Molecular characterization and phylogenetic utility of the rDNA external transcribed spacer region in <Emphasis Type="Italic">Stylosanthes</Emphasis> (Fabaceae)

Supplementary Material The nucleotide sequence of the ribosomal external transcribed spacer (ETS) region of Stylosanthes mexicana was determined and used to evaluate its potential for examination of intra- and inter-specific relationships in Stylosanthes, as compared to the use of the internal transcribed spacer (ITS) region. The entire ETS region comprises 1,145 bp and is composed of a region of non-repetitive sequences consisting of three subregions with organizational and nucleotide-sequence conservation, and a triplicated segment of about 100 bp. A primer designed in the second conserved subregion allowed us to amplify and sequence directly the 3' part (423-431 bp) of the ETS from 22 genotypes of 12 representative Stylosanthes species that were previously used in phylogenetic analysis of the genus. The study revealed that the right-hand part of Stylosanthes ETS contains approximately twice as much variable and informative characters than the ITS. Moreover, pairwise sequence-divergence values are twice as high, on average, when compared to the ITS. The ITS and ETS datasets are consistent in phylogenetic reconstruction of Stylosanthes, and combined parsimony analysis resulted in a strict consensus tree that is better resolved and generally better supported than trees obtained from separate analysis of the spacer regions.     

Relationships of Nematodirus species and Nematodirus battus isolates (Nematoda: Trichostrongyloidea) based on nuclear ribosomal DNA sequences

Nuclear ribosomal sequence data from the internal transcribed spacers (ITS-1 and ITS-2), 5.8S subunit, and regions of the 18S and 28S genes were used to investigate sequence diversity among geographic samples of Nematodirus battus, and to infer phylogenetic relationships among Nematodirus species. Phylogenetic analysis of these data yielded strong support for relationships among species, depicting Nematodirus helvetianus and Nematodirus spathiger as sister-taxa and a clade of these 2 species and Nematodirus filicollis. This tree is consistent with caprine bovids as ancestral hosts, with a subsequent host shift to Bovinae in N. helvetianus. Eleven of 14 N. battus sequences were unique, with 19 variable sites among sequences representing 5 geographic samples. The lowest number of variable nucleotide sites was observed in samples representing apparently recent introductions to the United States and Canada, which is consistent with a population bottleneck concomitant with translocation. Comparison of directly sequenced polymerase chain reaction products and clones revealed evidence for intraindividual variation at some of the sequence sites, and this pattern of variation and that within geographic samples indicates incomplete rDNA repeat homogenization within species. This pattern of variation is not conducive for inferring phylogenetic relationships among sequences representing N. battus or addressing the putative history of introduction.     

Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants

Sequences of the chloroplast trnC-trnD region and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were obtained for all species of Panax L. (the ginseng plant genus, Araliaceae) to reconstruct phylogenetic relationships. The trnC-trnD phylogeny is congruent with the ITS phylogeny for the diploid taxa of Panax. This study is the first use of the trnC-trnD sequence data for phylogenetic analysis at the interspecific level. We evaluated this DNA region for its phylogenetic utility at the lower taxonomic level for flowering plants. The trnC-trnD region includes the trnC-petN intergenic spacer, the petN gene, the petN-psbM intergenic spacer, the psbM gene, and the psbM-trnD intergenic spacer. The petN and psbM genes are small, 90 and 104-114 bp across angiosperms, respectively, and have conserved sequences. We have designed universal amplification and sequencing primers within these two genes. Using these primers, we have successfully amplified the entire trnC-trnD region for a diversity of flowering plant groups, including Aralia L. (Araliaceae), Calycanthus L. (Calycanthaceae), Corylus L. (Betulaceae), Hamamelis L. (Hamamelidaceae), Hydrocotyle L. (Apiaceae), Illigera Blume (Hernandiaceae), Nelumbo Adans. (Nelumbonaceae), Nolana L. ex L.f. (Solanaceae), Prunus L. (Rosaceae), and Staphylea L. (Staphyleaceae). In Panax, the trnC-trnD region provides a similar number of informative phylogenetic characters as the ITS regions and a slightly higher number of informative characters than the chloroplast ndhF gene. We thus demonstrate the utility of the trnC-trnD region for lower-level phylogenetic studies in flowering plants.     

Geographical diversification of the genus Cicer (Leguminosae: Papilionoideae) inferred from molecular phylogenetic analyses of chloroplast and nuclear DNA sequences

Cicer L. (Leguminosae: Papilionoideae) consists of 42 species of herbaceous or semi-shrubby annuals and perennials distributed throughout the temperate zones of the Northern Hemisphere. The origin and geographical relationships of the genus are poorly understood. We studied the geographical diversification and phylogenetic relationships of Cicer using DNA sequence data sampled from two plastid regions, trnK / matK and trnS - trnG , and two nuclear regions, the internal transcribed spacer (ITS) and external transcribed spacer (ETS) regions of nuclear ribosomal DNA, from 30 species. The results from the phylogenetic analyses of combined nuclear and chloroplast sequence data revealed four well-supported geographical groups: a Middle Eastern group, a West-Central Asian group, an Aegean–Mediterranean group, and an African group. Age estimates for Cicer based on methods that do not assume a molecular clock (for example, penalized likelihood) demonstrate that the genus has a Mediterranean origin with considerable diversification in the Miocene/Pliocene epochs. Geological events, such as mountain orogenesis and environmental changes, are major factors for the dispersal of Cicer species. The early divergence of African species and their geographically distinct region in the genus suggest a broader distribution pattern of the genus in the past than at present.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154 , 175–186.     

