Molecular phylogeny of mulberries reconstructed from ITS and two cpDNA sequences.

Data processing can mask biology: towards better reporting of fungal barcoding data?

The phylogenetic relationships of anastomosis groups (AG) of Rhizoctonia associated with Ceratobasidium and Thanatephorus teleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidium and Thanatephorus supported at least 12 monophyletic groupings within Ceratobasidium and Thanatephorus. There was strong support for separation of Ceratobasidium and Thanatephorus, however, six sequences representing different AG of Ceratobasidium grouped with certain sequences within the Thanatephorus clade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus (21 groups) and Ceratobasidium (10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidium may be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.

The phylogenetic relationships of anastomosis groups (AG) of Rhizoctonia associated with Ceratobasidium and Thanatephorus teleomorphs were determined by cladistic analyses of internal transcribed spacer (ITS) and 28S large subunit (LSU) regions of nuclear-encoded ribosomal DNA (rDNA). Combined analyses of ITS and LSU rDNA sequences from 41 isolates representing 28 AG of Ceratobasidium and Thanatephorus supported at least 12 monophyletic groupings within Ceratobasidium and Thanatephorus. There was strong support for separation of Ceratobasidium and Thanatephorus, however, six sequences representing different AG of Ceratobasidium grouped with certain sequences within the Thanatephorus clade. Phylogenetic analysis of ITS sequence data from 122 isolates revealed 31 genetically distinct groups from Thanatephorus (21 groups) and Ceratobasidium (10 groups) that corresponded well with previously recognized AG or AG subgroups. Although phylogenetic analysis of ITS sequences provided evidence that several AG of Ceratobasidium may be more closely related with some AG from Thanatephorus, these relationships were not as strongly supported by bootstrap analysis.

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.

Thirty isolates of Apiosporina morbosa, which were isolated from wild chokecherry (Prunus virginiana), an ornamental cultivar of chokecherry with purple foliage (P. virginiana ‘Shubert Select‘), and domestic plum (Prunus domestica), were compared for morphological and molecular characteristics, using culture techniques and data from internal transcribed spacers (ITS) I and II, restriction fragment length polymorphisms (RFLP), and sequence-related amplified polymorphism (SRAP) analyses. Strains of A. morbosa grew as olive-green colonies that changed in about 2 weeks to a dark-brown colour. Cultures produced single- or two-celled and cylindrical conidia with tapering ends. Conidia had a mean size of 4.51 μm × 10.97 μm. The overall colony and conidial morphology in cultures of the A. morbosa isolates was generally similar to that of an isolate of Cladosporium herbarum. Pseudothecia were globose and produced clavate asci with unequally two-celled and club-shaped ascospores having mean sizes of 6.68–7.37 μm × 16.42–16.78 μm. RFLP markers produced by endonucleases DdeI, Bst98I, and VspI in the ITS region differentiated the pathogen isolates from other gall-associated fungi, but no difference was observed among the A. morbosa isolates, using these markers. The sequences of the ITS region had >99% similarity among 30 isolates of A. morbosa, and a total of eight unique genotypes were found, based on ITS sequence alignment. The most common genotype was manifested in 21 of 30 A. morbosa isolates, based on the ITS sequences. Three A. morbosa isolates had an identical sequence to that of an isolate of C. herbarum. A comparison with an isolate of Cladosporium cladosporioides showed that A. morbosa differed from C. cladosporioides by 3%, based on ITS sequences. SRAP analysis detected 18 unique genotypes and a high genetic diversity (H = 0.256) among 25 isolates of A. morbosa. The pathogen population isolated from wild chokecherry produced more unique genotypes and higher genetic diversity (H = 0.283) than the population from ‘Shubert Select’ (H = 0.165). However, an exact test indicated no differentiation between these two populations (P = 0.334) with a gene flow rate of 4.28. Most of the isolates from ‘Shubert Select’ were clustered in a subgroup in a phylogenetic dendrogram. Phylogenetic analysis of ITS sequences of the A. morbosa isolates in our study and related taxa extracted from GenBank revealed that A. morbosa had the closest genetic distance with Cladosporium spp.

