Phylogenetic relationships in Elymus (Poaceae: Triticeae) based on the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences

To estimate the phylogenetic relationship of polyploid Elymus in Triticeae, nuclear ribosomal internal transcribed spacer (ITS) and chloroplast trnL-F sequences of 45 Elymus accessions containing various genomes were analysed with those of five Pseudoroegneria (St), two Hordeum (H), three Agropyron (P) and two Australopyrum (W) accessions. The ITS sequences revealed a close phylogenetic relationship between the polyploid Elymus and species from the other genera. The ITS and trnL-F trees indicated considerable differentiation of the StY genome species. The trnL-F sequences revealed an especially close relationship of Pseudoroegneria to all Elymus species included. Both the ITS and trnL-F trees suggested multiple origins and recurrent hybridization of Elymus species. The results suggested that: the St, H, P, and W genomes in polyploid Elymus were donated by Pseudoroegneria, Hordeum, Agropyron and Australopyrum, respectively, and the St and Y genomes may have originated from the same ancestor; Pseudoroegneria was the maternal donor of the polyploid Elymus; and some Elymus species showed multiple origin and experienced recurrent hybridization.     

Phylogenetic relationships of Empetraceae inferred from sequences of chloroplast gene matK and nuclear ribosomal DNA ITS region

We used sequences of nrDNA ITS and chloroplast gene matK to evaluate the monophyly of Empetrum and Corema and to examine phylogenetic relationships of the Empetraceae. Sequences of these two DNA markers were obtained for 11 plant samples, representing species of Empetrum from both the Southern and Northern Hemispheres, species and subspecies of Corema, and the monotypic Ceratiola. Sequences of four species of Rhododendron were used for rooting purposes. Our results show that species of Empetrum form a clade sister to the clade containing both Corema and Ceratiola. These two clades are strongly supported in both the matK and ITS trees, suggesting that Ceratiola is more closely related to Corema than to Empetrum, and is not of a hybrid origin between the ancestors of the latter two genera. In the matK tree, Corema conradii is more closely related to Ceratiola than to Corema album and C. album subsp. azoricum, whereas in the ITS tree, Ceratiola is allied with Corema album and C. album subsp. azoricum. This suggests that C. conradii might be a hybrid between ancestral populations of Ceratiola and C. album. The monophyly of Empetrum rejects the hypothesis of its independent origin in the two Hemispheres. Our trees also suggest the fact that the modern amphitropical distribution of Empetrum is the result of long distance dispersal, not of the vicarious events.     

Phylogenetic relationships in Leymus (Poaceae: Triticeae) revealed by the nuclear ribosomal internal transcribed spacer and chloroplast trnL-F sequences

Using the nuclear ribosomal internal transcribed spacer (ITS) sequences and the chloroplasttrnL-F sequence, phylogeneic analysis was performed on 57 accessions of species in the tribe Triticeae including 13 Leymus species (N(s)) with different ploidy levels and 40 diploid species from 18 genera. The ITS sequences revealed that ployploid Leymus has close phylogentic relationships with Psathyrostachys and an undefined genus in Triticeae. The trnL-F tree demonstrated close relationships between certain Leymus species and Psathyrostachys, and other Leymus species distributed in North America were far from Psathyrostachys. Based on these results, it is unlikely that the unknown genome in Leymus species originated from one of the sampled diploid species in the present study. The maternal donor of all the Leymus species with a natural distribution in Eurasia were N(s) genome. Furthermore, Elymus californicus should be transferred from the genus Elymus to Leymus.     