Identification of Nematodirus species (Nematoda: Molineidae) from wild ruminants in Italy using ribosomal DNA markers

The sequence of the second internal transcribed spacer of ribosomal DNA was determined for four species of Nematodirus (Nematodirus rupicaprae, Nematodirus oiratianus, Nematodirus davtiani alpinus and Nematodirus europaeus) from roe deer or alpine chamois. The second internal transcribed spacer of the four species varied in length from 228 to 236 bp, and the G + C contents ranged from 41 to 44%. While no intraspecific sequence variation was detected among multiple samples representing three of the taxa, sequence differences of 5.9-9.7% were detected among the four species, Nematodirus davtiani alpinus and N. rupicaprae were genetically most similar (94.1%), followed by N. oiratianus, N. europaeus and N. rupicaprae (91.1-91.5%), whereas N. oiratianus was genetically most different from N. davtiani alpinus. The interspecific sequence differences were exploited for the delineation of the four species by PCR-based restriction fragment length polymorphism (using two enzymes) and single-strand conformation polymorphism. The results have implications for diagnosis, epidemiology and for studying the systematics of the Nematodirinae.     

Species relationships between antifungal chitinase and nuclear rDNA (internal transcribed spacer) sequences in the genus Hordeum.

The sequences of the chitinase gene (Chi-26) and the internal transcribed spacer of 18S - 5.8S - 26S rDNA (ITS1) were determined to analyze the phylogenetic relationships among species representing the four basic genomes of the genus Hordeum. Grouping analysis based on data for Chi-26 gene sequences placed Hordeum secalinum (H genome) near the Hordeum murinum complex (Xu genome), and Hordeum bulbosum distant from the other species that carried the I genome. ITS sequence data showed the expected grouping based on the genome classification of the species studied. Different sequences of ITS were detected even in the genomes of the diploid species. The results are interpreted in terms of defective or unfinished concerted evolution processes in each taxon.     

Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.     

Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta:Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A reverse similar 1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5' end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.     

Re-evaluation of the phylogenetic relationship among species of the genus Tricholoma

The internal transcribed spacer sequences spanning the regions between the 17S and 25S rRNAs (ITS1 and ITS2) and including the complete sequence of the 5.8S rRNA were used for phylogenetic analyses. This approach to define phylogenetic relationships within the genus Tricholoma was tested using different isolates of T. terreum. Fruitbodies identified in nature were analysed in order to allow use of morphology for taxonomy. The isolates from different locations were closely related as could be expected for one species. Thus, the method could be applied to different Tricholoma species. Three clusters within the genus Tricholoma can be distinguished with four additional species not included in any of these clusters. Molecular analyses of two Cortinarius species confirm a phylogenetically distinct genus.     

Nuclear DNA and salmonid phylogenetics

There are many unresolved problems in salmonid systematics, both at the interspecific and sub-specific levels. Some of the major systematic problems in the subfamily Salmoninae are briefly reviewed along with the available molecular methods for their analysis. Nuclear DNA markers available for use in molecular systematics include localized and dispersed highly repetitive DNA sequences, moderately repetitive sequences such as the ribosomal RNA genes (rDNA), and single copy DNA sequences. Both coding and non-coding sequences can be examined in the rDNA and single copy DNA. The rDNA is especially suitable for use in phylogenetic analysis, since different regions evolve at different rates and can be used for comparisons at different taxonomic levels. Comparison of restriction maps of the entire rDNA repeating unit in 17 salmonid species from Hucho. Sahelinus, Salmo and Oncorhynchus has shown that the transcribed spacer regions are the most informative for interspecific comparisons and that the intergenic spacer has potential for use in intraspecific comparisons. Our current approach is to amplify selected regions from each of these spacers for analysis by DNA sequencing. DNA sequence analysis of the internal transcribed spacers should be very informative in elucidating interspecific relationships in Salvelinus and Oncorhynchus . Analysis of a hypervariable region in the intergenic spacer has potential for identification of geographically separated stocks. The relative utility of different types of nuclear DNA sequences for identification of stocks and subspecies is examined.     

Validity of species status of the parasitic mite Otodectes cynotis

A combined molecular and phenotypic approach was used to determine whether ear mites of the genus Otodectes (Acari: Psoroptidae) belong to a single species. The second internal transcribed spacer (ITS 2) of the rDNA of 16 isolates from 11 cats, two dogs, one arctic fox and two ferrets originating from four different continents was characterized. In addition, mites from dog, cat and arctic fox were investigated morphologically. Sequence comparisons revealed five different, but closely related genotypes which did not segregate according to host species or geographical origin. Morphologically, mites of the three host species did not differ significantly in their body or leg sizes. These investigations support the view that ear mites of the genus Otodectes from different hosts and geographical origins belong to a single species, Otodectes cynotis (Hering).     

