Ribosomal DNA Internal Transcribed Spacer Sequences Research Articles

Phylogenetic study of the endemic species Oxytropis almaatensis (Fabaceae) based on nuclear ribosomal DNA ITS sequences

BackgroundOxytropis almaatensis Bajt. is a rare, narrow endemic species of the Trans-Ili Alatau mountains in Kazakhstan. Up to now, no studies regarding the taxonomy and variation of key morphological traits of O. almaatensis were undertaken. The purpose of this analysis was to evaluate phenotypic variation of O. almaatensis and assess the position of the species within the genus based on nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region.ResultsTwo populations of O. almaatensis were collected in neighboring gorges of the Trans-Ili Alatau Mountains. The ITS sequences from the samples of two populations of O. almaatensis were identical. The phylogenetic analysis indicated that O. almaatensis is within Oxytropis genetically close to O. glabra as these species formed a separate subclade. The phenotypic variation of populations was assessed using nine morphological traits and compared to descriptions of O. glabra. The range of variation for the traits between two populations was established. A clear morphological difference of O. almaatensis and O. glabra was found in peduncle length to leaf length ratio. This was in O. almaatensis 1.56, while in O. glabra, it was 1.0.ConclusionsThe study provides the first phenotypic description and phylogenetic placement of the rare endemic species O. almaatensis. The morphological traits in two O. almaatensis populations showed a high level of phenotypic variability. Although clearly different from O. glabra, the ITS phylogeny grouped these species in a subclade within the genus.

Analysis of the relationship between ribosomal DNA ITS sequences and active components in Rhodiola plants.

Rhodiola plants are a valuable resource in traditional Chinese medicine. The objective of this study was to evaluate the correlation between ribosomal DNA internal transcribed spacer (ITS) sequences and the three active components in Rhodiola plants. For this, we determined ITS sequence polymorphisms and the concentrations of active components salidroside, tyrosol, and gallic acid in different Rhodiola species from the Tibetan Plateau. In a total of 23 Rhodiola samples, 16 different haplotypes were defined based on their ITS sequences. Analysis of the active components in these same samples revealed that salidroside was not detected in species with haplotypes H4, H5, or H10, tyrosol was not detected with haplotypes H3, H5, H7, H10, H14, or H15, and gallic acid was detected in with all haplotypes except H14 and H15. In addition, the concentrations of salidroside, tyrosol and gallic acid varied between samples with different haplotypes as well as those with the same haplotype, implying that no significant correlation exists between haplotype and salidroside, tyrosol or gallic acid concentrations. However, a statistically significant positive correlation was observed for among these three active components.

Paratylenchus shenzhenensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Anthurium andraeanum in China.

Paratylenchus shenzhenensis n. sp. was collected from the rhizosphere soil of Anthurium andraeanum in Shenzhen, Guangdong Province, China. The new species is characterized by having a female with a small body (249-302 μm), well developed stylet (17-21 μm), rounded head with four submedian lobes and lip-region with a slight depression at the oral area, small post-vulval uterine sac with a few vestigial cells; male with body dorsally curved behind the cloacal opening, stylet absent, pharynx degenerate, prominent penial sheath; and juveniles with a stylet. It is morphologically similar to P. minutus. The internal transcribed spacer sequences of ribosomal DNA (ITS-rDNA) of the new species only have 72-73% identity with P. minutus, confirming its status as a separate species. The D2/D3 region of 28S ribosomal DNA (28S rDNA) and 18S small subunit ribosomal DNA (18S rDNA) from P. shenzhenensis n. sp. were also amplified and sequenced in this study.

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.

