Molecular phylogeny and taxonomy of the Hydrangea serrata complex (Hydrangeaceae) in western Japan, including a new subspecies of H. acuminata from Yakushima

According to the contemporary classification of Hydrangea native to Japan, H. serrata is a polymorphic species including six varieties. We discovered a plant identified as H. serrata, but morphologically distinct from previously known varieties, in Yakushima island where approximately 50 endemic species are known. To determine the relationship of this plant with previously known varieties, we examined morphology and constructed a highly resolved phylogeny of H. serrata and its relatives using three chloroplast genomic regions, rbcL, trnL intron, psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, and Multiplex ISSR genotyping by sequencing (MIG-seq). Based on these morphological and phylogenetic observations, we describe Hydrangea acuminata subsp. yakushimensissubsp. nov. as a newly discovered lineage in Yakushima, Japan and propose Hydrangea minamitaniistat. nov. and Hydrangea acuminata subsp. australisstat. nov. which were previously treated as varieties of H. serrata.

Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae)

Background Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

Preliminary results of studying the genetic diversity of Malaxis monophyllos (Orchidaceae) in the Amur region

For research the plants of Malaxis monophyllos (L.) Sw. were sampled in the Amur region and takenfrom the herbarium collections of the same region (MW and MHA). The plants of M. monophyllos have a high degreeof vari-ability of quantitative morphological characters. First, we paid attention to the number of leaves, the height ofplants and peduncles, the number and size of leaves and the number of flowers. Therefore, the purpose of this workwas compara-tive molecular studies of individuals of the same species with different morphological characteristics.Internal transcribed spacers (ITS1 and ITS2) of the 18S–26S region of nuclear ribosomal DNA were chosen as amolecular marker. On the molecular phylogenetic tree, the analyzed M. monophyllos plants form clade with a highposterior probability (1.0). The ITS sequences of the analyzed samples are not identical. The level of geneticdifferentiation of nucleotide sequences was 0.12 %, which corresponds to the variability within the species, whichconfirms the belonging of the analyzed samples to the same species. Since the species has a large area, it is necessary toanalyze plant samples from other regions to obtain reliable results. The different regions of the nuclear and plastidgenomes should be added to the genetic analysis.

Fungal microbiota in seeds, seedlings and mature plants of raspberry (Rubus ideaus L.)

Presently, there is an intensive search for fungal endophytes to be used in agriculture for the protection and condition improvement of plants and in medicine. We screened for the presence of endophytes in raspberry, which occurs naturally in the Białowieża Forest. The fungal isolates representative of each morphotype were analysed using the molecular markers ITS1 and ITS2. In total, we found 34 taxa of endophytic fungi. The majority were potential pathogens. As many as 27 taxa were found in the leaves of mature plants. No fungi could be isolated from the surface sterilized seeds obtained from these plants. Seedlings were grown from the seeds deposited in the soil seed bank in the Białowieża Geobotanical Station of the University of Warsaw in Białowieża. 8 taxa of endophytic fungi were found in seedlings. It could be due to a possibility of seed infection with these endophytes in soil conditions.

Prevalence and molecular identification of Nematodirus helvetianus in camels in Iraq

Background and Aim: Camels from the central part of Iraq are infected with multiple parasitic diseases that have an economic impact by decreasing meat and milk production. This study aimed to evaluate Nematodirus spp. in camels (Camelus dromedarius). Materials and Methods: The study animals consisted of camels slaughtered in the central area of Iraq at the Al-Najaf slaughterhouse. All ages and sexes of camels were examined. Worms were recovered and identified microscopically. For molecular characterization, two Iraqi Nematodirus spp. partial ribosomal genes (ITS1 and ITS2) were sequenced and submitted to the NCBI database. Results: Of 160 camels tested, 29 were infected with Nematodirus spp. (18.13%). Twenty-one nematodes containing the Nematodirus genes were identified in the small intestines of naturally infected camels. BLAST analysis revealed 88.1% sequence similarity with that of Nematodirus helvetianus isolated in China and 87.2% similarity with N. helvetianus isolated in the United States. Conclusion: The prevalence of N. helvetianus warrants the use of anti-helminthic drugs for these animals and a rationale for future control strategies to prevent the transmission of this infection to other livestock.

Phylogeny, Global Biogeography and Pleomorphism of Zanclospora

Zanclospora (Chaetosphaeriaceae) is a neglected, phialidic dematiaceous hyphomycete with striking phenotypic heterogeneity among its species. Little is known about its global biogeography due to its extreme scarcity and lack of records verified by molecular data. Phylogenetic analyses of six nuclear loci, supported by phenotypic data, revealed Zanclospora as highly polyphyletic, with species distributed among three distantly related lineages in Sordariomycetes. Zanclospora is a pleomorphic genus with multiple anamorphic stages, of which phaeostalagmus-like and stanjehughesia-like are newly discovered. The associated teleomorphs were previously classified in Chaetosphaeria. The generic concept is emended, and 17 species are accepted, 12 of which have been verified with DNA sequence data. Zanclospora thrives on decaying plant matter, but it also occurs in soil or as root endophytes. Its global diversity is inferred from metabarcoding data and published records based on field observations. Phylogenies of the environmental ITS1 and ITS2 sequences derived from soil, dead wood and root samples revealed seven and 15 phylotypes. The field records verified by DNA data indicate two main diversity centres in Australasia and Caribbean/Central America. In addition, environmental ITS data have shown that Southeast Asia represents a third hotspot of Zanclospora diversity. Our data confirm that Zanclospora is a rare genus.

