Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

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.     

Herbarium tale: the utility of dry specimens for DNA barcoding Juncaceae

Phylogenetic and barcoding studies usually use fresh plant tissues as sources of DNA and have successfully amplified DNA for various loci. The use of dried samples, however, is often necessary due to the frequent inaccessibility of fresh rare plants or their parts for genetic analyses or barcoding. The difficulty in obtaining amplifiable DNA is a major restriction of the use of herbarium specimens for DNA analyses. Recent study has highlighted the crucial issues for comparing herbarium and fresh plants for barcoding. We analysed the performance of samples of the family Juncaceae from various herbarium specimens of different ages with fresh plant material in PCRs and the sequences of seven loci (rbcL, rpoC1, trnL-F intergenic spacer, trnL intron, and psbA-trnH from chloroplast DNA; atp1 from mitochondrial DNA; and ITS1-5.8S-ITS2 from nuclear DNA) using a combination of 28 primers. The herbarium specimens amplified well and may thus be successfully applied for both phylogenetic analyses and barcoding for the Juncaceae family. Amplifying DNA was more difficult from dried herbarium specimens than fresh samples but could be successful in most cases when appropriate internal primers were designed or methods were optimised. Using the set of universal primers recommended by the Consortium for the Barcode of Life and designing specific primers for a particular group of interest were both useful. Specimen age and amplicon length had limited detrimental effects on amplification success for most of the Juncaceae loci tested.     

Fungal root endophytes of tomato from Kenya and their nematode biocontrol potential

The significance of fungal endophytes in African agriculture, particularly Kenya, has not been well investigated. Therefore, the objective of the present work was isolation, multi-gene phylogenetic characterization and biocontrol assessment of endophytic fungi harbored in tomato roots for nematode infection management. A survey was conducted in five different counties along the central and coastal regions of Kenya to determine the culturable endophytic mycobiota. A total of 76 fungal isolates were obtained and characterized into 40 operational taxonomic units based on the analysis of ITS, β-tubulin and tef1α gene sequence data. Among the fungal isolates recovered, the most prevalent species associated with tomato roots were members of the Fusarium oxysporum and F. solani species complexes. Of the three genes utilized for endophyte characterization, tef1α provided the best resolution. A combination of ITS, β-tubulin and tef1α resulted in a better resolution as compared to single gene analysis. Biotests demonstrated the ability of selected non-pathogenic fungal isolates to successfully reduce nematode penetration and subsequent galling as well as reproduction of the root-knot nematode Meloidogyne incognita. Most Trichoderma asperellum and F. oxysporum species complex isolates reduced root-knot nematode egg densities by 35–46 % as compared to the non-fungal control and other isolates. This study provides first insights into the culturable endophytic mycobiota of tomato roots in Kenya and the potential of some isolates for use against the root-knot nematode M. incognita. The data can serve as a framework for fingerprinting potential beneficial endophytic fungal isolates which are optimized for abiotic and biotic environments and are useful in biocontrol strategies against nematode pests in Kenyan tomato cultivars. This information would therefore provide an alternative or complementary crop protection component.     

Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of <Emphasis Type="Italic">Vitis davidii</Emphasis> (Rom. Caill.) Foëx

Real-time quantitative polymerase chain reaction (RT-qPCR) is an effective method for detecting changes of gene expression in plant cell metabolic regulation. A set of 15 reference gene candidates were selected for the present study of anthocyanin biosynthesis regulation, and stability. The suitability of their expression was evaluated in eight different experimental treatments in spine grape (Vitis davidii [Rom. Caill.] Foëx.) cell cultures. The results indicated that SAND family protein (SAND) and V-type proton ATPase subunit G (VAG) were the most stable reference genes for culture duration, tubulin alpha-3/alpha-5 chain (α-tubulin) and tubulin beta-1 chain (β-tubulin) for illumination conditions, ubiquitin-conjugating enzyme E2-17 kDa (UBQ) and VAG for UVB treatment, VAG and 60S ribosomal protein L18-2 (60SRP) for temperature treatment, AP47, clathrin adaptor complex subunit mu (AP-2) and 60SRP for cinnamic acid treatment, α-tubulin and UBQ for chitosan treatment, actin and alcohol dehydrogenase 2 (ADH2) for kinetin treatment, and β-tubulin and elongation factor 1-α (EF1-α) for cell line. Finally, the reliability of the selected reference genes was confirmed by investigating the expression profiles of the target gene dihydroflavonol 4-reductase (DFR) in spine grape cell cultures. The results of the present study offer the most robust platform for the most precise and broad application of RT-qPCR to investigate gene expression associated with anthocyanin biosynthesis in spine grape cell cultures.     

