The phylogeny of Schistidium (Bryophyta, Grimmiaceae) based on primary and secondary structure study of nuclear rDNA internal transcribed spacers

The phylogeny of Schistidium (Bryophyta, Grimmiaceae) was studied on the basis of nucleotide sequences of internal transcribed spacers ITS1-2 of nuclear DNA and trnT-trnD region of chloroplast DNA. The consistency of phylogenetic trees constructed from nuclear and chloroplast sequences was shown. A basal grade and two large clades were resolved on the phylogenetic trees. Morphological characteristics specific for these clades were described. ITS1 and ITS2 secondary structures of Schistidium species were modeled using thermodynamic criteria. Four different structures of the longest ITS1 hairpin were identified. Possible paths of Schistidium evolution were considered based on the four types of ITS1 secondary structure and phylogenetic trees.     

Molecular phylogeny of Osmanthus(Oleaceae)based on non-coding chloroplast and nuclear ribosomal internal transcribed spacer regions

The phylogenetic relationships of Osmanthus Lour.were investigated using the nuclear ribosomal internal transcribed spacer(ITS)regions and non-coding chloroplast regions(psbA-trnH,trnL-F).The two datasets support the conclusion that Osmanthus is polyphyletic.with some species of the subtribe Oleinae nested within Osmanthus.Osmanthus didymopetalus P S.Green is nested within the clade formed by species of section Osmanthus in two trees.Osmanthus attenuatus P.S.Green.O.Funnanensis P.S.Green,and O gracilinervis R.L.Lu of traditional section Osmanthus are clearly divergent from other accessions,and do not form a monophyletic group with other Osmanthus accessions.Osmanthus marginatus Hemsl.is embedded in the clade formed by species of section Osmanthus in the ITS tree.In cpDNA trees all species of section Osmanthus are placed in the large clade and all species of section Leiolea formed a group.The taxonomic incongruence among trees for ITS and cpDNA indicate hybridization.as introgression may have occurred among some species of sections Osmanthus and Leiolea.Phylogeny of Osmanthus is discussed in light of molecular and morphological data,and a revised infrageneric classification with three sections(Leiolea,Siphosmanthu,and Osmanthus)is presented.The section Linocieroides is abandoned and united with section Osmanthus.     

Molecular phylogeny of Pottiaceae (Musci) based on chloroplast rps4 sequence data

Comparative sequencing of the chloroplast rps4 gene was used to reconstruct the phylogenetic relationships within the family Pottiaceae (Musci). The results confirm that Ephemerum spinulosum, Splachnobryum obtusum, Goniomitrium acuminatum and Cinclidotus fontinaloides are clearly positioned within the Pottiaceae and that Hypodontium dregei is not a member. At subfamily level, the data support the subfamily Pottioideae as being a monophyletic clade. The Trichostomoideae are probably paraphyletic. Neither the subfamily Chionolomideae, represented in this study by Pseudosymblepharis schimperiana, nor the subfamily Erythrophyllopsoideae, represented by both known species, Erythrophyllastrum andinum and Erythrophyllopsis fuscula, are supported by the sequence data. The Timmielloideae should be excluded from the Pottiaceae. The Merceyoideae, represented in this study by Scopelophila cataractae, might form a sister clade to all other Pottiaceae, but their position is not fully resolved. At the genus level, Barbula is clearly polyphyletic since Barbula bolleana and Barbula indica appear in a clade clearly separated from Barbula unguiculata. Pottiopsis caespitosa and Leptobarbula berica are placed within the Trichostomoideae. Likewise, the genera Gymnostomum and Anoectangium are excluded from the Pottioideae and placed within the Trichostomoideae. Leptophascum leptophyllum is closely related with Syntrichia; Aloina is not closely related to Tortula or Crossidium. Evidence of a clade within the Pottioideae, formed of Leptodontium and Triquetrella, is provided.     

Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA ofSaccharomyces

Polymorphism in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains ofSaccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding toS. bayanus andS. cerevisiae. The type culture ofS. carlsbergensis (ATCC 76529), which could not be separated from theS. cerevisiae group by small subunit (SSU) rDNA patterns, showed aScrfI profile that was distinct from all the other strains. The type culture ofs. intermedius var.turicensis (ATCC 76519) is assigned toS. bayanus on the basis of the combined results of SSU and ITS restriction analyses.S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.     