In order to resolve the relationships among species from Angelica s.s. and its allied genera in East Asia, ITS (Internal Transcribed Spacer) sequences of 44 taxa were used to analyze their sequence divergence and to construct three phylogenetic trees in this paper. Three taxa were used as the outgroup. Some conclusions could be achieved as follows. (1) Angelica s.s., Czernaevia, and Coelopleurum were closely related, but the relationship between Ostericum and them was ulterior. It was also supported by analyses on fruit anatomy and chemical constitutes. Ostericum was proposed as a relatively independent genus. (2) Results from ITS sequences supported the view that Angelica s.s. was monophyletic and could be divided into several sections. (3) Results from ITS sequences and chemical constitutes indicated that the relationship between Peucedanum and Angelica s.s. was very close. (4) Results from ITS sequences as well as from conformation and chemical constitutes showed that the divergence of A. sinensis from other taxa of Angelica s.s. was great. The taxonomic position of A. sinensis should be reconsidered. (5) Both analyses of ITS sequences and chemical constitutes revealed that Ligusticum was not a natural group.

To find the differences in the internal transcribed spacer(ITS) sequences and provide scientific data for the authentication of Potentilla chinensis and its related species, we extracted the genome DNA from the leaves of 5 common Potetilla species in Jilin Province, amplified the ITS region using ITS universal primers of angiosperm, and sequenced the purified PCR products directly. Polymorphism of ITS sequences was found within P. chinensis and the sequence data suggested that our samples of this species might be related to hybridization. Other 4 species showed intraspecies-stability in ITS sequence. The ITS sequences of these 5 Potentilla species are significantly different. So ITS sequence analysis and other methods derived from it can be used in authentication of Potentilla.

Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae).

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides , and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ∼ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ∼ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.

ABSTRACTSequence variation of ribosomal DNA internal transcribed spacers (ITS) among populations, species, and genera of the diatom genus Stephanodiscus was investigated. ITS 1 and ITS 2, including the 5.8S gene, were sequenced from geographically distant and nearby populations of S. niagarae Ehrenberg. In addition, repeats from S. hantzschii Grunow and Cyclotella meneghiniana Kützing were sequenced to determine the taxonomic range over which the ITS region could be used for diatom systematics. The morphologically distinct S. yellowstonensis Theriot & Stoermer, thought to have evolved from S. niagarae in Yellowstone Lake between 12,000 and 8000 years ago, also was sequenced to assess its relationship to nearby S. niagarae populations. The organization and relative sizes of ITS 1 and ITS 2 in Stephanodiscus species were similar to those reported for other eukaryotes. In general, ITS 2 was slightly larger and more variable than ITS 1. Cladistic analysis of ITS sequences did not resolve relationships of nearby S. niagarae and S. yellowstonensis populations. However, central North American S. niagarae populations were in a clade supported by two nucleotide changes. For Cyclotella, much of the ITS region was not alignable with that for Stephanodiscus species; therefore, generic‐level comparison within the Thalassiosiraceae may not be possible. The variation (95–96% similarity) between S. hantzschii and other Stephanodiscus species suggests that interspecific relationships could be assessed with ITS sequences. Although S. yellowstonensis is morphologically distinct from S. niagarae, no autapomorphic nucleotide sites were identified. Two S. niagarae populations (Heart and Lewis Lakes), however, did possess autapomorphic ITS sites.

Plants in the genus Croton (Euphorbiaceae) have been widely used in folklore medicine and are considered to be a rich source of bioactive compounds. In this study, genetic variation of fifteen Croton species existing in Thailand was evaluated for their phylogenetic relationship. The genes selected for the study included the internal transcribed spacer (ITS) of nuclear DNA and trnL-F gene of chloroplast DNA. The length of ITS sequences of various Croton species was from 620 to 627 bp with 52.5-57.1 % GC content. Genetic variation in the ITS region was mostly from substitutions. For the trnL-F gene, the Croton species had their sequences in the range from 1017 to 1041 bp with 31.0-31.9 % GC content. The genetic variation of this gene in Croton was caused mainly from insertions and deletions. Phylogenetic analysis revealed that the relationship of all fifteen Croton species was monophyletic type. In terms of variation in population, the two well known Thai medicinal plants C. stellatopilosus (Plau-noi) and C. roxburghii (Plau-yai) were extensively investigated for their genetic variation. Both species were studied not only on the ITS region and trnL-F gene, but also on the trnK intron of chloroplast DNA. Based on the obtained ITS sequences of C. stellatopilosus, two groups of individual sequences, designated as Type A and Type B were found as well as various nucleotide additive sequences which were arised from these two groups and designated as “putative hybrids”. These putative hybrids of C. stellatopilosus showed their trnK intron sequences identical to the Type B but different from the Type A. Thus, it was suggested that Type A was paternal parent and Type B was maternal parent of the hybridization of C. stellatopilosus. For C. roxburghii, it showed high genetic variation in their ITS, trnL-F and trnK intron sequences but could be systematically divided into two groups. In conclusion, the technique of direct sequencing was successfully used in this case as a tool for estimating the phylogenetic relationship of Croton and identifying the definite sites of genetic variation in the important Croton species of C. stellatopilosus and C. roxburghii.