Novel fungi from an ancient niche: lachnoid and chalara-like fungi on ferns

A survey was conducted in Brazil to collect fungi on ferns. Based on morphology and inferred phylogeny from DNA sequences of two loci, namely the internal transcribed spacer (ITS) regions and the large subunit nuclear ribosomal RNA gene (LSU), several species belonging to chalara-like genera and lachnoid fungi were recognized. Eighteen fungal isolates, collected from five host species, representing 10 different localities were studied. Three novel genera (Lachnopsis, Scolecolachnum and Zymochalara), and six novel species (Bloxamia cyatheicola, Lachnopsis catarinensis, Lachnopsis dicksoniae, Scolecolachnum pteridii, Zymochalara lygodii and Zymochalara cyatheae) are introduced. Furthermore, two new combinations (Erioscyphella euterpes and Erioscyphella lushanensis) are proposed. Two novel taxa (Lachnopsis catarinensis and Lachnopsis dicksoniae) may be included in the list of potentially endangered fungal species in Brazil, if proven to be restricted to their tree-fern host, Dicksonia sellowiana, which is included in the official list of endangered plant species in Brazil.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Disentangling <Emphasis Type="Italic">Phialophora</Emphasis> section <Emphasis Type="Italic">Catenulatae</Emphasis>: disposition of taxa with pigmented conidiophores and recognition of a new subclass,Sclerococcomycetidae (Eurotiomycetes)

A new genus Rhopalophora is described for Phialophora clavispora, a lignicolous species formerly placed in Phialophora section Catenulatae that possesses pigmented conidiophores, phialides with a single conidiogenous locus that occasionally appear as schizophialides, and clavate, aseptate conidia arranged in chains or sometimes in heads. Sexual morphs are not known for this taxon. Phylogenetic analysis of DNA sequences from five loci (nucSSU, ITS, nucLSU, mitSSU, rpb1 and rpb2) of this and related fungi supports the introduction of a new family, Sclerococcaceae, for which we establish the order Sclerococcales. This order belongs to the new subclass Sclerococcomycetidae, a strongly supported clade within the Eurotiomycetes that is basal to a lineage containing the Chaetothyriomycetidae, Coryneliomycetidae and Eurotiomycetidae. Rhopalophora clavispora fits in this new family and is closely related to an isolate of Fusichalara minuta. The Sclerococcales also encompass marine, lignicolous species of Dactylospora, two species of the lichenicolous genus Sclerococcum, and a lineage comprised of strains from the digestive tracts of Neotropical wood-inhabiting beetles. We confirm that Dactylospora is polyphyletic; the phylogenetic placement of D. parasitica, the generic type, remains unknown.     

Phylogenetic study of clavicipitaceous fungi using acetaldehyde dehydrogenase gene sequences

Aciculosporium and Heteroepichloë (Clavicipitaceae) are characteristic bambusicolous fungi in east Asia. In this study, we examined their intergeneric relationships based on the ALDH1-1 gene, which encodes a member of the aldehyde dehydrogenase family. In the clavicipitaceous fungi examined in this study, the nucleotide sequence of the third exon of ALDH1-1 (Exon-3) is 889 bp in length and has no insertion/deletion. A phylogenetic tree based on Exon-3 indicated that the clavicipitaceous fungi could be divided into two large groups: Cordyceps, Nomuraea, and Ustilaginoidea species formed a paraphyletic group, and the other grass biotrophic species formed a monophyletic group. This monophyletic group was further divided into three groups with high bootstrap support: i.e., species with Neotyphodium anamorphs (e.g., Epichloë), species with Ephelis anamorphs (e.g., Heteroepichloë), and Aciculosporium-Claviceps species. We discuss the relationships among Aciculosporium, Heteroepichloë, and other clavicipitaceous fungi.     

<Emphasis Type="Italic">Pattersoniomyces tillandsiae</Emphasis> gen. et comb. nov.: linking sexual and asexual morphs of the only known smut fungus associated with Bromeliaceae