Molecular characterization of the non-costate morphotypes of buliminid foraminifers based on internal transcribed region of ribosomal DNA (ITS rDNA) sequence data

We performed molecular phylogenetic analyses of four morphotypes of the benthic foraminiferal genus Bulimina (B. aculeata, B. marginata f. marginata, B. marginata f. denudata, and B. elongata) based on sequences of internal transcribed spacer region of ribosomal DNA (ITS rDNA). Six genetically distinct phylotypes were revealed by our phylogenetic analyses. The six phylotypes basically correspond to the fundamental morphotypes: clades A + B (B. aculeata); clade C (B. elongata); clade D (B. marginata f. denudata); clade E (B. marginata f. marginata genotype 1); and clade F (B. marginata f. marginata genotype 2). All six phylotypes are well distinguished, except phylotype B, which shows only little sequence divergence compared to clade A, possibly indicating that genetic differentiation is in progress. Morphological characters including the direction, placement, and shape of spines, the angle of undercutting of chamber periphery, and the roundness of the chambers were stable among specimens of each clade. In contrast, the length and density of spines, and chamber size, were variable within each clade. These intermediate morphological characters may reflect ecophenotypic variation. Our study clearly shows that the examined B. acuelata, elongata, and denudata morphospecies are genetically separated and that B. marginata is a species complex comprising several genotypes. A novel phylotype represent different morphotype compare to B. marginata f. marginata that can be distinguished based on differences of chamber angularity, the direction, placement, and shape of spines, and test dimensions.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

Molecular phylogeny of silk producing insects based on internal transcribed spacer DNA1

Silk moths are the best studied silk secreting insects and belong to the families Bombycidae and Saturniidae. The phylogenetic relationship between eleven silk producing insects was analyzed using the complete DNA sequence of the internal transcribed spacer DNA 1 locus. The PCR amplification and sequence analysis showed variation in length ranging from 138 bp (Antheraea polyphemus) to 911 bp (Hyalopora cecropia). Microsatellite sequences were found and was be used to distinguish Saturniidae and Bombycidae members. The nucleotide sequences were aligned manually and used for construction of phylogenetic trees based on Maximum parsimony and Maximum likelihood methods. The topology in both the approaches yielded a similar tree that supports the ancestral position of the Antheraea assama.     

Molecular evolution of rDNA external transcribed spacer and phylogeny of sect. Petota (genus Solanum)

The 5(') external transcribed spacer (ETS) region of ribosomal DNA of 30 species of Solanum sect. Petota and the European Solanum dulcamara were compared. Two structural elements can be distinguished in the ETS: (i). a variable region (VR), demonstrating significant structural rearrangements and (ii). a conservative region (CR), evolving mainly by base substitutions. In VR, a conservative element (CE) with similarity to the ETS of distantly related Nicotiana is present. The ancestral organization of ETS (variant A) was found for non-tuber-bearing species of ser. Etuberosa, tuber-bearing wild potatoes of Central American ser. Bulbocastana, Pinnatisecta, and Polyadenia and S. dulcamara. Duplication of CE took place in the ETS of species from ser. Commersoniana and Circaeifolia (variant B). South American diploids and Mexican polyploids from superser. Rotata also possess two CE, and additionally two duplications around CE1 are present in VR (variant C). Three major lineages could be distinguished: non-tuber-bearing species of ser. Etuberosa, tuber-bearing Central American diploids and all South American species radiated from a common ancestor at early stages of evolution, indicating a South American origin of the tuber-bearing species. Later, Central and South American diploids evolved further as independent lineages. South American species form a monophyletic group composed of series with both stellata and rotata flower morphology. Solanum commersonii represents a sister taxon for all rotata species, whereas ser. Circaeifolia diverged earlier. Two main groups, C1 and C2, may be distinguished for species possessing ETS variant C. C1 contains ser. Megistacroloba, Conicibaccata, Maglia, and Acaulia, whereas all diploids of ser. Tuberosa are combined into C2. A closer relationship of Solanum chacoense (ser. Yungasensa) to the C2 group was found. The origin of polyploid species Solanum maglia, Solanum acaule, Solanum tuberosum, Solanum iopetalum, and Solanum demissum is discussed.     

Nucleotide sequence and presumed secondary structure of the internal transcribed spacers of rDNA of the pea aphid, Acyrthosiphon pisum.

