Phylogeny of Neolitsea (Lauraceae) inferred from Bayesian analysis of nrDNA ITS and ETS sequences

ITS and ETS-based sequence analyses of 29 Neolitsea, six Actinodaphne and five outgroup `core' Laureae taxa show that Neolitsea is monophyletic with two large subclades, whereas most of the sampled Actinodaphne are paraphyletic below it. Inflorescence features appear to be among the more reliable morphological characters for explaining relationships between Neolitsea and other genera within the `core' Laureae, with the Neolitsea/Actinodaphne clade defined by inflorescences lacking vegetative terminal buds in the main axis. Although the relationships within Neolitsea are still poorly resolved, there is enough structure to suggest that the genus seems to divide into two groups based on fruit shape: elliptic or ovoid, versus globose, although more evidence (both molecular and morphological) and wider taxon sampling are required to confirm this.     

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.     

Phylogenetic analysis of Leucojum and Galanthus (Amaryllidaceae) based on plastid matK and nuclear ribosomal spacer (ITS) DNA sequences and morphology

Phylogenetic analyses of the monocotyledonous genera Leucojum and Galanthus (Amaryllidaceae, Asparagales), using plastid (trnL-F and matK) and largely non-coding nuclear ribosomal (ITS) DNA sequences show the two to be closely related to Lapiedra, Narcissus, Vagaria, Pancratium and Sternbergia. We compare the results obtained with a combined parsimony analysis of these nucleotide sequences with that of a matrix of morphological characters. The sampling included all species of Leucojum and most species of Galanthus (representing all series and subseries of the genus) and used as outgroup the above mentioned genera of Amaryllidaceae shown to be close relatives. The plastid, nuclear and morphological data were analysed independently and in combination, showing that the boundaries between the two genera are not appropriate. Galanthus is monophyletic but embedded in Leucojum. On the basis of chromosome numbers and floral characters Leucojum has been previously divided into four subgenera, which have been accepted as genera by some authors. In our phylogenetic analyses (separate as well as combined), Leucojum species are separated in two primary clades corresponding to L. subgenera Ruminia + Acis and L. Leucojum + Aerosperma. The taxonomic implications of this pattern are discussed, and an alternative classification is proposed. Finally, biogeographic relationships of species of both Leucojum and Galanthus are discussed, emphasising the possible origin of the narrowly distributed taxa of Leucojum relative to the widespread species.     

RAPD patterns,nrDNA ITS sequences and morphological patterns inSilene sectionSedoideae (Caryophyllaceae)

Hierarchical patterns inSilene sect.Sedoideae were investigated using random amplified polymorphic DNA (RAPD), nucleotide sequences of the internal transcribed spacer (ITS) regions of the 18S–28S nuclear ribosomal DNA, and discrete morphological characters. All data sets firmly supported the species recognized. The RAPD data offered the best resolution at the intraspecific level, supporting the current intraspecific classifications ofS. sedoides andS. integripetala. The ITS sequences and the morphological data gave poor resolution within species, and the three data sets disagreed about the relationships among species. The signal from the RAPD data was strongest and remained when the total data set was analysed. The three data sets all support an amphiploid origin ofS. aegaea, with the strongest evidence from the ITS sequences. Incongruences among data sets as well as merits and shortcomings of each are discussed. The robustness of the results can be evaluated using perturbations of data, i.e., bootstrap and jackknife of taxa and characters. These methods should not be taken as methods of statistical inference at the taxonomic level, because unbiased sampling appears impossible. RAPD data, however, come close to being suitable for statistical estimation of hierarchies at the genome level, but several methodological problems have to be solved.     

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.     

