ITS Data Research Articles

Molecular phylogeny of the sect. Adonis (Genus adonis L., Ranunculaceae)

The ornamentally important genus Adonis L. (Ranunculaceae) consists of about 30 species. In Iran, 8 species are recognized. The new classification system recognizes two subgenera, six sections, and six series for Adonis. The species differentiation within the genus is controversial due to morphological overlaps in different species and extensive morphological variation. Therefore, the aims of present study were: 1- Species delimitation within the Sect. Adonis based on morphological and molecular evidences and, 2- Studying the species relationship based on molecular phylogeny. Multivariate analyses of morphological and molecular (ITS and ISSR) data differentiated the Adonis species. PCA biplot analysis of morphological characters revealed morphological characters such as leaf arrangement; leaf length, flower diameter, petal length and width, calyx length, and pedicel length are of taxonomic implication in Adonis. Bayesian tree of ITS sequences revealed that the sect. Adonis is differentiated from the sect. Adonanthe. Based on ITS data, A. microcarpa showed close affinity to A. scrobiculata, while A. aestivalis, A. icrocarpa and A.dentata were close to each other.

Additions to Chaetothyriaceae (Chaetothyriales): Longihyalospora gen. nov. and Ceramothyrium longivolcaniforme, a new host record from decaying leaves of Ficus ampelas.

A novel ascomycete genus, Longihyalospora, occurring on leaf litter of Ficus ampelas in Dahu Forest Area in Chiayi, Taiwan is described and illustrated. Longihyalospora is characterized by dark mycelium covering the upper leaf surface, elongate mycelial pellicle with ring of setae, pale brown to brown peridium, broadly obovoid, short pedicellate asci and hyaline, fusiform, elongated (tapering ends) and multi-septate ascospores with a thin mucilaginous sheath. Phylogenetic analyses of combined ITS, LSU and SSU sequence data revealed Longihyalospora as a distinct genus within the Chaetothyriaceae with high bootstrap support. Moreover, based on morphological similarities, Chaetothyrium vermisporum transferred to the new genus. In addition, Ceramothyrium longivolcaniforme is reported for the first time on Ficus ampelas. Newly added species are compared with other similar species and comprehensive descriptions and micrographs are provided.

Molecular strategies for identification and characterization of some wild edible mushrooms of Nagaland, India.

Nagaland has a rich macro fungal flora but not many works has been carried out till today. Present investigation deals with molecular characterization and phylogenetic analysis of six popular wild edible mushrooms (WEMs) species of Nagaland, India viz., Lentinula edodes, Lentinus squarrosulus, L. sajor-caju, L. tigrinus, Schizophyllum commune, Termitomyces heimii and one variety of L. squarrosulus based on molecular markers (ITS, 18S rRNA and 28S rRNA genes) data. The use of DNA markers for identification of mushrooms is highly desirable and practical because it is reliable and quick. This approach could resolve successfully the identity and interrelationship of six WEM species with respect to their infrageneric groups. The high CI values of the mushrooms species indicated the low homoplasy nature. The ITS and 28S rRNA data sets were found to be more informative then the 18S rRNA datasets. The molecular data generated for each mushroom species in the present investigation will help in correct identification and conservation of these widely consumed WEM of the region. Additionally assessment of bioactive molecules indicates that studied species are rich in pro-health bioactive compounds. The study hence throws light on the potential and importance of mushrooms especially the edible mushrooms as an economically valuable crop.

What Can Integrated Analysis of Morphological and Genetic Data Still Reveal about the Anastrepha fraterculus (Diptera: Tephritidae) Cryptic Species Complex?

The South American fruit fly Anastrepha fraterculus (Wiedemann) is a complex of cryptic species, the so-called “Anastrepha fraterculus complex”, for which eight morphotypes are currently recognized. A previous analysis of ITS1 in samples of the Anastrepha fraterculus complex, while revealing high distinctiveness among samples from different localities of South America, Central America, and Mexico, no direct association was made between sequence type and morphotype. In the present report, a correlated analysis of morphometry and ITS1 data involved individuals belonging to the same population samples. Although showing a low level of intra-populational nucleotide variability, the ITS1 analysis indicated numerous inter-population sequence type variants. Morphotypes identified by morphometric analysis based on female wing shape were highly concordant with ITS1 genetic data. The correlation of genetic divergence and morphological differences among the tested samples gives strong evidence of a robust dataset, thereby indicating the existence of various taxonomic species within the A. fraterculus complex. However, the data revealed genetic and morphological variations in some regions, suggesting that further analysis is still required for some geographic regions.