ITS secondary structure reconstruction to resolve taxonomy and phylogeny of the Betula L. genus

The taxonomy and phylogeny of the Betula L. genus remain unresolved and are very difficult to assess due to several factors, especially because of frequent hybridization among different species. In the current study, we used nucleotide sequences of two internal transcribed spacer regions (ITS1 and ITS2), which are commonly used as phylogenetic markers. In addition to their nucleotide variation we reconstructed their secondary structure and used it to resolve phylogenetic relationships of some birch species. We explored whether consideration of secondary structure in phylogenetic analyses based on neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods would help us obtain more solid support of the reconstructed phylogenetic trees. The results were not unambiguous. There were only a few clades with higher support when secondary structure was included into analysis. The phylogenetic trees generated using different methods were mostly in agreement with each other. However, the resolving power of these markers is still insufficient to reliably discriminate some closely related species. To achieve this aim more reliably there is a need for application of modern genomic approaches in combination with traditional ones.

The Use of DNA Barcoding and Phylogenetic Analysis to Improve Identification of Usnea spp. Based on ITS rDNA

Lichen of the genus Usnea is quite common being used as a traditional herbal remedy. This genus is characterized by thallus, which is very similar among the species, leads to some difficulties in distinguishing them. In Indonesia, such research report on the availability of this genus based on their morphological characteristic is minimal. This might be due to too high morphological similarities among them. The molecular character, which is based on the DNA Barcode of Internal Transcribed Spacer (ITS) rDNA sequences, with its conserved region (5.8S) and varied region (ITS1 and ITS2), are becoming essential characters on identifying as well as analyzing the phylogenetic. The current study then proposed to identify and draw the species dendrogram of species within the Usnea genus obtained from Mount Lawu Forest of Central Java and Turgo Forest of Yogyakarta based on their phylogenetic and phenetic analysis. The dendrogram was constructed with UPGMA using the simple matching coefficient, whereas the phylogenetic tree was constructed with Maximum Likelihood (ML) using Kimura-2 parameter with 1000 bootstrap. The data were unable to draw phenetic relationships among the subgenus Usnea and Eumitria members. The phylogenetic tree shows the primary two clades, distinguishing the subgenus Usnea and Eumitria. The ITS rDNA sequence was able to identify most of the Usnea species.

Characterization of New Molecular Markers of Three Botflies Parasitizing Cervid Hosts

Specific identification of oestrid larvae is usually problematic not only when using morphobiometric features, but also when applying molecular criteria, since very few molecular markers have been described for this group of flies. New molecular markers for oestrid are needed for more reliable species identification, diagnostic purposes, and epidemiological surveys; moreover, they can help in phylogenetic reconstruction. Here, we report the characterization of COI, 28S rDNA, ITS1, and ITS2 in Cephenemyia stimulator from roe deer and in Cephenemyia auribarbis and Pharyngomyia picta from red deer. The COI and 28S rDNA are very uniform in length, while the ITSs sequences are highly variable at both intraspecific and interspecific levels. The described ITSs sequences were longer than those described for other dipteran species by the presence of simple repeats and tandem repeat sequences. In C. auribarbis both ITS1 and ITS2 appeared as two variants, one short and the other long. In general, the analyzed markers present low intraspecific genetic variation and high interspecific variation. ITSs showed the greatest amount of intraspecific and interspecific variation. Phylogenetic analysis demonstrated that the characterized sequences differentiate the species and genera of Oestridae.

In Silico Study Suggesting the Bias of Primers Choice in the Molecular Identification of Fungal Aerosols

This paper presents an in silico analysis to assess the current state of the fungal UNITE database in terms of the two eukaryote nuclear ribosomal regions, Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2), used in describing fungal diversity. Microbial diversity is often evaluated with amplicon-based high-throughput sequencing approaches, which is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of the primers used for amplification. The goal of this study is to validate if the mismatches of the primers on the binding sites of the targeted taxa could explain the differences observed when using either ITS1 or ITS2 in describing airborne fungal diversity. Hence, the choice of the pairs of primers for each barcode concur with a study comparing the performance of ITS1 and ITS2 in three occupational environments. The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2. However, the database contains an equal number of unidentified taxa from ITS1 and ITS2 regions in the six taxonomic levels employed (phylum, class, order, family, genus, species). The chosen ITS primers showed differences in their ability to amplify fungal sequences from the UNITE database. Eleven taxa consisting of Trichocomaceae, Dothioraceae, Botryosphaeriaceae, Mucorales, Saccharomycetes, Pucciniomycetes, Ophiocordyceps, Microsporidia, Archaeorhizomycetes, Mycenaceae, and Tulasnellaceae showed large variations between the two regions. Note that members of the latter taxa are not all typical fungi found in the air. As no universal method is currently available to cover all the fungal kingdom, continuous work in designing primers, and particularly combining multiple primers targeting the ITS region is the best way to compensate for the biases of each one to get a larger view of the fungal diversity.