Molecular systematics of the Old World Astragalus (Fabaceae) as inferred from nrDNA ITS sequence data

This study represents a nuclear rDNA ITS-based phylogenetic analyses of a greater sampling of the Old WorldAstragalus compared to our previous work (212 vs. 134 taxa). Phylogenetic relationships among 212 species (213 accessions) of the Old WorldAstragalus, including newly segregated monotypic genusPodlechiella, the two aneuploid New WorldAstragalus, and five related genera, were inferred from analyses of nuclear rDNA ITS sequences using maximum parsimony. A total of 658 nucleotide sites and four binary characters for indels were analyzed. The results of phylogenetic analyses suggest sect.Phyllolobium, comprising mostly the Chinese species, is placed outside of the so-calledAstragalus s. str. and is a well-supported monophyletic group. The monotypic annual segregate genusThlaspidium (≡Astragalus sect.Thlaspidium, A. thlaspi), is clearly nested withinAstragalus s. str. Among the many sections analyzed here, only sects.Cenanthrum, Caraganella, Eremophysa, Incani, Laxiflori, andLotidium are strongly supported as monophyletic. Our analysis, in agreement with previous studies, shows that the North American euploidAstragalus species are scattered throughout the Old World groups of the genus.     

Phylogenetic relationships and hybrid origin of Potamogeton species (Potamogetonaceae) distributed in China: insights from the nuclear ribosomal internal transcribed spacer sequence (ITS)

Hybridization and polyploidization are important evolutionary processes in higher plants and have greatly enriched the diversity of the genus Potamogeton (Potamogetonaceae). To study the phylogenetic relationships and hybrid origin of Potamogeton species, 35 accessions representing 20 species, including diploids, tetraploids and hexaploids, and three hybrids were collected in China and their ribosomal internal transcribed spacers (ITS) were cloned, sequenced and statistically analyzed. The data showed that ITS sequences were informative to analyze the phylogeny of Potamogeton, and the phylogenetic tree revealed that Potamogeton species examined could be mainly divided into two groups (Group I and II), corresponding to subgenus Potamogeton and subgenus Coleogeton, respectively. Then, the evolutionary mechanism on the polyploidy of Potamogeton species was discussed. P. natans probably was an allotetraploid and one of its parent might result from aneuploidy change of species with 2n=28. P. hubeiensis might be derived from the hybridization between P. octandrus and P. cristatus. We suggested that both P. lucens and P. maackianus probably were allotetraploids, and P. obtusifolius might be a diploid hybrid between P. compressus and P. pusillus. Moreover, P. malainoides might have undergone biased concerted evolution toward one of its parent P. wrightii, and P. intortusifolius might be a synonymy of P. × anguillanus.     

Phylogenetic relationship and taxonomic position of Xylanthemum tianschanicum (Krasch.) Muradyan (Compositae,Anthemideae) as inferred from nrDNA ITS data

The central Asian endemic Xylanthemum tianschanicum (Krasch.) Muradyan (Compositae, Anthemideae) is the only species of Xylanthemum Tzvelev with radiate capitula. While the species was formerly found to be closely related to members of the subtribe Handeliinae, other representatives of the genus were considered to be similar to members of Tanacetum. In order to assess its phylogenetic relationships and taxonomic classification, a molecular analysis based on ITS sequences of 13 representatives of the subtribe Handeliinae and 17 species of three other subtribes (Anthemidinae, Artemisiinae and Matricariinae) was performed. Due to the close relationship of the X. tianschanicum to the type species of Richteria (Richteria pyrethroides) in a well supported clade (PP = 1, BS = 100%), which is supported by morphological features concerning the indumentum, capitulum morphology, and pappus structure, the combination Richteria tianschanica (Krasch.) Sonboli & Oberpr. is proposed based on the basionym Pyrethrum tianschanicum Krasch.     

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.     

nrDNA internal transcribed spacer data reveal that Rhodoplagiochila R.M. Schust. (Marchantiophyta: Jungermanniales) is a member of Plagiochila sect. Arrectae Carl

The monospecific genus Rhodoplagiochila, previously known only from the type collected in the Venezuelan Andes, has been collected at a second site close to the type locality. Up to now, generic status of Rhodoplagiochila has been accepted by most authors, but the taxon had been alternately assigned to Plagiochilaceae or Lophoziaceae. Phylogenetic analyses of nrITS sequences from Rhodoplagiochila and other representatives of the Plagiochilaceae clearly reveal the former to be in a clade with members of the Neotropical–Atlantic European Plagiochila sect. Arrectae. Within the Arrectae, Rhodoplagiochila is positioned in a robust clade made up of several accessions of Plagiochila bifaria. Morphologically, Rhodoplagiochila differs from other phenotypes of P. bifaria by the papillose leaf surface and a leaf margin which is ciliately toothed all-around. As an outcome of the molecular and morphological investigations, Rhodoplagiochila rosea is treated as a variety of P. bifaria, and the new combination P. bifaria var. rosea comb. et stat. nov. is proposed. In tropical America the family Plagiochilaceae seems to be represented only by Plagiochila.     