New species and new genus of Pseudohaploporinae (Digenea): Pseudohaploporus pusitestis sp. n. and Parahaploporus elegantus n. g., sp. n. (Digenea: Pseudohaploporinae) from Vietnamese mullet fish

Two new species of Pseudohaploporinae, Pseudohaploporus pusitestis sp. n. and Parahaploporus elegantus n. g., sp. n., are described from intestines of the Vietnamese mullet fish Moolgarda seheli and Osteomugil cunnesius, respectively. Pseudohaploporus pusitestis sp. n. differs from two known Pseudohaploporus species, P. vietnamensis and P. planiliza, by the absence of a diverticulate hermaphroditic duct and muscular sphincters at the proximal end of the hermaphroditic sac. Metrically, P. pusitestis sp. n. is close to P. vietnamensis and differs from this species and from P. planilizum by lower maximum sizes of most parameters. Parahaploporus elegantus n. g., sp. differs from representatives of Pseudohaploporus by the presence of a single testis and the armament of hermaphroditic duct and is morphologically close to trematodes of the genus Haploporus. However, P. elegantus n. g differs from all known Haploporus species from mugilids of the Indo-West Pacific by the structure of the armament of the hermaphroditic duct and also by size of body, organs and eggs. The validity of designating two new species and a new genus of trematodes is supported by ITS and 28S rDNA sequence data. Phylogenetic reconstructions showed that the new trematodes belong to the Pseudohaploporinae, which formed a well-supported cluster within the monophyletic Haploporidae.

<p><strong><em>Tubeufia sahyadriensis </em></strong><strong>(<em>Tubeufiaceae</em>),<em> </em>a new dictyosporous<em> </em>anamorph<em> </em>from the Western Ghats, India</strong></p>

An unusual anamorphic dictyosporous species, Tubeufia sahyadriensis, was collected on decaying Bambusa bambos litter in the Tamhini Ghats in the northern Western Ghats of Maharashtra, India. This new species can be easily differentiated from allied Tubeufia species by its dark brownish to umber, maize corncob-like dictyoseptate conidia with reduced, simple cylindrical conidiogenous cells. Phylogenetic analyses using ITS, LSU and TEF1α sequence data delineated the new species within the genus Tubeufia under family Tubeufiaceae.

<p><strong><em>Cymbidium atrolabium</em></strong><strong> (Orchidaceae; Epidendroideae), a new species from China: evidence from morphological and molecular data</strong></p>

A new orchid species, Cymbidium atrolabium, from Yunnan Province, China, is described and illustrated based on morphological and molecular analyses. A detailed comparison between the newly discovered orchid and other members of Cymbidium was conducted. The new plant is similar to C. tortisepalum but differs by having a dark-purple inflorescence with smaller flowers, sepals that are pale green-yellow with a dark-purple broad midrib, petals that are dark-purple with pale green-yellow margins and a smaller, dark purple, densely papillose lip. Phylogenetic analyses based on nuclear ribosomal ITS and plastid (matK, rbcL) sequence data support the status of C. atrolabium as a new species.

Molecular Identification and Evaluation of Genetic variability among Some Aflatoxigenic Aspergillus flavus Strains Isolated from Fish Feed in Egypt