1. Internal transcribed spacer (ITS) 1 and ITS 2 of rDNA of the pea aphid, Acyrthosiphon pisum consisted of 229 and 280 nucleotides, whose G+C contents were 70 and 74%, respectively. 2. Secondary structure models constructed for the ITS 1 and ITS 2 suggested that certain structural motifs have been conserved in these regions despite extensive divergence in nucleotide sequence due to species.     

A phylogeny of Packera (Senecioneae; asteraceae) based on internal transcribed spacer region sequence data and a broad sampling of outgroups

A phylogeny of Packera is presented based on sequence data from the internal transcribed spacer region of nuclear ribosomal DNA of 26 species (28 populations) of Packera and 23 outgroup taxa, including representatives of all three subtribes of the Senecioneae. The results support a Mexican origin for Packera, with its closest relatives found among Old World taxa in the subtribe Senecioninae, such as Senecio jacobaea and Pericallis. Packera species from the west coast of the United States, previously included in the section Bolanderi of Greenman, are part of a basal assemblage including species of Greenman's Mexican section Sanguisorboidei. The rest of Packera separates into two sister groups, one containing species from the Arizona-New Mexico region and the other containing more geographically diverse taxa. Among the outgroups, New World Senecio species are monophyletic and two Tussilaginoid assemblages are strongly supported; the Tephroseroid group (Tephroseris and Sinosenecio) plus Petasites combine with the Luina complex to form a clade of north temperate taxa, and the four Mexican genera (Psacalium, Robinsonecio, Barkleyanthus, and Pittocaulon) form a monophyletic group.     

Molecular phylogeny of Dipterocarpaceae in Indonesia based on chloroplast DNA

In order to construct a molecular phylogeny of Indonesian Dipterocarpoideae (Dipterocarpaceae), PCR-RFLP of the chloroplast regions rbcL, petB, psbA, psaA, and trnL-F was performed with seven restriction enzymes in 129 samples including 58 species from nine genera. In the strict consensus tree with Monotes kerstingii as outgroup Indonesian Dipterocarpaceae were divided into two major clades. One clade (bootstrap value=71) consisted of Upuna, Cotylelobium, Anisoptera, Vatica, Dipterocarpus (tribe Dipterocarpeae, bootstrap value=83) and Dryobalanops (tribe Shoreae, bootstrap value=99) in a basal position. The second clade consisted of Hopea, Parashorea, and Shorea (tribe Shoreae) with 95% bootstrap support. Tribe Dipterocarpeae is monophyletic, tribe Shoreae is polyphyletic since Dryobalanops is sister to tribe Dipterocarpeae. In the neighbour-joining tree the sister group position of Dryobalanops to tribe Dipterocarpeae is not supported by the bootstrap analysis. Alternatively, we used Upuna borneensis as outgroup. The effect of outgroup selection on tree topology, taxonomic classification and the interpretation of character evolution is discussed.     

Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data

Lathyrus (Leguminosae; Papilionoideae) is the largest genus in tribe Fabeae and exhibits an intriguing extratropical distribution. We studied the systematics and biogeography of Lathyrus using sequence data, from accessions representing 53 species, for the internal transcribed spacer plus 5.8S-coding region of nuclear ribosomal DNA as well as the trnL-F and trnS-G regions of chloroplast DNA. Our results generally supported recent morphology-based classifications, resolving clades corresponding to sections Lathyrus and Lathyrostylis, but question the monophyly of the large, widespread section Orobus sensu Asmussen and Liston. Sections Orobus, Aphaca, and Pratensis form a predominantly northern Eurasian-New World clade. Within this clade, the North American and eastern Eurasian species, including both Holarctic species (L. palustris and L. japonicus), form a transberingian clade of relatively recent origin and diversification. The South American Notolathyrus group is distant from this transberingian lineage and should be reinstated as a distinct section within the northern Eurasian-New World clade. The Notolathyrus lineage reached the New World most probably through long-distance dispersal from Eurasia. The remaining sections in the genus are centered on the Mediterranean region.     

Additional data for a new Theileria sp. from China based on the sequences of ribosomal RNA internal transcribed spacers

Theileria sinensis was recently isolated and named as an independent Theileria species that infects cattle in China. To date, this parasite has been described based on its morphology, transmission and molecular studies, indicating that it should be classified as a distinct species. To test the validity of this taxon, the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were cloned and sequenced from three T. sinensis isolates. The complete ITS sequences were compared with those of other Theileria sp. available in GenBank. Phylogenetic analyses based on sequence data for the complete ITS sequences indicate that T. sinensis lies in a distinct clade that is separate from that of T. buffeli/orientalis and T. annulata. Sequence comparisons indicate that different T. sinensis isolates possess unique sizes of ITS1 and ITS2 as well as species-specific nucleotide sequences. This analysis provides new molecular data to support the classification of T. sinensis as a distinct species from other known Theileria spp. based on ITS sequences.     

