovering ten main clades. Within core Calosphace, the most inclusive of these main clades, 17 new subclades were identified. Of the 42 sections for which more than one species was analysed, only 12 are monophyletic. Our biogeographical analysis identified at least twelve migrations to South America from Mexican and Central American lineages, in agreement with previous suggestions of multiple origins of South American Calosphace diversity. This analysis also confirmed a colonization of the Antilles by Andean lineages. The reconstruction of ancestral states of pollination syndromes showed multiple shifts to ornithophily from melittophily and one reversal to the latter.     

First report on cystacanths of <Emphasis Type="Italic">Sphaerirostris lanceoides</Emphasis> (Petrochenko, 1949) (Acanthocephala: Centrorhynchidae) from the Asiatic toad <Emphasis Type="Italic">Bufo gargarizans</Emphasis> Cantor (Amphibia: Anura) in China

Everted cystacanths of Sphaerirostris lanceoides (Petrochenko, 1949) Golvan 1956 are reported from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) for the first time. The prevalence was 1.96% and the intensity ranged between 1.0–3.0 acanthocephalans. SEM observations revealed the morphology of the gonopore and the presence of a flat, bare region on the apical part of the proboscis. Moreover, S. lanceoides was characterised using molecular approaches by sequencing the ribosomal ITS1-5.8S-ITS2 region and the mitochondrial cox1 gene. The resulting ITS sequences were identical and the cox1 sequences showed a divergence of 0–0.75%. Sphaerirostris lanceoides is the first species of the genus for which the ITS1-5.8S-ITS2 and cox1 loci have been sequenced to aid species identification.     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.     

Taxonomy and molecular phylogeny of Diatrypaceae (Ascomycota,Xylariales) species from the Brazilian semi-arid region,including four new species

Members of the Diatrypaceae are predominantly saprotrophic on the decaying wood of angiosperms worldwide and the family has received little attention due to its difficult taxonomy. However, the recent detection of several pathogenic species, once considered saprotrophic, associated with the wood of diseased grapevines has increased interest in this family. The diversity of tropical species is less well known and more poorly sampled in phylogenetic studies than temperate species. In the present study, we investigated the diversity of diatrypaceous fungi from three areas in the Brazilian semi-arid region and performed phylogenetic analyses of the family based on the entire internal transcribed spacer (ITS) region and partial ß-tubulin gene. Twenty-eight new ITS and 19 new ß-tubulin sequences were generated, representing eight species distributed in five clades. Diatrypella atlantica, Eutypa guttulata, Eutypella cearensis, and Peroneutypa diminutispora are proposed here as new species, while Eutypella microtheca and P. curvispora are new records for Brazil. All eight species are described, illustrated, and discussed.     

Analysis of the ITS1/ITS2 nuclear spacers and the secondary structure of <Emphasis Type="Italic">5.8S</Emphasis> rRNA gene in endemic species <Emphasis Type="Italic">Bellevalia sarmatica</Emphasis> (Pall. ex Georgi) Woronow and related species of the subfamily scilloideae

Sequence variability of the ITS spacers and 5.8S rRNA gene was examined in 11 accessions of the subfamily Scilloideae, including seven accessions of rare and endangered species Bellevalia sarmatica from Volgograd region. The intraspecific polymorphism level of the examined ITS1–5.8S–ITS2 sequence of B. sarmatica accessions constituted 1.3%. The phylogenetic position of B. sarmatica within the genus Bellevalia was determined. It was demonstrated that B. sarmatica belonged to the section Nutantes, and the most closely related species were B. webbiana and B. dubia. Nucleotide substitutions in the 5.8S rRNA gene sequence of the analyzed Scilloideae accessions were identified and studied. The predicted secondary structure of 5.8S rRNA gene was constructed. It was demonstrated that in the examined accessions, mutations in the 5.8S rRNA gene were mainly localized in the third hairpin region and had no effect on the secondary structure of the 5.8S rRNA molecule.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Relationships within <Emphasis Type="Italic">Capitotricha bicolor</Emphasis> (Lachnaceae,Ascomycota) as inferred from ITS rDNA sequences,including some notes on the <Emphasis Type="Italic">Brunnipila</Emphasis> and <Emphasis Type="Italic">Erioscyphella</Emphasis> clades