The enigmatic species Ustilago tillandsiae is the only known smut fungus associated with Bromeliaceae. Its generic position is evaluated by morphological, physiological, and molecular phylogenetic analyses using large subunit rDNA sequences. Phylogenetic analyses resolved U. tillandsiae as a member of the Ustilaginales in a sister relationship to the lineage containing Tranzscheliella species. However, U. tillandsiae differs from Tranzscheliella species by the development of sori in flowers, a different structure of sori and a different type of spore ornamentation. Consequently, a new genus Pattersoniomyces is described to accommodate U. tillandsiae. The new combination Pattersoniomyces tillandsiae is substantiated. In the sexual stage (teleomorph), this species infects bromeliads: Tillandsia flabellata, Tillandsia leiboldiana, and Tillandsia sp. in Central America between southern Mexico and Costa Rica. The yeast stage (anamorph) of P. tillandsiae was found associated with the phylloplane of Canistrum improcerum and in water tanks (phytotelmata) of Vriesea minarum, two bromeliads occurring in northeast and southeast Brazil, respectively. The link between the teleomorph and anamorphic strains is supported by identical sequences of the D1/D2 domains of the large subunit rDNA. Pattersoniomyces represents the tenth endemic smut genus to the Americas, but the only one that occurs in both North and South America, being a truly neotropical genus. The host plant families of Ustilaginales are extended to the Bromeliaceae. As far as we know, Pattersoniomyces represents the single event of a host jump from Cyperaceae or Poaceae to Bromeliaceae, apparently without further species radiation on multiple bromeliad species and genera growing in South America.     

<Emphasis Type="Italic">BrRxLR11</Emphasis> – a new phylogenetic marker with high resolution in the downy mildew genus <Emphasis Type="Italic">Bremia</Emphasis> and related genera

The genus Bremia (Peronosporaceae; Oomycete) is a widespread pathogen of Eurasian Asteraceae (Compositae). In addition to several species infecting weeds, it includes the economically important species, B. lactucae, which causes downy mildew of lettuce (Lactuca sativa). A few loci have already been successfully sequenced for this genus, but the resolution of the resultant phylogenies is insufficient to resolve the evolutionary history of Bremia. Therefore, there is a need to develop additional loci that can provide high resolution in phylogenetic inference. In this study, we report a new locus, BrRxLR11, derived from genomic data, which shows high resolving power within the genus Bremia, similar to phylogenies based on three different loci. This gene encodes a protein of 204 aa with unknown function, a below-threshold secretion signal, and an RxLR-EER motif in the c-terminal half. As only one site seems to be under positive selection, it has potential for future application in phylogenetic investigations in the economically important genus Bremia and related genera.     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

A revision of <Emphasis Type="Italic">Sphaeria pilosa</Emphasis> Pers. and re-evaluation of the Trichosphaeriales

Sphaeria pilosa, the basionym of the type of Trichosphaeria, T. pilosa, was insufficiently described by Persoon. The current interpretation of T. pilosa comes from Fuckel and is based on his own material published in the exsiccatal series Fungi Rhen. Exs. no. 946. The examination of the type and other authentic material of S. pilosa and T. pilosa revealed several different fungi. Fresh material of T. pilosa is not available and Fuckel’s historical specimens have never been sampled for DNA. In order to reconcile Persoon’s concept of S. pilosa with Fuckel’s concept of T. pilosa, we designate a neotype in our study. The species is a unitunicate ascomycete characterized by perithecial ascomata, persistent paraphyses, and cylindrical short-stipitate asci without visible apical annulus containing eight hyaline, ellipsoidal ascospores. The genus Trichosphaeria includes 87 described species and the Trichosphaeriales with the single family Trichosphaeriaceae recently accommodate 17 genera of apparently diverse phylogenetic affinity. Although the relationship of Trichosphaeria with other members of the Sordariomycetes is unknown, the family and order based on it are widely used to label incertae sedis clades inferred in phylogenetic analyses. Based on these findings, the Trichosphaeriales are re-evaluated and their use in phylogenetic studies is recommended to be abandoned until recognition of T. pilosa by molecular data.     

Diversity of Ukrainian winter common wheat varieties with respect to storage protein loci and molecular markers for disease resistance genes

Diversity of Ukrainian winter common wheat varieties was studied with respect to the storage protein loci Gli-A1, Gli-B1, Gli-D1, Glu-A1, Glu-B1, Glu-D1, Gli-A3, Gli-B5, and Gli-A6 (362 varieties) and markers for the Lr34/Yr18/Pm38/Sr57/Bdv1 gene conferring moderate resistance to a number of biotrophic pathogens, the Tsn1 gene for sensitivity to the toxins A of the necrotrophic fungi Pyrenophora tritici-repentis and Stagonospora nodorum, the Tsc2 gene for sensitivity to the toxin B of P. tritici-repentis, and the TDF_076_2D gene for moderate resistance to Fusarium head blight (181 varieties). Significant differences in frequencies of alleles at these marker loci between groups of varieties developed in different soil and climatic zones were revealed. The retention of a set of predominant alleles in groups of varieties of a certain zone in different periods of breeding was confirmed. At the same time, the appearance of new allele associations in the groups of varieties of the Steppe (in particular Gli-A1g and Glu-B1al) and the Central Forest-Steppe (1AL/1RS and Glu-B1d) in the last two decades has been noted. Nonrandom associations between alleles of disease resistance genes as well as alleles of disease resistance genes and storage protein alleles were revealed.     