Phylogenetic relationships in Lupinus (Fabaceae: Papilionoideae) based on internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 44 taxa of the genus Lupinus and five outgroup taxa were used for phylogenetic analysis. Lupinus appears as a strongly supported monophyletic genus, which is unambiguously part of the Genisteae. The lupines are distributed into five main clades in general accordance with their geographical origin. In the Old World, almost all the recognized taxonomic units are well resolved. The ITS data reveal an unexpectedly close relationship between the diverse sections Angustifoli and Lutei. The ITS results suggest a geographical division between the western New World lupines and the eastern ones. They also indicate the presence of some moderately to strongly supported groups of taxa, such as the Microcarpi-Pusilli group, the L. spariflorus-L. arizonicus group, the L. mexicanus-L. elegans group in the western New World, and the notable L. multiflorus-L. paraguariensis group in the eastern New World. The latter group strongly suggests that the eastern South American compound- and simple-leaved perennial lupines derive from a common ancestor. However, apart from some exceptions, relationships within the genus still remain largely unresolved based on ITS data. The lack of resolution at the base of the genus is suggestive of a rapid initial radiation of the lupines subsequent to the dispersal of their common ancestor. Relative rate tests demonstrate the presence of rate heterogeneity of ITS sequences within Lupinus. In many pairwise comparisons between taxa, substitution rate inequalities are correlated with the habit (annual, perennial), suggesting some role for the generation time effects in the evolutionary history of lupines.     

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.     

Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

A 650-bp portion of the nuclear ribosomal DNA internal transcribed spacer region was sequenced in 47 species of Pinus, representing all recognized subsections of the genus, and 2 species of Picea and Cathaya as outgroups. Parsimony analyses of these length variable sequences were conducted using a manual alignment, 13 different automated alignments, elision of the automated alignments, and exclusion of all alignment ambiguous sites. High and moderately supported clades were consistently resolved across the different analyses, while poorly supported clades were inconsistently recovered. Comparison of the topologies highlights taxa of particularly problematic placement including Pinus nelsonii and P. aristata. Within subgenus Pinus, there is moderate support for the monophyly of a narrowly circumscribed subsect. Pinus (=subsect. Sylvestres) and strong support for a clade of North and Central American hard pines. The Himalayan P. roxburghii may be sister species to these "New World hard pines," which have two well-supported subgroups, subsect. Ponderosae and a clade of the remaining five subsections. The position of subsect. Contortae conflicts with its placement in a chloroplast DNA restriction site study. Within subgenus Strobus there is consistent support for the monophyly of a broadly circumscribed subsect. Strobi (including P. krempfii and a polyphyletic subsect. Cembrae) derived from a paraphyletic grade of the remaining soft pines. Relationships among subsects. Gerardianae, Cembroides, and Balfourianae are poorly resolved. Support for the monophyly of subgenus Pinus and subgenus Strobus is not consistently obtained.     

Phylogeny of Rubus (rosaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

We used nuclear ribosomal DNA internal transcribed spacer region (ITS 1 - 5.8S - ITS 2; ITS) sequences to generate the first phylogeny of Rubus based on a large, molecular data set. We sampled 57 taxa including 20 species of subgenus Rubus (blackberries), one to seven species from each of the remaining 11 subgenera, and the monotypic and closely related Dalibarda. In Rubus, ITS sequences are most informative among subgenera, and variability is low between closely related species. Parsimony analysis indicates that Rubus plus Dalibarda form a strongly supported clade, and D. repens may nest within Rubus. Of the subgenera with more than one species sampled, only subgenus Orobatus appears monophyletic. Three large clades are strongly supported: one contains all sampled species of nine of the 12 subgenera; another includes extreme Southern Hemisphere species of subgenera Comaropsis, Dalibarda, and Lampobatus; and a third clade consists of subgenus Rubus plus R. alpinus of subgenus Lampobatus. Rubus ursinus appears to be a hybrid between a close relative of R. macraei (subgenus Idaeobatus, raspberries) and an unidentified subgenus Rubus species. ITS sequences are generally consistent with biogeography and ploidy, but traditionally important morphological characters, such as stem armature and leaf type, appear to have limited phylogenetic value in Rubus.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences

The phylogenetic relationships within the genus Betula (Betulaceae) were investigated using a part of the nuclear ADH gene and DNA sequences of the chloroplast matK gene with parts of its flanking regions. Two well-supported phylogenetic groups could be identified in the chloroplast DNA sequence: one containing the three American species B. lenta, B. alleghaniensis, and B. papyrifera and the other including all the other species studied. The ADH gene displayed more variation, and three main groups could be identified. In disagreement with the classical division of the genus Betula, B. schmidtii and B. nana grouped with the species in subgenus Betula, and B. ermanii grouped with species in subgenus Chamaebetula, including B. humilis and B. fruticosa. The ADH phylogeny suggests that several independent polyploidizations within the genus Betula could have taken place. The ADH and chloroplast phylogenies were in part incongruent due to the placement of B. papyrifera. The most likely reason for this seems to be cytoplasmic introgression.     