ITS 염기서열에 의한 한국산 담배풀속(Carpesium L.)의 계통분류학적 연구

한국산 담배풀속(Carpesium L.) 7분류군과 3개의 외군(Inula britannica L., Inula germanica L., Rhanteriopsis lannginosa(DC.) Rauschert)을 대상으로 유연관계를 파악하기 위하여 nuclear ribosomal DNA(nrDNA) 중 ITS(internal transcribed spacer) 지역의 계통분류학적 분석을 수행하였다. 계통분류학적 연구방법은 maximum parsimony, neighbor-joining와 maximum likelihood 방법을 사용하였다. 정렬된 계통분의 총 길이는 731 bp이며, ITS1, ITS2와 5.8S 부위의 길이는 각각 284~297 bp, 264~266 bp와 164 bp로 나타났다. 계통분류학 변이를 보이는 site는 111개로 확인 되었으며, 그 중 64개의 site가 계통학적으로 유효한 것으로 나타났고, ITS1 지역이 ITS2 지역보다 염기 변이가 다양하게 나타나는 것으로 확인되었다. 그 결과, 한국산 담배풀속은 단계통을 형성하였으며, 담배풀(C. abrotanoides L.)이 가장 기저부에 위치하였다. 여우오줌(C. macrocephalum Franch. & Sav.)와 두메담배풀(C. triste Maxim.)은 가까운 유연관계를 나타냈으며, 애기담배풀(C. rosulatum Miq.)와 천일담배풀(C. glossophyllum Maxim.) 그리고 좀담배풀(C. cernuum L.)와 긴담배풀(C. divaricatum Siebold & Zucc.)도 유연관계가 가깝게 나타났다. 이와 같은 결과로 담배풀속 nrDNA의 ITS 지역 염기서열에 기초한 분자 계통학적 연구는 계통분류를 이해하는데 유용한 방법으로 판단된다. Phylogenetic analyses were conducted to evaluate relationships of 7 taxa of Korean Carpesium including three outgroup (Inula britannica L., Inula germanica L., Rhanteriopsis lannginosa (DC.) Rauschert) by using ITS (internal transcribed spacer) sequences of nuclear ribosomal DNA. Phylogenetic studies used maximum parsimony, neighbor-joining and maximum likelihood methods analysis. The length of the ITS sequences was 731 bp, and the lengths of the ITS1, ITS2 and 5.8S regions were 284~297 bp, 264~266 bp and 164 bp, respectively. The total number of variable sites was 111 for the entire sequences, and a parsimony informative sites of 64 are valid. Base change appeared variously in ITS1 rather than in ITS2. As the result, Korean Carpesium were formed monophyletic group and C. abrotanoides situated as the most basal clade. The results show that C. macrocephalum is closely related with C. triste. C. rosulatum has the closest relationship with C. glossophyllum. C. cernuum is close to C. divaricatum. These results suggest that the ITS data used in this study could be useful for the phylogenetic analysis of Korean Carpesium.

Phylogeny and systematics of Indian Polygonum sensu lato in the subfamily Polygonoideae based on ITS sequences of nuclear ribosomal DNA

The nuclear ribosomal DNA internal transcribed spacer (ITS) sequences from 44 Indian Polygonum taxa were examined to investigate relationships among various sections proposed by earlier researchers. The maximum parsimony trees obtained from analysis of the ITS sequences suggested eight major groups of the Indian Polygonum spp. The relationships among different sections were largely congruent with those inferred from morphological characters as described by Hooker. Also, the treatment of the Persicaria suggested by Haraldson on the basis of anatomical characters proved to be nearly in line with that based on our molecular data. We provide a high resolution of phylogeny of the Himalayan Polygonum sensu lato and support merger of the section Amblygonon in the section Persicaria. Moreover, we made the first phylogenetic analysis of many of the less known Himalayan Polygonums, including Polygonum microcephalum, P. assamicum, P. recumbens, and P. effusum. Molecular differences were detected among Persicaria barbata collected from different geographical locations of India, although these were not differentiated at the morphological level.

Comparisons of ribosomal DNA-internal transcribed spacer sequences and biological features among Olpidium bornovanus isolates from Cucurbitaceae-cultivating soil in Japan

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.