DNA sequences of Capitotricha bicolor from Quercus, Fagus sylvatica, Alnus alnobetula, and Nothofagus, and C. rubi from Rubus idaeus were obtained from apothecia to establish whether specimens from different hosts belong to separate species. The obtained ITS1–5.8S–ITS2 rDNA sequences were examined with Bayesian and parsimony phylogenetic analyses. Intra- and interspecific variation was also investigated based on molecular distances in the ITS region. The phylogenetic analyses supported the specific distinctness of Capitotricha rubi and the Capitotricha from Nothofagus, but also suggest specific distinctness between samples from Quercus, Fagus, and Alnus. The interspecific distances were larger than intraspecific distances for all examined units. The smallest distance was found between the “Alnus alnobetula” and “Fagus sylvatica” units. Two new sequences of Brunnipila are published. Capitotricha, Lachnum, and Erioscyphella are compared to each other based on hair and excipulum characteristics.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. isolated from chili pepper fruit exhibiting symptoms of anthracnose in Thailand

Colletotrichum spp. are causal agents of anthracnose disease in chili fruits and other tropical crops. The disease is increasing in chili fruits in Thailand and significantly reduces fruit quality and fruit production. Forty-eight isolates of Colletotrichum spp. associated with chili anthracnose were collected from different areas of Thailand during 2010–2015. Based on morphological characteristic identification, 10 isolates were shown to belong to the C. gloeosporioides species complex, 24 isolates belong to the C. acutatum species complex and 14 isolates to C. capsici. For molecular identification, two primer sets, ITS1/ITS4 and ACT528/ACT738, were used for amplification of the internal transcribed spacer of rRNA gene (ITS1–5.8S–ITS2) and partial region actin gene (ACT), respectively. The phylogenetic analysis of individual and combined ITS region and actin nucleotide sequences identified the collected isolates into 4 species: C. gloeosporioides, C. siamense, C. acutatum and C. capsici. The pathogenicity test demonstrated that all four species were pathogenic on intact unwounded and healthy fruits. These results indicated that C. capsici, C. acutatum, C. gloeosporioides and C. siamense were the causal agents of chili anthracnose disease.     

Polymorphic sites in transcribed spacers of 35S rRNA genes as an indicator of origin of the <Emphasis Type="Italic">Paeonia</Emphasis> cultivars

Region ITS1–5.8S rDNA–ITS2 is sequenced in 27 varieties of cultivated ornamental peonies, ten of which presumably originate from Paeonia lactiflora, one from P. officinalis, 13 from hybridization of P. lactiflora and P. peregrina, or P. officinalis, and three are Itoh hybrids. Comparative analysis of distribution patterns of polymorphic sites (PS) for the obtained DNA sequences and data from GenBank is carried out. Hypotheses of origin of the studied varieties, except for two, which, as previously assumed, originate from hybridization of P. lactiflora and P. peregrina, are confirmed. It is shown that the sequence ITS1–5.8S rDNA–ITS2 is a good genetic marker for cultivars of the P. lactiflora group and Itoh hybrids, and that the PS distribution patterns in these sequences can provide valuable information on the kinship and origin of individual varieties. However, insufficient knowledge of wild species from the P. officinalis kinship group limits the use of this marker in the study of varieties obtained through interspecific hybridization within the Paeonia section.     

Morphology and molecules reveal the alien <Emphasis Type="Italic">Posthodiplostomum centrarchi</Emphasis> Hoffman, 1958 as the third species of <Emphasis Type="Italic">Posthodiplostomum</Emphasis> Dubois, 1936 (Digenea: Diplostomidae) in Europe

