A phylogenetic analysis of the Illiciaceae based on sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were determined for 15 species ofIllicium (Illiciaceae) to examine phylogenetic relationships. The ITS trees show a major dichotomy between the two North American species (I. floridanum andI. parviflorum) and the remaining east Asian species. This suggests that the existing division between two sections (sect.Illicium and sect.Cymbostemon) ofIllicium based on tepal characters in unnatural. The ITS phylogeny shows congruence with palynology: of the species examined, the three species (I. angustisepalum, I. anisatum andI. fargesii) from sect.Illicium that possess trizonocolpate pollen consistently form a clade, although nesting within a clade consisting of the species of sect.Cymbostemon, which generally have trisyncolpate pollen. The low ITS sequence divergence and the close relationship among east Asian species suggest a recent diversification of this group of species or an unusual slowdown of sequence mutations.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Infrageneric phylogeny of the genus Gentiana (Gentianaceae) inferred from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacers (ITSs) of nuclear ribosomal DNA have been sequenced for 20 species of Gentiana. By incorporating previously released sequence data of eight species, phylogenelic analyses using Fitch parsimony and character-state weighted parsimony were carried out. The length of ITS 1 in the taxa surveyed ranged from 223 to 238 bp and ITS2 from 216 to 234 bp. Sequence divergence between pairs of species ranged from 5.0% to 48.9% in ITS1, from 1.1% to 45.3% in ITS2, and from 3.2% to 46.1% in combined data of ITS1 and ITS2. The ITS phylogeny was generally congruent with morphological classifications except that G. asclepiadea was revealed to be closely related to section Gentiana instead of section Pneumonanthe and section Stenogyne was shown to be a paraphyletic group of the genus Gentiana that would be better excluded from the genus. A divergence among the three European endemic sections and the remaining sections of the genus other than section Stenogyne was revealed. Thus the European species of the genus together do not form a monophyletic group. A close relationship between the sections Chondrophyllae s. l. (including section Dolichocarpa), Cruciata and Pneumonanthe was suggested. The section Frigidae s. l. (including sections Monopodiae, Isomeria, Microsperma, and Phyllocalyx) contained two well-supported clades: section Frigidae s. str. and all others together. The monophyly of the typically dysploid group section Chondrophyllae s. l. was confirmed. Optimization of chromosome numbers on the ITS phylogeny suggested that 2/1 = 26 is a plesiomorphic state for the clade comprising sections Frigidae s. l., Cruciata, Pneumonanthe, and Chondrophyllae s. l., and probably 2n = 20 is a plesiomorphic state for the dysploid group, section Chondrophyllae s. l.     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

GC balance in the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes

Summary The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, andCucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5 region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes.The two investigated Cucurbitaceae species show a G+C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G+C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G+C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G+C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.     

Phylogenetic relationships in the Lejeuneaceae (Hepaticae) inferred using ITS sequences of nuclear ribosomal DNA

Sequences of the ITS1–5.8S–ITS2 region of nuclear ribosomal DNA were generated for 12 species from 9 genera of Lejeuneaceae and a single species of Jubulaceae (outgroup). The taxon sampling of Lejeuneaceae included representatives of the two widely recognized subfamilies, Lejeuneoideae and Ptychanthoideae. The molecular dataset was analysed independently and in combination with a morphological dataset. The nrITS dataset and the combined dataset resulted in identical topologies. The genus Bryopteris, sometimes treated as a separate family Bryopteridaceae, is nested within the Lejeuneaceae subfamily Ptychanthoideae. Lejeuneaceae subfamily Lejeuneoideae proved to be paraphyletic with the tribe Lejeuneeae sister to Ptychanthoideae, albeit without significant bootstrap support. The tribes Brachiolejeuneeae and Cheilolejeuneeae of Lejeuneoideae, established recently based on morphological evidence, are well supported in bootstrap analyses both of the ITS and the combined molecular–morphological datasets. The results support classifications of Lejeuneaceae based on morphological data and demonstrate the usefulness of the ITS region for phylogenetic studies within or among closely related genera of Lejeuneaceae.     

Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

The genus Alyssum consists of about 195 species native to Europe, Asia and northern Africa. All species were assigned to six sections. Previous molecular phylogeny studies indicate that Alyssum is polyphyletic. However, the divergence time and dispersal of the genus are not well studied. In this study, the phylogenetic relationships within the genus Alyssum were studied with nrDNA ITS sequences obtained from five sections. The divergence time was estimated by fossil calibration and the biogeography was examined by spread analysis. The phylogeny indicated two main lineages: lineage 1 includes the section of Alyssum, Gamosepalum and Psilonema; lineage 2 includes the section of Odontarrhena, Meniocus and Clypeola. The phylogenetic relationship was not congruent with the previous sectional classifications. The age of Alyssum was dated to the upper Miocene. Molecular data suggested the diversification of Alyssum in Mediterranean areas and wide-ranging distribution such as North Africa, eastward into Central Asia and immigration into North America. Climatic aridification and arid/semiarid areas established in the Pliocene/Pleistocene could have provided favourable conditions for the migration and diversification of Alyssum.     

Molecular phylogeny ofCheirolophus (Asteraceae:Cardueae-Centaureinae) based on ITS sequences of nuclear ribosomal DNA

The genusCheirolophus has an interesting western Mediterranean and Macaronesian distribution. Here we investigate the delimitation of the genus and its exclusion from the large genusCentaurea, the systematic position of the related genusPaleocyanus, the delimitation of some species and the phylogeny of the group. We have carried out a phylogenetic analysis of the PCR-generated sequences of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. The results suggest that the genus, includingPaleocyanus crassifolius is monophyletic; thus, a new combination of this species underCheirolophus is proposed. The Macaronesian group of species is also monophyletic, indicating a single colonization of the archipelago. The poor resolution of microspecies in the Macaronesian group reinforces the hypothesis of a very recent differentiation of the group.     

应用ITS序列分析探讨偏花报春的系统位置

对于偏花报春Primula secundiflora Franch. 的系统位置,主要有两种意见,一种认为偏花报春具有典型的钟状花冠,应置于钟花报春组Sect．Sikkimensis;而另一种意见则依据其他特征将其置于灯台报春组sect．Proliferae。通过对偏花报春、灯台报春组4种植物和钟花报春组3种植物核糖体DNA中的内转录间隔区(ITS)的序列测定及分析,并结合形态学及染色体特征的比较论证,认为偏花报春应置于灯台报春组。     

Phylogenetics of Miscanthus,Saccharum and related genera (Saccharinae,Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers

DNA sequences were used to assess the monophyly and inter-relationships of Miscanthus, Saccharum and related genera in the Saccharum complex. Three DNA regions were sequenced, including the trnL intron and the trnL-F intergenic spacer of the plastid genome and the ITS region of nuclear ribosomal DNA (nrDNA). Because it was more variable, the ITS region proved most suitable for phylogenetic reconstruction at this level, and the results indicate that Miscanthus s.l. and Saccharum s.l. are polyphyletic. A set of species from Saccharum section Ripidium (clade a) do not group closely with any members of Saccharum s.l.. A number of Miscanthus species from eastern or south-eastern Asia represent a monophyletic group with a basic chromosome number of 19 (clade b), but the other species from Africa and the Himalayas are clearly excluded. There is support for a monophyletic Saccharum s.s. clade including S. officinarum and S. spontaneum that is sister to Miscanthus s.s (clade c). There is no evidence to support the division of some Saccharum s.l. into the genera currently known as Erianthus and Narenga. Saccharum contortum (=Erianthus contortus), S. narenga (=Narenga porphyrocoma) and Erianthus rockii, group more closely with Miscanthus fuscus, a species from the Himalayas and also with the African Miscanthus s.l. species (=Miscanthidium, clade d). Electronic Publication     

Delimitation of Sauropus (Phyllanthaceae) based on plastid matK and nuclear ribosomal ITS DNA Sequence data

Background and Aims

A recent molecular phylogenetic study showed that Sauropus is deeply embedded within Phyllanthus together with its allies, Breynia and Glochidion. As relationships within Sauropus are still problematic and the relationship with Breynia has long been doubted, more molecular data are needed to test/corroborate such a broad definition of Phyllanthus. This study aims to clarify the status and delimitation of Sauropus and establish its position within Phyllanthaceae.

Methods

Plastid matK and nuclear ribosomal ITS DNA sequence data for Sauropus and its allies were used to construct phylogenetic trees using maximum parsimony and Bayesian methods.

Key Results

Within Phyllanthus, Sauropus can be split into the mainly south-east Asian Sauropus sensu stricto (s.s.) plus Breynia and the mainly Australian Sauropus (formerly Synostemon). Sauropus s.s. plus Breynia comprise two distinct clades; one is the combination of Sauropus sections Glochidioidei, Sauropus and Schizanthi and the other is the combination of Sauropus sections Cryptogynium and Hemisauropus and the monophyletic genus Breynia.