Molecular identification of Chinese cultivated Porphyra (Bangiaceae, Rhodophyta) based on the rDNA internal transcribed spacer-1 sequence and random amplified polymorphic DNA markers

Porphyra yezoensis Ueda and P. tenera Kjellman are two common commercial seaweeds cultivated in China, but it is difficult to identify them routinely because they are morphologically and systematically very close. The nuclear ribosomal internal transcribed spacer-1 (ITS-1) region of nine selected Porphyra cultivars from China were sequenced and determined. Combined with some ITS-1 data from GenBank, the phylogenetic analysis inferred from the neighbour-joining method indicated that the Porphyra species in this study exhibited clear taxonomic relationships and could be identified clearly to species. Based on the results, the random amplified polymorphic DNA (RAPD) technique was applied to distinguish 12 Porphyra cultivars, some of which turn out to be different lineages of the same Porphyra species. Eight specific RAPD markers were scored and used to construct a fingerprint that could distinctly identify different Porphyra cultivars. The results suggest that both the rDNA ITS-1 sequence and RAPD markers are useful methods to identify Porphyra cultivars, and may also be valid for phylogenetic and taxonomic studies.     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

Phylogeny of the genus trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species of Trichoderma sect. Pachybasium. To this end, 85 strains-including all the available ex-type strains-were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 including T. polysporum, T. piluliferum, and T. minutisporum, while A2 included T. hamatum, T. pubescens, and T. strigosum in addition to species previously included in sect. Trichoderma (i.e., T. viride, T. atroviride, and T. koningii). The ex-type strain of T. fasciculatum formed a separate branch basal to clade A. Clade B contained the sect. Pachybasium members T. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale, and the anamorphs of Hypocrea semiorbis and H. cf. gelatinosa. Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them and T. longibrachiatum, which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e., T. virens and T. flavofuscum; T. strictipile and H. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g., T. crassum and T. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile, and H. semiorbis; T. hamatum and T. pubescens; and T. viride, T. atroviride, and T. koningii) differences were in the range of 1-3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group of T. harzianum strains (Th2) was strikingly different from that of the ex-type strain of T. harzianum and closely related species and is likely to be a separate species. Copyright 1998 Academic Press.     

Nucleotide sequence of intergenic and external transcribed spacers of rDNA of diploid wheat Triticum urartu Thum. ex Candil

The primary structure of intergenic non-transcribed and external transcribed spacers of rDNA of diploid wheat Triticum urartu, cloned in pTu3 plasmid 2402 b.p. long was determined. The intergenic non-transcribed rDNA spacer of Tr. urartu was shown to consist of 8 subrepeats with an average of 133 b.p. long, heterogeneous in length and nucleotide sequence. A number of repeated sequences was revealed within each subrepeat. While comparing nucleotide sequences of rDNA subrepeats of Tr. urartu and Tr. aestivum a high homology was found (up to 82%). A high similarity between these plant species was also found in the promoter region and in the external transcribed rDNA spacer. Suppression of the nucleolar organizer of 1A chromosome in the presence of 1B and 6B chromosomes of Tr. aestivum is supposed to be connected with the existence of a great number of subrepeats in the intergenic non-transcribed rDNA spacer of B genome donors in polyploid wheat species of turgidum-aestivum row.     

The phylogeny of <Emphasis Type="Italic">Schistidium</Emphasis> (Bryophyta,Grimmiaceae) based on the primary and secondary structure of nuclear rDNA internal transcribed spacers

Phylogeny of Schistidium (Bryophyta, Grimmiaceae) was studied by comparing the nucleotide sequences of internal transcribed spacers ITS1-2 of nuclear rDNA and the trnT-trnD region of chloroplast DNA. Phylogenetic trees constructed based on nuclear and chloroplast sequences were consistent, comprising a basal grade and two large clades. Morphological characteristics specific for these clades were described. Secondary structures of ITS1 and ITS2 Schistidium species were modeled using thermodynamic criteria. Four different structures of the longest ITS1 hairpin were identified. These results were used to analyze possible paths of Schistidium evolution. Characteristics of the ITS2 secondary structure support the two major clades recognized in the phylogenetic trees.