Mating tests, internal transcribed spacer (ITS) sequence analysis, intergenic spacer 1-restriction fragment length polymorphism (IGS1-RFLP), IGS1 sequence analysis, and IGS2-RFLP analysis were carried out on isolates of 17 morphologically different Pleurotus mushrooms collected on Ferula sinkiangensis. The isolates were divided, based on mating tests and ITS sequence analysis, into two groups identical to P. eryngii var. ferulae and P. nebrodensis, respectively. Single spores from these two groups were incompatible, but those from P. eryngii var. ferulae and P. eryngii were compatible and combined to produce 56.25% dikaryon mycelia with clamp connections. The ITS of P. eryngii var. ferulae and P. nebrodensis (GenBank accession no. AY311408) were both 638 bp in size but differed by 3% in sequence. P. eryngii var. ferulae and P. eryngii (GenBank accession no. AY368658) were identical in ITS size and sequence. P. nebrodensis was the dominant population of Pleurotus mushroom growing on F. sinkiangensis. It exhibited genetic diversity. The two species could also be distinguished by IGSI-RFLP, similar to identification by mating tests and ITS sequence analysis. Difference in IGS1-RFLP existed between P. eryngii var. ferulae and P. nebrodensis. The sequence difference reached 2.28%. Both IGS1 size and IGS1-RFLP were similar among the different samples of P. nebrodensis. The 17 isolates were separated into five types based on IGS2 size and IGS2-RFLP, with both interspecies and extraspecies differences. P. nebrodensis exhibited polymorphism and was divided into four types. These results agreed with macroscopic differences. IGS2 might be the effective domain of genetically polymorphic ribosomal DNA in P. nebrodensis mushrooms found in Xinjiang, China.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.

The genus Ranunculus L., with a worldwide distribution, is the largest member of the Ranunculaceae. Here, nuclear ribosomal internal transcribed spacer (ITS) sequence data and chloroplast trnLF sequence data were used to analyze phylogenetic relationships among members of the annual and perennial (Group Praemorsa, Group Rhizomatosa, Group Grumosa and Group non-Grumosa) species of Ranunculus in Iran. In the strict consensus tree of nrDNA ITS sequence analyses, seven sub-clades were described, based on morphological, karyological, palynological, and ecological features. Within each clade, there were species belonging to more than one Group, and species of a single Group may fall into different clades, revealing that a classification based on underground system characters does not show natural interspecific relationships and must be revised. This is also apparent from trnL-F sequence analyses. Based on our results, the Praemorsa and Rhizomatosa Groups can be merged. The ITS sequence data show interspecific relationships more clearly than trnL-F sequence data. © 2011 Progress in Biological Sciences, Vol. 1, No.1, 41-47.

The genus Eustrongylides Jägerskiöld, 1909 includes parasitic nematodes (Dioctophymatidae) affecting various fish species and piscivorous birds of freshwater ecosystems. Currently, there is little information on the molecular characterization of E. excisus based on nuclear ribosomal internal transcribed spacer (ITS) rDNA regions. However, before the present study, there had been no reports of characterizing the E. excisus using nuclear small subunit ribosomal RNA (SSU rRNA) and mitochondrial cytochrome c oxidase subunit I (COI) genes sequences. In the present study, Eustrongylides spp. larvae were collected from pike-perch Sander lucioperca (L.) in Northern Turkey, and characterized by sequencing of ITS regions, SSU rRNA, and COI markers. Larvae herein morphologically identified as the fourth stage of Eustrongylides spp. were genetically identified as E. excisus based on the ITS sequence analysis. This study is the first record of SSU rRNA and COI sequences for E. excisus in GenBank. This is also a molecular characterization of E. excisus for the first time in Turkey. The ITS, SSU rRNA, and COI sequences of E. excisus can be used to establish the phylogenetic relationships of Eustrongylides species from Turkey and worldwide for further studies.