AFLATOXINS are among the most potent carcinogenic, mutagenic and teratogenic natural compounds occurring in food and feed. In this study, molecular typing based on sequence variations in the ITS1-5.8S-ITS2 region was used to identify and genetically discriminate five selected strains of A. flavus, four strains (AUMC13909, AUMC13910, AUMC13911 and AUMC13917) were aflatoxigenic isolates recovered from fish feed. A. flavus AUMC10311 is a non-aflatoxigenic reference strain which was used as a negative control to investigate the molecular variability of the studied strains concerning their aflatoxigenic potential. The ITS data of the studied strains were submitted to NCBI GenBank with the accession numbers: MK491621, MK491622, MK491623, MK491624 and MK491625. Cluster analysis of these strains was performed depending on their ITS sequences, which discriminate them into three different sub-clusters. The highest genetic similarity (100%) was observed between AUMC13911 and AUMC10311. Proteins banding patterns of the examined strains were analyzed using SDS-PAGE, which resolved six polymorphic protein bands out of a total of 22 bands, ranged from 14 to 125kDa. Interestingly, a protein band (~ 40kDa) was relatively overexpressed in case of the aflatoxigenic strains while was hardly detected in the non-aflatoxigenic strain, suggesting that this protein might be involved in aflatoxin biosynthesis. Cluster analyses based on this banding pattern grouped the tested strains into two major clusters. The highest similarity index (97%) was between AUMC13911 and AUMC10311. These results confirmed the genetic variation among the examined A. flavus strains and the necessity for greater insight into cluster assembly using different Omics approaches.

<p><strong><em>Sporidesmium guizhouense</em></strong><strong> sp. nov. (Sordariomycetes<em> incertae sedis</em>), a new species from a freshwater habitat in Guizhou Province, China</strong></p>

During a survey of freshwater fungi in Guizhou Province, China, a collection from a submerged decaying twig in Baihua Lake was identified as a new species of Sporidesmium sensu lato based on morphological characters and phylogenetic analyses of combined LSU, SSU, ITS, TEF1α and RPB2 sequence data. Phylogenetic analyses supported its placement in Sordariomycetes but the fungus grouped distant from Sporidesmium sensu stricto, and its ordinal or familial position within the class remained inconclusive. Sporidesmium guizhouense sp. nov. is introduced with an illustrated account and notes on its taxonomy, phylogeny and systematic position are provided.

Additions to the knowledge of Ganoderma in Thailand: Ganoderma casuarinicola, a new record; and Ganoderma thailandicum sp. nov.

Ganoderma is a cosmopolitan genus of mushrooms, which can cause root and butt rot diseases on many tree species. Members of this genus are particularly diverse in tropical regions. Some Ganoderma spp. are medicinally active and therefore are used to treat human diseases or as a dietary supplement. In this study, three Ganoderma strains were collected in tropical southern Thailand. Phylogenetic analyses of combined ITS, LSU, TEF1α and RPB2 sequence data indicated that the three strains grouped in a distinct lineage within laccate Ganoderma. One strain was collected from Surat Thani Province clustered in the G. casuarinicola clade with high statistical support (MLBS = 100% / MPBS = 98% / PP = 0.96), while the other two strains of Ganoderma, collected from Nakhon Si Thammarat Province, formed a distinct well-supported clade (MLBS = 100% / MPBS = 100% / PP = 1.00) and are described here as a new species. Ganoderma casuarinicola is reported here as a new record to Thailand. Morphological differences of the two taxa and their closely related taxa are discussed. Colour photographs of macro and micro morphological characteristics and a phylogenetic tree to show the placement of the new record and new species are provided.

<p><strong><em>Iodosphaeria honghense </em></strong><strong>sp. nov<em>. </em>(Iodosphaeriaceae<em>, </em>Xylariales) from Yunnan Province, China</strong></p>

Iodosphaeria honghense sp. nov., collected from dead twigs of an unknown host in Honghe, Yunnan Province, China, is introduced based on morphological characteristics and phylogenetic analyses. Iodosphaeria honghense is distinct from other similar taxa in having cylindrical to allantoid, guttulate ascospores without a mucilaginous sheath. Maximum likelihood and Bayesian analyses based on combined LSU, ITS and RPB2 data matrix indicate that I. honghense forms a distinct lineage within the Iodosphaeriaceae. This study provides a detailed description and illustrations with morphological comparisons, and includes a key to accepted Iodosphaeria species.

<p><strong><em>Phaeosphaeria chinensis</em></strong><strong> <em>sp. nov</em>. (<em>Phaeosphaeriaceae</em>) with an asexual/sexual morph connection from GuangDong Province, China</strong></p>

Phaeosphaeria chinensis sp. nov. was found on dead leaves, collected from Guangdong Province, China. Morphology of the new species was compared with other Phaeosphaeria species and related genera of Phaeosphaeriaceae. Phylogenetic analyses of combined ITS, LSU, SSU and TEF-1 sequence data based on maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) revealed that P. chinensis as a distinct species within the Phaeosphaeria with high bootstrap support. The comparison of the new species with other Phaeosphaeria species and a comprehensive description and micrographs are provided. The linkage of sexual and asexual morphs of the new species is also showed.