ITS2 sequence–structure phylogeny reveals diverse endophytic <Emphasis Type="Italic">Pseudocercospora</Emphasis> fungi on poplars

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence–secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence–structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi’s evolution and function in ecology.     

DNA barcoding of selected UAE medicinal plant species: a comparative assessment of herbarium and fresh samples

It is commonly difficult to extract and amplify DNA from herbarium samples as they are old and preserved using different compounds. In addition, such samples are subjected to the accumulation of intrinsically produced plant substances over long periods (up to hundreds of years). DNA extraction from desert flora may pause added difficulties as many contain high levels of secondary metabolites. Herbarium samples from the Biology Department (UAE University) plant collection and fresh plant samples, collected from around Al-Ain (UAE), were used in this study. The three barcode loci for the coding genes matK, rbcL and rpoC1-were amplified. Our results showed that T. terresteris, H. robustum,T. pentandrus and Z. qatarense were amplified using all three primers for both fresh and herbaium samples. Both fresh and herbarium samples of C. comosum, however, were not amplified at all, using the three primers. Herbarium samples from A. javanica, C. imbricatum, T. aucherana and Z. simplex were not amplified with any of the three primers. For fresh samples 90, 90 and 80% of the samples were amplified using matK, rbcL and rpoC1, respectively. In short, fresh samples were significantly better amplified than those from herbarium sources, using the three primers. Both fresh and herbarium samples from one species (C. comosum), however, were not successfully amplified. It is also concluded that the rbcL regions showed real potentials to distinguish the UAE species under investigation into the appropriate family and genus.     

Identification and evaluation of potential bio-control fungal endophytes against <Emphasis Type="Italic">Ustilagonoidea virens</Emphasis> on rice plants

False smut disease of rice is posing an increasing concern for production, not only because of the hiking epidemic occurrence in rice production, but also because of the challenging specific pathogenesis of the disease. The aim of this work was to evaluate the potential of five fungal endophytes to reduce negative effects of rice false smut fungus (Ustilagonoidea virens) on rice plants, in both the laboratory and greenhouse. Though all the fungal isolates showed the ability to inhibit the growth of U. virens with varying degrees, isolate E337 showed significant antagonistic activity against the pathogenic fungi. The isolate E337 was identified as Antennariella placitae by molecular and morphological data analysis including 18S rDNA sequence analysis. This isolate showed a significant in vitro inhibition of mycelial growth of U. virens by dual culture method and it was subsequently tested for its in vivo biocontrol potential on false smut disease on rice plants. Greenhouse experiments confirmed that applications of conidia of A. placitae protected rice plants by improving rice yield and by decreasing the severity of false smut disease on susceptible rice plants. This is the first report where A. placitae has been identified as a biocontrol organism.     

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.     

<Emphasis Type="Italic">Viola woosanensis</Emphasis>, a recurrent spontaneous hybrid between <Emphasis Type="Italic">V. ulleungdoensis</Emphasis> and <Emphasis Type="Italic">V. chaerophylloides</Emphasis> (Violaceae) endemic to Ulleung Island,Korea

Ulleung Island is an oceanic volcanic island in Korea, which has never been connected to the adjacent continent. Previous studies highlighted Ulleung Island as an excellent system to study the pattern and process of early stages of flowering plant evolutions on oceanic island. The predominant mode of speciation in flowering plants on Ulleung Island appears to be anagenesis. However, the potentially important role of hybrid speciation among incompletely reproductively isolated lineages cannot be ruled out. Viola woosanensis (Violaceae) is of purportedly hybrid origin between V. ulleungdoensis (i.e., formerly recognized as V. selkirkii in Ulleung Island) and V. chaerophylloides, based on morphology. To examine the origin of V. woosanensis, we sampled a total of 80 accessions, including V. woosanensis and its putative parental species and sequenced nrDNA ITS, and four highly variable chloroplast noncoding regions (trnL-trnF, rpl16 intron, atpF-atpH, and psbA-trnH). Representative species of Viola from Korea were also included in the phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). Additive polymorphic sites in the nrDNA ITS regions were confirmed by cloning amplicons from representative species. The molecular data strongly supported the hybrid origin of V. woosanensis, and the maternal and paternal parent were determined to be V. ulleungdoensis and V. chaerophylloides, respectively. The presence of two parental ribotypes in V. woosanensis (with the exception in one population) was confirmed by cloning, suggesting V. woosanensis is primarily the F₁ generation. No trace of backcrossing and introgression with its parents was detected due to low fertility of hybrid species. We found a multiple and unidirectional hybrid origin of V. woosanensis. Additional studies are required to determine which factors contribute to asymmetric gene flow of Viola species in Ulleung Island.