Metacercariae of two species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) were subjected to morphological and molecular studies: P. brevicaudatum (von Nordmann, 1832) from Gasterosteus aculeatus (L.) (Gasterosteiformes: Gasterosteidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Perca fluviatilis L. (Perciformes: Percidae), Czech Republic (morphology, cox1, ITS1-5.8S-ITS2 and 28S); and P. centrarchi Hoffman, 1958 from Lepomis gibbosus (L.) (Perciformes: Centrarchidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Slovakia (cox1 and ITS1-5.8S-ITS2). In addition, cercariae of P. cuticola (von Nordmann, 1832) from Planorbis planorbis (L.) (Mollusca: Planorbidae), Lithuania (morphology and cox1) and metacercariae of Ornithodiplostomum scardinii (Schulman in Dubinin, 1952) from Scardinius erythrophthalmus (L.) (Cypriniformes: Cyprinidae), Czech Republic, were examined (morphology, cox1, ITS1-5.8S-ITS2 and 28S). These represent the first molecular data for species of Posthodiplostomum and Ornithodiplostomum Dubois, 1936 from the Palaearctic. Phylogenetic analyses based on cox1 and ITS1-5.8S-ITS2, using O. scardinii as the outgroup and including the three newly-sequenced Posthodiplostomum spp. from Europe and eight published unidentified (presumably species-level) lineages of Posthodiplostomum from Canada confirmed the distinct status of the three European species (contrary to the generally accepted opinion that only P. brevicaudatum and P. cuticola occur in the Palaearctic). The subspecies Posthodiplostomum minimum centrarchi Hoffmann, 1958, originally described from North America, is elevated to the species level as Posthodiplostomum centrarchi Hoffman, 1958. The undescribed “Posthodiplostomum sp. 3” of Locke et al. (2010) from centrarchid fishes in Canada has identical sequences with the European isolates of P. centrarchi and is recognised as belonging to the same species. The latter parasite, occurring in the alien pumpkinseed sunfish Lepomis gibbosus in Europe, is also supposed to be alien for this continent. It is speculated that it colonised Europe long ago and is currently widespread (recorded in Bulgaria, Slovakia and Spain); based on the cox1 sequence of an adult digenean isolate from the Ebro Delta, Spain, only the grey heron (Ardea cinerea L.) (Ciconiiformes: Ardeidae) is known to be its definitive host in Europe.     

Phylogeny of anaerobic fungi (phylum <Emphasis Type="Italic">Neocallimastigomycota</Emphasis>), with contributions from yak in China

The phylum Neocallimastigomycota contains eight genera (about 20 species) of strictly anaerobic fungi. The evolutionary relationships of these genera are uncertain due to insufficient sequence data to infer their phylogenies. Based on morphology and molecular phylogeny, thirteen isolates obtained from yak faeces and rumen digesta in China were assigned to Neocallimastix frontalis (nine isolates), Orpinomyces joyonii (two isolates) and Caecomyces sp. (two isolates), respectively. The phylogenetic relationships of the eight genera were evaluated using complete ITS and partial LSU sequences, compared to the ITS1 region which has been widely used in this phylum in the past. Five monophyletic lineages corresponding to six of the eight genera were statistically supported. Isolates of Caecomyces and Cyllamyces were present in a single lineage and could not be separated properly. Members of Neocallimastigomycota with uniflagellate zoospores represented by Piromyces were polyphyletic. The Piromyces-like genus Oontomyces was consistently closely related to the traditional Anaeromyces, and separated the latter genus into two clades. The phylogenetic position of the Piromyces-like genus Buwchfawromyces remained unresolved. Orpinomyces and Neocallimastix, sharing polyflagellate zoospores, were supported as sister genera in the LSU phylogeny. Apparently ITS, specifically ITS1 alone, is not a good marker to resolve the generic affinities of the studied fungi. The LSU sequences are easier to align and appear to work well to resolve generic relationships. This study provides a comparative phylogenetic revision of Neocallimastigomycota isolates known from culture and sequence data.     

Diversity within the <Emphasis Type="Italic">Hygrophorus agathosmus</Emphasis> group (Basidiomycota,Agaricales) in Northern Europe

The North European species of the Hygrophorus agathosmus group in subsection Tephroleuci were studied. Three new species are identified based on morphology, ecology and sequence data. Two species are associated with Pinus spp. One of these is described here as H. suaveolens, while the other one is only known from one locality in the Nordic countries and seems to have a more South European distribution range. A closely related sister species to H. agathosmus is identified based on ITS sequence data, H. cf. agathosmus. It is confirmed to have an intercontinental distribution range and to be associated with Picea spp. probably on more acidic to neutral soil, whereas H. agathosmus s.s. has a more limited North-East European distribution range and occurs in older and rich Picea abies forests. A neotype for H. agathosmus is here selected from South Sweden. Hygrophorus agathosmus f. albus and H. agathosmus f. aureofloccosus are confirmed as forms. No genetic differences in the ITS region between specimens with grey cap colour and the two forms were observed. A key to the species in Northern Europe is provided.     