<p><strong>Contribution to the phylogeny of Caucasian <em>Nonea </em>(Boraginaceae) inferred from nrDNA ITS and <em>trnL-F</em> data</strong></p>

Nonea (Boraginaceae) with nearly 35 species was divided into four sections based on the shape of mericarpids and the position of the anthers in the corolla tube. Although several comprehensive taxonomic studies have been performed on Turkish and European Nonea taxa, Caucasian ones have not been studied well. Therefore Caucasian Nonea need close attention with regard to molecular systematics. In this study, 15 Caucasian Nonea including N. cyanocalyx and N. bakuensis were evaluated with nrDNA ITS and cpDNA trnL-F sequence data using Maximum Parsimony and Bayesian Inference to reconstruct phylogeny. All examined members of Nonea were grouped at three main clades with weak to good support values based on nrITS and at two main clades with moderate to good support values based on trnL-F. Both trees did not coincide with the traditional sub-generic delimitation of Nonea, but nrITS tree supported monophyly of Nonea. On the other hand, Pulmonaria is deeply nested in Nonea in the trnL-F tree. Moreover, present findings support treating N. cyanocalyx and N. bakuensis as distinct species rather than subspecies and revealed a preliminary phylogenetic structure of little known Caucasian Nonea.

<p><strong>Morphological and phylogenic evidences reveal a new <em>Seiridium </em>species in China</strong></p>

During the fungal collection trip in China, three Seiridium isolates were obtained from cankered tissues of the Chinese windmill palm (Trachycarpus fortunei) branches. Identification of these isolates was conducted by morphological characters on PDA plates, and combined ITS, RPB2, TEF and TUB sequences data. Hence, Seiridium chinense sp. nov. was described herein, which represents a new member from Arecaceae plants. Seiridium chinense is distinguished from its phylogenetically close species, S. venetum, by having wider conidia.

<p><strong>Multigene phylogenetic characterisation of<em> Colletotrichum artocarpicola sp. nov.</em> from <em>Artocarpus heterophyllus</em> in northern Thailand</strong></p>

A new species, Colletotrichum artocarpicola, on Artocarpus heterophyllus from Chiang Rai, Thailand, is introduced using both morphological and molecular approaches. Combined phylogenetic analysis of ITS, GAPDH, CHS-1, ACT and TUB2 sequence data demonstrate that Colletotrichum artocarpicola is a distinct species within the gloeosporioides species complex. The new species is illustrated and compared with related taxa, and evidence of its pathogenicity is provided.

<p><strong><em>Murispora aquatica sp. nov.</em> and <em>Murispora fagicola</em>,<em> </em>a new record from freshwater habitat in China</strong></p>

We are currently studying lignicolous freshwater fungi in China. In this paper, we introduce two taxonomic accounts to the family Amniculicolaceae in Pleosporales viz. Murispora aquatica sp. nov (from Yunnan) and a new record of Murispora fagicola (from Tibet). The conclusions were derived from both morphological comparisons and the results of phylogenetic analyses (concatenated ITS, LSU, SSU and TEF1-α sequence data). The new species is compared with other existing species of Murispora and comprehensive descriptions and micro-photographs are provided.

Revision of Alnus (Betulaceae) in Iran using molecular ITS markers and morphological characteristics

Alnus (Alder) is a genus of Betulaceae, which comprises more than 29 species in the world. According to different classifications of flora, eight taxa of the genus naturally grow in Iran and are distributed along the northern slope of Albourz Mountains in Iran (Hyrcanian forests). In the current research, we investigated morphological characteristics and phylogenetic relationships of 31 populations of the genus in Iran. Twenty-eight qualitative and quantitative traits were studied for morphological evaluation and ITS molecular marker was investigated for molecular study. We used MVSP, SPSS, MrBayes, RAXMLGUI, Mesquite 2.71, Modeltest 3.7 software packages for statistical analyses. Morphological features varied widely among the studied taxa and ANOVA test revealed significant difference for most of them. Moreover, the angle of leaf base, the leaf margin shape, the length and width of the leaf blade, fruits length, and the presence of hair on the petioles, young twigs, and buds were more variable morphological traits. The studied taxa were clustered separately in the UPGMA tree, PCA and PCO plots using morphological traits. In addition, CA-joined plot showed each taxon to have distinct morphological trait(s), which is useful in the identification of the taxa. Phylogeny analysis revealed that Alnus genus is a monophyletic group. Furthermore, the studied taxa were clustered separately in phylogenetic dendrogram. According to morphological and ITS data, we listed ten taxa of the genus in Iran and introduced A. hyrcana and A. longiflorescentia as two new species from Hyrcanian forests of Iran.