Conclusions

Molecular data indicate that Synostemon should be reinstated at the same level as Sauropus s.s. and that Sauropus s.s. should be united with Breynia under the latter, older name. The molecular data corroborate only two of the five infrageneric groups of Sauropus recognized on the basis of morphological data.Key words: Breynia, DNA sequence data, Euphorbiaceae, ITS, matK, molecular phylogenetics, Phyllanthaceae, Sauropus, Synostemon     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA

The phylogenetic relationships of the genus Sorghum and related genera were studied by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS). DNA was extracted from 15 Sorghum accessions, including one accession from each of the sections Chaetosorghum and Heterosorghum, four accessions from Parasorghum, two accessions from Stiposorghum, and seven representatives from three species of the section Sorghum (one accession from each of S. propinquum and S. halepense, and five races of S. bicolor). The maize (Zea mays) line, H95, and an accession from Cleistachne sorghoides were also included in the study. Variable nucleotides were used to construct a strict consensus phylogenetic tree. The analyses indicate that S. propinquum, S. halepense and S. bicolor subsp. arundinaceum race aethiopicum may be the closest wild relatives of cultivated sorghum; Sorghum nitidum may be the closest 2n=10 relative to S. bicolor, the sections Chaetosorghum and Heterosorghum appear closely related to each other and more closely related to the section Sorghum than Parasorghum; and the section Parasorghum is not monophyletic. The results also indicate that the genus Sorghum is a very ancient and diverse group.This research was partially supported by a Third Country Scholarship from Pioneer Hi-Bred International Incorporated Contribution 94-182-J from Kansas Agricultural Experiment Station     

Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences

The nuclear ITS region of 19 species of Alnus was amplified and sequenced. The inferred molecular phylogeny shows that all species of the genus Alnus form a monophyletic group close to Betula and that the fundamental dichotomy within the genus lies between the subgenera Alnaster and Gymnothyrsus, sensu Murai (1964). The subgenus Alnaster appears to be basal in the genus, based on archaism of morphological features, and branching close to the root of the trees due to low ITS divergence from genus Betula. The monophyly of the section Clethropsis is not supported by the present data: Alnus nepalensis is positioned in the subgenus Gymnothyrsus, away from A. nitida and A. maritima. Surprisingly, A. formosana sect. Japonicae is closely tied to A. maritima sect. Clethropsis, with which it shares few morphological traits, and is separate from A. japonica sect. Japonicae with which it shares many traits. An increase in substitution rate is noted in the group comprising A. formosana, A. maritima and A. nitida relative to the rest of the genus, which appears to have had, on the average, a very slow mutation rate. Alnus glutinosa, the designated type for the genus, appears to be representative of the genus both for morphological characters and evolutionary rate. North-East Asia is comforted in its position of origin of the genus since not only does it have the highest number of species and representatives in all deep branching lineages, there are also fewer transcontinental migrations when a North-East Asian ancestor is postulated than when a North American ancestor is postulated.     

Origin and evolution of the endemic Macaronesian Inuleae (Asteraceae): evidence from the internal transcribed spacers of nuclear ribosomal DNA

The tribe Inuleae (Asteraceae) has 10 species endemic to the Macaronesian islands, including the three endemic genera Allagopappus, Schizogyne, and Vierea. Phylogenetic analyses of DNA sequence data from the internal transcribed spacers (ITS) of the nuclear ribosomal DNA of 47 taxa were performed using all Macaronesian endemics and representative species from 21 of the 36 genera of the Inuleae. The resulting ITS phylogeny reveals that Allagopappus is sister to a large clade that contains all genera with a predominantly Mediterranean distribution. This finding suggests that Allagopappus may represent an ancient lineage that found refuge in the Canary Islands following the major climatic and/or geologic changes in the Mediterranean basin after the Tertiary. The Macaronesian endemic genus Schizogyne is sister to Limbarda from the Mediterranean. The third Macaronesian endemic genus, Vierea, is sister to Perralderia, which is restricted to Morocco and Algeria. Pulicaria canariensis is sister to P. mauritanica, a species endemic to Morocco and Algeria. In contrast, P. diffusa from the Cape Verde Islands is sister to a broadly distributed species, P. crispa, that occurs from North Africa to the Arabian peninsula. Based on the ITS data, the genera Blumea, Inula, and Pulicaria are not monophyletic. The ITS trees suggested that Blumea mollis belongs to the tribe Plucheeae, a finding that is congruent with recent morphological evidence. A possible southern African origin for the core of the Laurasian taxa of the Inuleae is also suggested.