Morphology, Molecular Phylogeny and Pathogenicity of Colletotrichum panacicola Causing Anthracnose of Korean Ginseng

Colletotrichum panacicola isolates were obtained from anthracnose lesions of Korean ginseng and compared with four Colletotrichum species in morphology, molecular phylogeny and pathogenicity. Based on morphological characteristics, C. panacicola was easily distinguished from Colletotrichum gloeosporioides but not from Colletotrichum higginsianum, Colletotrichum destructivum and Colletotrichum coccodes. A phylogenetic tree generated from ribosomal DNA-internal transcribed spacer sequences revealed that C. panacicola is remarkably distinguished from C. gloeosporioides and C. coccodes but not from C. higginsianum and C. destructivum. However, molecular sequence analysis of three combined genes (actin + elongation factor-<TEX>$1{\alpha}$</TEX> + glutamine synthatase) provided sufficient variability to distinguish C. panacicola from other Colletotrichum species. Pathogencity tests showed that C. panacicola is pathogenic to Korean ginseng but not to other plants. These results suggest that C. panacicola is an independent taxon distin-zguishable from C. gloeosporioides and other morphologically similar Colletotrichum species.

Phylogenetic Relationships of Sugarcane Related Genera and Species Based on ITS Sequences of Nuclear Ribosomal DNA

Sugarcane related genera and species are important germplasm resources in sugarcane breeding. The internal transcribed spacer (ITS) sequence of 120 Saccharinae accessions that belong to 8 genera and 37 species were used to analyze their genetic relationships and construct phylogentic relationships with Pennisetum purpureum as an outgroup. The results showed that the sequence lengths were 200–208 bp for ITS1, 215–221 bp for ITS2, and 164 bp for 5.8sDNA with variable sites of 91, 93, and 18, respectively. The informative sites were 70, 68, and 9 for ITS1, ITS2, and 5.8sDNA, and the GC contents were 60.4%–69.1%, 66.1%–73.4%, and 54.1%–58.0%, respectively. According to the ratio of the variable sites and the ratio of informative sites to all sites, ITS sequence had richer variance than 5.8sDNA sequence, and the variance of ITS1 sequence was richer than that of ITS2 sequence. The result of genetic distance analysis revealed that that Miscanthus and Triarrhena were the closest genera to Saccharum, followed by Erianthus and Narenga, and genera Microstegium, Spodiopogon, and Imperata represented the furthest relationships with Saccharum. In the phylogenic tree constructed using neighbor-joining and maximum parsimony methods, the sugarcane related genera and species were separated into 10 groups. Erianthus arundinaceus should be classified into Erianthus genus rather than in Saccharum genus. Triarrhena is likely to be involved in Miscanthus. The 2 species from Narenga were sepearted in different groups, of which N. porphyrocoma was grouped with E. rockii, and N. allax was in an independent group. The Erianthus and Miscanthus species went to different groups, indicating the complex genetic relationships within genera.

Phylogeny of Isatis (Brassicaceae) and allied genera based on ITS sequences of nuclear ribosomal DNA and morphological characters

Systematics of the genus Isatis (Brassicaceae) is difficult and controversial, and previous studies were based solely on morphological characters. Sequence variation of the internal transcribed spacer (ITS) regions and the 5.8S gene of nuclear ribosomal DNA (nrDNA) were analyzed using parsimony and Bayesian methods. Twenty-eight taxa of Isatis and related genera of the tribe Isatideae were sampled, including 20 Isatis species representing almost all major morphological lineages, all three species of Pachypterygium, two of nine species of Sameraria, and monospecific Boreava, Myagrum, and Tauscheria. Two well-supported clades were resolved in the ITS tree, and they demonstrate the artificiality of the present delimitation of the tribe. One clade includes I. emarginata, I. minima, I. trachycarpa, P. brevipes, P. multicaule, P. stocksii, and T. lasiocarpa. The second clade includes I. buschiana, the polymorphic I. cappadocica with five subspecies, I. gaubae, I. kotschyana, I. leuconeura, I. pachycarpa, I. takhtajanii, I. tinctoria, and S. armena. Pachypterygium is polyphyletic and, together with Boreava, Sameraria, and Tauscheria, all are nested within Isatis. This study is a continuation of our recent systematic survey based on seed-coat microsculpturing ( Moazzeni et al., 2007. Flora 202, 447–454) and reveals that fruit characters mapped onto the molecular tree show considerable convergence. The reliance on fruit characters alone in the delimitation of genera may well lead to erroneous phylogenetic results and thus to incorrect taxonomic conclusions.