<p><strong>Taxonomic status of three controversial species of <em>Cephalanthera</em> (Orchidaceae) from Korea based on morphological and molecular data</strong></p>

The taxonomic status of three of the seven Cephalanthera species in Korea is controversial: C. erecta var. oblanceolata, C. subaphylla, and C. shizuoi. To clarify their taxonomic status, we used molecular data of ITS of nuclear DNA, and three regions of chloroplast DNA (matK, rpl16 and trnL-F), in addition to morphological characters. We analyzed 82 accessions collected from 26 sites including the other four taxa: C. erecta, C. falcata, C. longibractea, and C. longifolia. The analysis of ITS, matK, rpl16 and trnL-F data showed that Cephalanthera erecta var. oblanceolata should be treated as a synonym of C. erecta. Molecular data supported that C. subaphylla is an independent species rather than a variety or forma of C. erecta. Cephalanthera shizuoi was currently treated as a synonym of C. erecta or C. longifolia based on morphology and the molecular data supported that C. shizuoi should not be recognized as distinct species; but C. shizuoi should be a synonym of C. longifolia, not a synonym of C. erecta.

Playing the Taxonomic Cupid: Matching Pistillate and Staminate Conspecifics in Dioecious Clusia (Clusiaceae)

Abstract—Dioecious reproduction, in which unisexual flowers are developed in separate individuals, is an uncommon characteristic that has evolved multiple times independently in angiosperms. Nearly all species in the neotropical genus Clusia are dioecious and most of them are incompletely known because only one sexual morph, either the staminate or the pistillate individual, has been described. Here we explored the variability in chloroplast regions atpF intron, matK, ndhA intron, petL-psbE, rpl16 intron, as well as the nrDNA ITS to evaluate conspecificity between staminate and pistillate individuals within the genus Clusia. Our results indicate that phylogenetic analyses using maximum likelihood and Bayesian inference based on nrDNA ITS data is a suitable approach to assess whether sexual morphs are conspecifics and to estimate species phylogenetic position within the genus. Staminate and pistillate individuals were effectively identified for Clusia araracuarae, C. lundellii, C. stenophylla, and C. uvitana. Distribution maps as well as full morphological descriptions for these species are provided along with comments on their phylogenetic relationships.

Population genetic analysis of trematode Parasaccocoelium mugili Zhukov, 1971 (Haploporidae Nicoll, 1914) from the Russian Far East and Vietnam based on ribosomal ITS and mitochondrial COI gene partial sequence data.

Intraspecific variation of Parasaccocoelium mugili collected from mullet fish of the south of Russian Far East and Vietnam has previously been estimated on the basis of two molecular markers: ribosomal internal transcribed spacer 1 (ITS1) rDNA and mitochondrial cytochrome oxidase I (COI) gene sequences. In the present study, molecular identification of this species from the Kievka River, Primorye and from Vietnam was performed by analysis of 28S rDNA sequences. Analysis of ITS1 rDNA sequences variation revealed two highly differentiated main groups, representing trematode specimens from the two regions. Genetic variation within each region was relatively low. Mitochondrial COI gene sequence data analysis revealed fixed nucleotide and amino acid substitutions, and supported the existence of two genetically different groups associated with geographical origin. Analysis of the COI gene fragments showed extremely high variation within Russian and Vietnamese P. mugili samples. Our results for P. mugili most probably represent a case of initial step of allopotric speciation for this trematode, caused by living strategy of its definitive host at evolutionary scale. Mitochondrial DNA sequence data show that existence of gene flow between local populations of P. mugili in the Primorye Region caused by definitive hosts can be proposed.