Polymorphic Sites in ITS1-5.8S rDNA-ITS2 Region in Hybridogenic Genus × <Emphasis Type="Italic">Elyhordeum</Emphasis> and Putative Interspecific Hybrids <Emphasis Type="Italic">Elymus</Emphasis> (Poaceae: Triticeae)

The genus Elymus L. is a complicated aggregate of ecological and geographical races, species, subspecies, varieties, and hybrids. We suggest that comparative analysis of intragenomic polymorphism of internal transcribed spacers ITS1 and ITS2 of 35S rRNA genes in the supposed hybrids and their possible “parents” can be one of the approaches to verification of hybrid origin of the samples collected in nature to confirm or reject the hypotheses about their possible “parents.” Polymorphic sites (PS) in ITS of 23 Elymus species, as well as in two supposed interspecific Elymus hybrids and in a supposed intergeneric hybrid between Elymus × Hordeum determined as × Elyhordeum sp., were analyzed in the work. We collected all hybrids in the Altai. There were 2 and 5 PS in two samples of E. dahuricus and 1 and 4 PS in two studied samples of E. schrenkianus in the ITS1-5.8S rDNA-ITS2 region. From 0 to 4 (modes 0 and 3) PS were detected in 32 samples relating to 21 tetraploid Elymus species. More PS (14) were found in the × Elyhordeum sp. sample. A large number of single nucleotide substitutions were found in 5.8S rRNA in × Elyhordeum. It was shown that about half of them do not change the secondary structure of the 5.8S rRNA molecule, so these molecules probably retain the ability to work as a component of large subunit of a ribosome. On the other hand, the absence or weakening of 5.8S rDNA homogenization in × Elyhordeum indirectly suggests that a significant part of 5.8S rDNA is not transcribed. Paradoxically, ITS sequences of × Elyhordeum sp. are less polymorphic than 5.8S rDNA. There are no ITS sequences derived from Hordeum among × Elyhordeum ITS sequenced by Sanger method. No traces of the H subgenome and a subgenome originating from Agropyron (P-subgenome) are seen in the Alt 10–278 plant genome (a chimera, combining the morphological traits of Elymus, Elytrigia, and Agropyron). In this plant, as well as in the supposed intersectional hybrid Alt 11–60 distinguished by a mosaic of the traits typical for the E. caninus × E. mutabilis species, only 4 and 5 PS, respectively, are detected when sequencing by Sanger method. The comparison of ITS sequences of the supposed Elymus Alt 10–278 hybrid and its probable “parents” demonstrates that one of the species of the Elymus macrourus kinship circle, as well as the Elytrigia geniculata, could be one of its ancestors. The comparison of the ITS sequence of the supposed parental species with ITS of Alt 11–60 samples and five PS of the supposed Alt 11–60 hybrid does not contradict the hypothesis that this is an intersectional hybrid of the first generation that emerged with the involvement of E. caninus and E. mutabilis common in the Altai.     

Phylogenetic and morphological analysis of a new cliff-dwelling species reveals a remnant ancestral diversity and evolutionary parallelism in <Emphasis Type="Italic">Sonchus</Emphasis> (Asteraceae)

We describe a new cliff-dwelling species within Sonchus (Asteraceae): Sonchus boulosii and analyze its systematic position and evolutionary significance; in addition, we provide a key to the species of Sonchus in Morocco. Both morphological and ecological characteristics suggest a close relationship of S. boulosii with taxa of section Pustulati. However, ITS nrDNA and cpDNA matK markers indicate its uncertain position within the genus, but clear genetic differentiation from the remaining major clades. ITS phylogenetic trees show that likely evolutionary shifts to rocky habitat took place at least five times within genus Sonchus and that sect. Pustulati and S. boulosii clades have a clearly independent evolutionary origin. We postulate that the strong resemblance of S. boulosii to other rocky species reflects a phenomenon of homoplasy, probably driven by parallel evolutionary adaptations to the severe ecological constraints of its cliff face habitat. Therefore, a new section is also described, which includes S. boulosii as its sole representative: section Pulvinati. According to phylogenetic trees, the new clade may share its common ancestor with the clade comprising sections Maritimi and Arvenses, from which it is widely divergent in morphology and ecology, with the exception of Sonchus novae-zelandiae. However, the latter is a derived taxon, with high level of polyploidy unlike S. boulosii that shows 2n?=?18, basal chromosome number of the genus. Since sections Pulvinati and Pustulati seem to be quite old in Sonchus, we also hypothesize that some similarities, such as fruit morphology, may reflect the persistence of some primitive characteristics.     