Hybrid status of Tibouchina urvilleana Cogn. revealed by ITS sequences of nuclear ribosomal DNA

Tibouchina urvilleana Cogn. is native to southern Brazil and currently cultivated as an important ornamental shrub in frost free areas around the world. Its rapid vegetative growth and sterility suggests that it might be of hybrid origin. In this study, Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA and chloroplast trnL- trnF spacer from T. urvilleana and three other congeneric species were sequenced to test its hybrid status. Cloning sequencing revealed two distinct types of ITS sequences from T. urvilleana, with one type almost identical with Tibouchina aspera. Genetic distance between the two types was much larger than the average interspecific genetic distances calculated from other Tibouchina species. Sequencing of chloroplast trnL- trnF spacer showed that T. urvilleana has identical sequence with T. aspera, but differed from other congeneric species by one nucleotide substitution and two indels. Molecular data demonstrated clearly that T. urvilleana indeed was a hybrid, with T. aspera or closely related species acting as the maternal parent.

Taxonomic study of Angelica from East Asia: Inferences from ITS sequences of nuclear ribosomal DNA

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.

Molecular Distinctions Between Phytophthora capsici and Ph. tropicalis Based on ITS Sequences of Ribosomal DNA

AbstractAmong four species of Phytophthora tested, only Ph. capsici and Ph. tropicalis showed the same length for DNA sequence for both internal transcribed spacer (ITS)1 and ITS2 of ribosomal DNA. Phytophthora palmivora and P. nicotianae have lengths different from each other, and from the other two species. Although A1 and A2 types of Ph. capsici differ from each other by only one nucleotide, there are 10 different nucleotides between A1 and A2 types of Ph. tropicalis. Phylogenetic analysis of combined ITS sequences identified four clades each consisting A1 and A2 mating types of same species. The neighbor‐joining and maximum parsimony trees show that Ph. tropicalis (A2) is clustered with the clade of two isolates of Ph. capsici before joining the clade of A1 and two other isolates of Ph. tropicalis from GenBank. Our results support the separation of Ph. tropicalis and demonstrate the need to sequence more than a single isolate of a species in the study of molecular phylogeny of Phytophthora. The phylogenetic trees also suggest that Ph. tropicalis (A2) may represent a transitional isolate in the process of species evolution.

Phylogenetic relationships of Mangifera species revealed by ITS sequences of nuclear ribosomal DNA and a possibility of their hybrid origin

The phylogenetic relationships among 14 Mangifera L. species of Thailand were analyzed by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Parsimony and neighbor joining (NJ) analyses revealed that the common mango (M. indica L.) was closely related to M. laurina Bl., M. sylvatica Roxb., and M. oblongifolia Hook. f. Mangifera foetida Lour. and M. odorata Griff. were also related to M. indica in both parsimonious and NJ trees, although these two species are classified into a different subgenus (subgenus Limus) from the subgenus Mangifera to which M. indica belongs. ITS sequence analysis revealed that several species have nucleotide additivity (two different nucleotides at the same locus) at several sites in the ITS region. Also, M. indica had several polymorphisms among cultivars. This finding may suggest a possibility of hybrid origin of Mangifera species, although Mangifera species are all assumed to be diploid having chromosome number of 2n=2x=40.