Phylogenetic relationships of Korean Sparassis latifolia based on morphological and ITS rDNA characteristics

Recent studies based on morphological characteristics and molecular analyses have revealed that the characteristics of Sparassis crispa from Asia are not concordant with those of collections from Europe and North America. Consequently, the Asian isolate was redefined as Sparassis latifolia. This study is the first report of Sparassis latifolia collected in Korea. The taxonomic relationships and replacement of Sparassis species were inferred from a comparison of the morphological characteristics and by molecular sequence analysis of the internal transcribed spacer (ITS) rDNA regions. In particular, this study focused on the phylogenetic relationships inferred from the biogeographical distribution of isolates within the genus Sparassis.     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

A phylogenetic analysis of Doronicum (Asteraceae, Senecioneae) based on morphological, nuclear ribosomal (ITS), and chloroplast (trnL-F) evidence.

A phylogenetic analysis of the Old World genus Doronicum (26 species, 4 subspecies) based on sequence data of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, the chloroplast spacer trnL-F, and morphology is presented. Congruence among the three data sets was explored by the computing of several indices, all of which suggest homogeneity between only the two molecular matrices. We argue that the morphological data set contains poor phylogenetic signal and advocate simultaneous analysis of the three data sets (total evidence approach) so that morphological characters are tested for homology by congruence with molecular data. The resulting phylogenetic hypothesis allows several well-supported conclusions including the placement of a Corsican endemic (D. corsicum), sister to the remainder of the genus, and the inference that an early southern European or Mediterranean diversification took place in the genus. Shifts in morphological characters (e.g., homocarpy to heterocarpy) are confirmed to have evolved several times. Results from comparative studies of sequence data of the chloroplast gene ndhF support inclusion of Doronicum in tribe Senecioneae.     

The phylogeny of Gaura (Onagraceae) based on ITS, ETS, and trnL-F sequence data

Gaura (Onagraceae: Onagreae) is a small North American genus of 21 species consisting mostly of night-blooming, moth-pollinated annuals and perennials. The current infrageneric classification based on differences in habit, floral symmetry, and fruit morphology recognizes eight sections within the genus. We examine the phylogenetic relationships of all 21 species of Gaura using DNA sequence data from the internal transcribed spacer region (ITS), the external transcribed spacer region (ETS), and the plastid trnL-F region. Combined analysis of these regions indicate Gaura is monophyletic only if it includes Stenosiphon, a monotypic genus comprised of S. linifolius. Within Gaura, our studies indicate that sections Gauridium, Schizocarya, Campogaura, Stipogaura, Xenogaura, and Gaura are monophyletic, but sections Xerogaura and Pterogaura are not and should be reevaluated. In addition, molecular data provide support for the hypothesis that G. sinuata and G. drummondii arose via interspecific hybridization followed by genome doubling; their influence on phylogenetic reconstruction is discussed.     

Two new ballistoconidium-forming yeast species, Bullera melastomae and Bullera formosana, found in Taiwan

Two yeast strains, the cells of which contained xylose and Q-10 as the major ubiquinone, were isolated from a plant leaf collected in Taiwan. These yeasts were found to represent two new species of the genus Bullera in the Hymenomycetes. Identification was based on the sequence analysis of the 18S rDNA, the internal transcribed spacer (ITS) regions and the D1/D2 domain of 26S rDNA. The yeasts are named Bullera melastomae sp. nov. and Bullera formosana sp. nov. In the phylogenetic trees based on 18S rDNA and D1/D2 domain of 26S rDNA sequences, these two species constitute a cluster connected with Dioszegia cluster in the Cryptococcus luteolus lineage.     

A novel clade of sporocarp-forming species of glomeromycotan fungi in the <Emphasis Type="Italic">Diversisporales</Emphasis> lineage

In the early times of taxonomy of arbuscular mycorrhizal fungi (Glomeromycota), exclusively sporocarpic species were described. Since then the focus has mainly shifted to species forming spores singly. For many of the sporocarpic species, no molecular data have been made available, and their phylogenetic position has remained unclear. We obtained small subunit ribosomal rDNA and internal transcribed spacer data from specimens of glomeromycotan sporocarps from tropical areas that were assigned to three morphospecies. The complete sequence of the 18S small rDNA subunit sequence, internal transcribed spacers (ITS) 1 and 2 and 5.8S rDNA subunit, was determined from a sporocarp of Glomus fulvum. Partial sequences of the small subunit and the other regions were obtained from Glomus pulvinatum and the newly described species Glomus megalocarpum. Molecular phylogenetic analyses placed all species analyzed as a monophyletic sister group to the Diversispora spurca/Glomus versiforme clade group (“Glomus group C”) within the Diversisporales. The phylogenetic divergence from other known species suggests that this clade may constitute a new genus. These findings will have important consequences for taxon definition in the Diversisporales. They will facilitate identification of these fungi using rDNA sequences within colonized roots or the environment. Taxonomic novelties: Glomus megalocarpum D. Redecker