Novel <Emphasis Type="Italic">Collophorina</Emphasis> and <Emphasis Type="Italic">Coniochaeta</Emphasis> species from <Emphasis Type="Italic">Euphorbia polycaulis</Emphasis>, an endemic plant in Iran

During a study on the biodiversity of yeasts and yeast-like ascomycetes from wild plants in Iran, four strains of yeast-like filamentous fungi were isolated from a healthy plant of Euphorbia polycaulis in the Qom Province, Iran (IR. of). All four strains formed small hyaline one-celled conidia from integrated conidiogenous cells directly on hyphae and sometimes on discrete phialides, as well as by microcyclic conidiation. Two strains additionally produced conidia in conidiomata that open by rupture. The internal transcribed spacer (ITS) sequences suggested the placement of these strains in the genera Collophorina (Leotiomycetes) and Coniochaeta (Sordariomycetes), respectively. Blast search results on NCBI GenBank and phylogenetic analyses of ITS, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translation elongation factor 1α (EF-1α) sequences, and the nuclear large subunit ribosomal gene (LSU), partial actin (ACT), and β-tubulin (TUB) sequences, respectively, revealed the isolates to belong to three new species, that are described here as Collophorina euphorbiae, Coniochaeta iranica, and C. euphorbiae. All three species are characterised by morphological, physiological, and molecular data.     

Revision of the taxonomic status of the genus <Emphasis Type="Italic">Gloeoporus</Emphasis> (Polyporales,Basidiomycota) reveals two new species

Gloeoporus Mont. is characterized by an easily separated gelatinous hymenophore and a continuous hymenium over the pore mouth. Recent molecular taxonomic and phylogenetic research showed that morphological grouping of Gloeoporus is polyphyletic. The lack of comprehensive phylogenetic studies of Gloeoporus exacerbates confusion in determining the taxonomic position of the genus. To delimit the genus Gloeoporus, we performed multi-locus phylogenetic analysis using the internal transcribed spacer (ITS) region, the nuclear large subunit ribosomal DNA (LSU), and the second-largest subunit of RNA polymerase II (rpb2). The phylogenetic analyses revealed that current delimitation of Gloeoporus is not monophyletic. Gloeoporus s.s. includes mostly clamped species lacking cystidia. Some species of Gloeoporus featuring simple septa and cystidia are proposed to be renamed to Meruliopsis. Two new species of Gloeoporus were also observed and they are named Gloeoporus africanus and Gloeoporus orientalis.     

Characterisation of seven <Emphasis Type="Italic">Inocybe</Emphasis> ectomycorrhizal morphotypes from a semiarid woody steppe

Ectomycorrhizas (ECM) of Inocybe species (Inocybaceae, Basidiomycota) formed by three host plant species (Populus alba, Salix rosmarinifolia and Pinus nigra) in a semiarid woody steppe of Hungary were studied. To identify the fungal partners, we performed phylogenetic analyses of nucleotide sequences for the internal transcribed spacer region of nuclear DNA (nrDNA ITS) together with sequences gained from public databases. Seven Inocybe ectomycorrhiza morphotypes were morpho-anatomically characterised. Five morphotypes were identified (I. phaeoleuca, I. psammophila, I. semifulva, I. splendens and I. subporospora), whereas two morphotypes represented unidentified Inocybe species. Differences were discernible among the morphotypes, and they showed general anatomical characteristics of Inocybe ECM, such as the slightly organised plectenchymatic mantle (types A, B and E and the gelatinous C). The ECM of I. subporospora and I. phaeoleuca were detected from the introduced Pinus nigra. These two fungi are probably native to the area but capable of forming a novel ectomycorrhizal association with the invasive host.