Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

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.     

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 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.     

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

Abstract 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. yunnanensis 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 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.     

Molecular phylogeny and evolution of Monilinia (Sclerotiniaceae) based on coding and noncoding rDNA sequences

The nuclear internal transcribed spacers, the 5.8S subunit, ~560 bp of the small subunit, and ~320 bp of the large subunit of the nuclear ribosomal DNA repeat from 17 species of Monilinia and eight species of closely related genera were sequenced. Phylogenies were constructed using maximum parsimony. The results support the hypothesis that Monilinia is not monophyletic. A fundamental distinction was found between the section Junctoriae and the section Disjunctoriae. Four evolutionary lineages were identified within the Disjunctoriae: one species on Crataegus, one group of species on dry stone fruits of rosaceous hosts, one group of species on capsular fruits of ericaceous hosts, and one group of species on sweet berry fruits of ericaceous hosts. Comparisons between branching topologies of hosts and Monilinia species suggest that although cospeciation among hosts and parasites has been the rule, several host jumps have taken place. Sclerotinia pirolae was determined to be a true member of the Disjunctoriae. The closest taxon groups to the Junctoriae were found to be Botrytis and Sclerotinia, with Ciborinia being the closest taxon group to the Disjunctoriae. There is evidence of an increased rate of ssrRNA evolution in the lineage of species that attack ericaceous berries.     

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.     

rDNA internal transcribed spacer sequence analysis of Craterellus tubaeformis from North America and Europe

The basidiomycete Craterellus tubaeformis (Fries) Quélet is an important widespread ectomycorrhizal basidiomycete found in the Northern Hemisphere. In this study, 12 samples of C. tubaeformis from North America and Europe were analyzed using internal transcribed spacer (ITS) sequences to reveal the correlation between ITS genotypes and geographic locations and to provide molecular evidence for the identification of C. tubaeformis from different habitats in North America and Europe. The analyses identified abundant sequence variations within C. tubaeformis. The length of the ITS region varied from 571 to 640 bp. The proportion of variable sites was 17.6%, and the proportion of parsimony information sites was 16.7%. Phylogenetic analysis showed some correlations between the ITS genotypes and geographic locations of C. tubaeformis; however, some discrepancies between geographical location and affinity were also found. The results indicated that C. tubaeformis from different habitats in North America and Europe underwent genetic drifting and evolved into 2 different species. nrDNA ITS could be a good markers for distinguishing among C. tubaeformis from different habitats, but rational affinity should be determined by associating the available ITS data with other information sources.     

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.     

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.