A molecular phylogeny of Arceuthobium (Viscaceae) based on nuclear ribosomal DNA internal transcribed spacer sequences

Ribosomal DNA (rDNA) internal transcribed spacer (ITS) and 5.8S rDNA sequences were obtained from 22 species of dwarf mistletoes (Arceuthobium — Viscaceae) to test phylogenetic relationships. Interspecific distances ranged from 0 to 21.4% between New World species, values two to five times higher than those measures for the ITS region in other plants. One Old World species (A. oxycedri) and one New World species (A. abietis-religiosae) were remarkably similar to each other but exhibited up to 41% sequence divergence from the remaining species. Minimum length trees support the concept of a verticillately branched subgenus Arceuthobium; however, interspecific distances indicate this group is extremely heterogeneous. Subgenus Vaginata, Section Vaginata, is centered in Mexico and encompasses all the taxa previously placed in this group but is expanded to include several species previously classified in Section Campylopoda (e.g., A. divaricatum, A. rubrum, and A. strictum). The sister group relationship between A. divaricatum and A. douglasii, first seen following isozyme analysis, is supported by ITS sequence data. Section Campylopoda s. s. is now composed of 13 mainly U.S. species that show a high degree of morphological and genetic similarity. The eastern dwarf mistletoe, A. pusillum, is not closely related to A. douglasii but rather with A. bicarinatum from Hispaniola, which suggests that these taxa represent highly modified relicts that shared an ancestor in the early Tertiary. Two endemic species from Mexico and Central America (A. guatemalense and A. pendens) formed a sister group and have been placed in a new Section (Penda). Rapid molecular evolution in Arceuthobium may be associated with the adaptive radiation of this genus on numerous conifer hosts.     

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA

Summary Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.     

Molecular phylogeny of JapaneseAmanita species based on nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal DNA

The molecular phylogeny of 36 specimens of JapaneseAmanita species was studied based on nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. The phylogenetic tree obtained supported the traditional classification systems of Bas (1969) and Singer (1986), which are based on morphological characters, in the division of the genusAmanita is divided into subgeneraAmanita andLepidella by the amyloidity of basidiospores. However, at section-level, we suggest that subgenusAmanita should be divided into three sections (Amanita, Vaginatae, andCaesareae). Our results also showed the necessity to modify the taxonomic treatments at section-level in the subgenusLepidella. It appears that the establishment ofA. muscaria andA. pantherina from a common ancestral species might be a very recent event, or these might be lower taxa of same species. As for three subspecies ofA. hemibapha and three varieties ofA. vaginata, it is necessary to grade up their taxonomical ranks from subspecies/variety to species. A new combination,A. javanica, is proposed forA. hemibapha subsp.javanica.     

Molecular phylogeny and genetic variation in the genus Lilium native to China based on the internal transcribed spacer sequences of nuclear ribosomal DNA

We present a comprehensive phylogeny derived from nuclear ribosomal DNA (nrDNA) for 214 samples representing 98 species and five varieties, including 44 species and five varieties native to China. Our collection of 25 species and five varieties (44 samples) covering all five sections of the genus (Comber) distributed in China also were included in the internal transcribed spacer (ITS) database. This study incorporates previous research with an emphasis on Chinese species, including the controversial subsection, Sinomartagon 5c Comber. In the phylogenetic tree obtained by maximum parsimony (PAUP) and maximum likelihood (RAxML) analyses, the samples were divided into four major groups. Our results suggest that the subsection (subsect.) 5c Comber should be classified into the true subsect. 5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum I, subsect. Lophophorum II, and subsect. Lophophorum III). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, we report that L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion 6a. Our results support Comber’s subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.     

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.     

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.     

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

Background

Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns.

Methodology/Principal Findings

In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level.

Conclusions

Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.