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.     

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.     

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.     

Molecular genetic markers of intra- and interspecific divergence within starfish and sea urchins (Echinodermata)

A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1–5.8S rDNA–ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions–Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster–were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1–5.8S rDNA–ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Taxonomy and phylogenetic position of <Emphasis Type="Italic">Phyllactinia takamatsui</Emphasis>, a newly described powdery mildew on cotoneaster,based on molecular and morphological data

The newly recognised powdery mildew species Phyllactinia takamatsui on Cotoneaster nummularius (Rosaceae) is described and illustrated. This species, collected in Kerman Province, Iran, is well characterised by its conidial morphology and rDNA ITS sequences clearly different from allied species. Conidia are broadly ellipsoid to subcylindrical, i.e. they are not clavate-spathuliform as in most Phyllactinia species. The rDNA ITS sequence analysis showed that this species is closely allied to other species described on hosts belonging to Rosaceae, such as Ph. mali and Ph. pyri-serotinae. The ITS sequence of P. takamatsui was 92 to 94 % similar to that of the closest known relatives. The new species is described in detail, illustrated and compared with other similar taxa.     

Two new and one known species of <Emphasis Type="Italic">Tergestia</Emphasis> Stossich, 1899 (Trematoda: Fellodistomidae) with novel molecular characterisation for the genus

Combined morphological and molecular analyses are employed to characterise three species of Tergestia Stossich, 1899 (Digenea: Fellodistomidae) from fishes of Moreton Bay, Queensland, Australia. Tergestia clonacantha Manter, 1963 is reported here for the first time from the halfbeak (Beloniformes: Hemiramphidae) species Arrhamphus sclerolepis krefftii (Steindachner), Hyporhamphus australis (Steindachner), H. quoyi (Valenciennes) and H. regularis ardelio (Whitley). Two new species, both infecting trevally (Perciformes: Carangidae) species, are described: T. maryae n. sp. from Alepes apercna Grant and T. henryi n. sp. from Pantolabus radiatus (MacLeay). Complete ITS2 and partial 28S ribosomal DNA data were generated for each of the new taxa. The three species differ from each other by 47–58 base pairs (bp) in the ITS2 rDNA region. Phylogenetic analysis of 28S rDNA supports Tergestia as a reliable generic concept, with our analyses showing that some species of the genus form a well-supported clade to the exclusion of all other fellodistomids for which sequence data are available.     

Genetic diversity of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> (Acari: Ixodidae) in western China

Haemaphysalis qinghaiensis as an endemic species in China mainly infests domestic animals and causes great harm to animals and humans in Northwestern plateau. However, there is no information about genetic diversity within the recently established populations of this tick species. Therefore, the present study analyzed the fragments of mitochondrial 16S rDNA, COI and the nuclear gene ITS1 of 56 H. qinghaiensis ticks across four regions of China which are main endemic areas of this species. Analysis showed 98.1–100% (16S rDNA), 97.9–100% (COI), 99.7–100% (ITS1) identity within individuals. For these sequences, 9, 15 and 8 haplotypes were found for 16S rDNA, COI and ITS1, respectively. Ticks from Yongjing were the most variable group, followed by Lintan, Huangyuan, and Tianzhu. Based on parallel analysis of the mitochondrial and nuclear genetic diversity of H. qinghaiensis, our results indicated that mitochondrial markers (especially COI) were much more useful than nuclear ITS for intraspecific genetic variability analysis.     

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.     

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.     

Secondary structure prediction of ITS rRNA region and molecular phylogeny: an integrated approach for the precise speciation of <Emphasis Type="Italic">Muscodor</Emphasis> species

Muscodor is a non-sporulating, volatile organic compounds producing endophytic fungi that has been extensively explored as a bio-fumigant and bio-preservative. Novel species of this genus have been mainly identified using ITS sequences. However, the ITS hyper-variability hinders the creation of reproducible alignments and stable phylogenetic trees. Conserved structural data of the ITS region represents as a vital auxiliary information for accurate speciation of fungi. In the present study, secondary structural data of ITS1, 5.8S, and ITS2 region of all Muscodor species were generated using LocaRNA web server. The predicted secondary structural data displayed greater variability in ITS1 region in comparison to ITS2. The structural data of all sequences exhibited characteristic conserved features of eukaryotic rRNA. Evolutionary conserved motifs were found among all 5.8S and ITS2 sequences. Profile neighbor joining (PNJ) tree based on combined sequence-structural information of ITS region was generated in ProfDists. The PNJ tree resolved into four major groups whereby M. fengyangenesis and M. albus species formed monophyletic clades. However, three M. albus species along with other Muscodor species emerged as sister branches to the existing clades, thereby, improving the precision of phylogenetic analysis for identification of novel species of Muscodor genus. Hence, the results indicated that structural analysis along with primary sequence information can provide new insights for precise identification of Muscodor species.     

<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.     

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.     

FISH-aimed karyotype analysis in <Emphasis Type="Italic">Aconitum</Emphasis> subgen. <Emphasis Type="Italic">Aconitum</Emphasis> reveals excessive rDNA sites in tetraploid taxa

The location of 5S and 35S rDNA sequences in chromosomes of four Aconitum subsp. Aconitum species was analyzed after fluorescence in situ hybridization (FISH). Both in diploids (2n?=?2x?=?16; Aconitum variegatum, A. degenii) and tetraploids (2n?=?4×?=?32; A. firmum, A. plicatum), rDNA repeats were localized exclusively on the shorter arms of chromosomes, in subterminal or pericentromeric sites. All analyzed species showed similar basal genome size (Cx?=?5.31–5.71 pg). The most striking features of tetraploid karyotypes were the conservation of diploid rDNA loci and emergence of many additional 5S rDNA clusters. Chromosomal distribution of excessive ribosomal sites suggests their role in the secondary diploidization of tetraploid karyotypes.     

<Emphasis Type="Italic">Salinispora</Emphasis><Emphasis Type="Italic">arenicola</Emphasis> from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.