Phylogeography of the ectomycorrhizal Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences

Summary The fungal genus Pisolithus is cosmopolitan in warm temperate regions and forms ectomycorrhizal associations with a wide range of woody plants. To delimit phylogenetic Pisolithus species and identify their geographical distribution, 102 collections were made worldwide and their rDNA internal transcribed spacer (ITS) was sequenced. Phylogenetic analyses of these sequences, together with 46 additional GenBank accessions, identified 11 species. A strong phylogeographical pattern was observed related to the native geographical origin of the host plants. In the Holarctic, P. tinctorius was widely distributed, associated with Pinus and Quercus. It has been co‐introduced with pines to other biogeographical regions. Several Pisolithus lineages, including P. aurantioscabrosus, occurred in restricted biogeographical regions associated with endemic plants, such as Afzelia in eastern Africa. Pisolithus albus, P. marmoratus and P. microcarpus were associated with Australasian hosts (Eucalyptus, Acacia) and were distributed with their hosts worldwide. By contrast, two additional unnamed species were restricted to Australia. The present study shows that evolutionary lineages within Pisolithus are related to the biogeographical origin of the hosts. In addition, regional floras and endemic plants could act as hosts of endemic species of Pisolithus.

THE PHYLOGENY OF CAULERPA BASED ON RDNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Phylogenetic hypotheses for the pantropical marine green algal genus, Caulerpa, were inferred based on analyses of nuclear‐encoded rDNA internal transcribed spacer (ITS) sequences. Results of these analyses were used to assess the correspondence between rDNA phylogeny and traditional sectional taxonomy, to identify synapomorphic morphological characters (including assimilator morphology and chloroplast ultrastructure), and to examine marine biogeographic hypotheses for the genus. Ribosomal DNA ITS sequences were aligned for thirty‐three species and intraspecific taxa of Caulerpa. Results indicate limited correspondence between phylogeny and sectional taxonomy for the genus, (e.g., the sections Filicoideae and Sedoideae were not monophyletic). In contrast, chloroplast morphology could be mapped to the tree topology with limited homoplasy. Pantropical isolates of the filicoidean species, Caulerpa sertularioides and Caulerpa mexicana each formed monophyletic groups. Caulerpa reyesii was included as a derived taxon within the Caulerpa taxifolia clade, suggesting that these species were conspecific and affirmed the lack of correspondence between phylogeny and assimilator morphology. Isolates and various intraspecific taxa of Caulerpa racemosa did not form a monophyletic group. Instead, these taxa formed a heterogeneous assemblage with other sedoidean and filicoidean taxa. Within the C. sertularioides clade, Caribbean and Atlantic isolates formed a basal paraphyletic group, whereas eastern and western Pacific isolates formed a more derived monophyletic group. Therefore, these results are not consistent with an Indo‐West Pacific origin of this species.

Biogeographic patterns in Mediterranean and Macaronesian species of Saxifraga (Saxifragaceae) inferred from phylogenetic analyses of ITS sequences

A biogeographic study of Saxifraga section Saxifraga was performed based on phylogenetic analyses of ITS (internal transcribed spacer) sequences of nuclear ribosomal DNA. ITS sequences from 21 species and 31 populations were examined to identify colonization patterns for the two species of Saxifraga occurring in Macaronesia and for S. globulifera in the west Mediterranean basin. Phylogenetic analysis of the sequence data yield a single most parsimonious tree with many of the major clades well supported by bootstrap and decay values. The ITS tree provided resolution at specific and populational levels that points to two biogeographic patterns within the genus. In contrast to the molecular evidence provided by other authors for a Mediterranean origin of several Macaronesian genera of angiosperms, our results indicate that the Madeiran archipelago was colonized a single time by a species of Saxifraga originating from the Eurosiberian region. On the other hand, the molecular evidence also suggests that populations of S. globulifera from North Africa have been isolated for a long time from populations occurring in the Iberian Peninsula, and that the endemic S. reuteriana has evolved from the Iberian populations of S. globulifera. The Mediterranean Sea has probably been an effective isolating barrier for some plant groups that occur in Europe and North Africa.