Phylogenetic relationships of species in <Emphasis Type="Italic">Pseudoroegneria</Emphasis> (Poaceae: Triticeae) and related genera inferred from nuclear rDNA ITS (internal transcribed spacer) sequences

To evaluate the phylogenetic relationships of species in Pseudoroegneria and related genera, the nuclear ribosomal internal transcribed spacer (ITS) sequences were analyzed for eighteen Pseudoroegneria (St), two Elytrigia (E ^e St), two Douglasdeweya (StP), three Lophopyrum (E ^e and E ^b ), three Agropyron (P), two Hordeum (H), two Australopyrum (W) and two Psathyrostachys (Ns) accessions. The main results were: (i) Pseudoroegneria gracillima, P. stipifolia, P. cognata and P. strigosa (2x) were in one clade, while P. libanotica, P. tauri and P. spicata (2x) were in the other clade, indicating there are the differentiations of St genome among diploid Pseudoroegneria species; (ii) P. geniculata ssp. scythica, P. geniculata ssp. pruinifera, Elytriga caespitosa and Et. caespitosa ssp. nodosa formed the E ^e St clade with 6-bp indel in ITS1 regions; and (iii) Douglasdeweya wangii, D. deweyi, Agropyron cristatum and A. puberulum comprised the P clade. It is unreasonable to treat P. geniculata ssp. scythica and P. geniculata ssp. pruinifera as the subspecies of P. geniculata, and they should be transferred to a new genus Trichopyrum, which consists of species with E ^e St genomes. It is also suggested that one of the diploid donor of D. wangii and D. deweyi is derived from Agropyron species, and it is reasonable to treat tetraploid species with StP genomes into Douglasdeweya.     

Genetic heterogeneity in internal transcribed spacer genes of Balantidium coli (Litostomatea, Ciliophora)

The species Balantidium coli is the only ciliate that parasitizes humans. It has been described in other primates, and it has been proposed that the species B. suis from pigs and B. struthionis from ostriches are synonyms of B. coli. Previous genetic analysis of pig and ostrich Balantidium isolates found a genetic polymorphism in the ITS region but its taxonomic relevance was not established. We have extended the genetic analysis to Balantidium isolates of pig, gorilla, human and ostrich origin. We have PCR-amplified and sequenced the ITS region of individual Balantidium cells. The predicted ITS secondary structures of the sequences obtained were transferred by homology modelling to the sequences of other Trichostomatia ciliates (Isotricha, Troglodytella, Lacrymaria and Spathidium) and compared to determine the importance of the differences in the primary sequences. The results show that the ITS2 secondary structure of the species considered follows the general pattern of other ciliates, although with some deviations. There are at least two main types of ITS sequence variants in B. coli which could be present in the same cell and they are common to the mammal and avian hosts studied. These data do not support B. suis and B. struthionis as distinct species.     

Phylogenetic relationships of Scaphyglottis and related genera (Laeliinae: Orchidaceae) based on nrDNA ITS sequence data

Sequences of the nuclear ribosomal internal transcribed spacer regions 1 & 2 (nrDNA ITS) including the intervening 5.8S region were analyzed cladistically for 43 individuals of 35 species ofScaphyglottis s.l. plus two outgroup taxa. Low levels of sequence divergence do not allow estimation of relationships among most clades, but the analyses indicate that four segregate genera (Hexisea Lindl.,Reichenbachanthus Barb. Rodr.,Hexadesmia Brogn., andPlatyglottis coriacea L.O. Williams) are embedded within a broad paraphyleticScaphyglottis. This broadly definedScaphyglottis sensu Dressler is characterized within Laeliinae by the usual presence of superposed growth habit and the presence of a column foot. In order to accommodate species formerly placed inPlatyglottis andReichenbachanthus, three new combinations are made inScaphyglottis:Scaphyglottis brasiliensis (Schltr.) Dressler,S. coriacea (L. O. Williams) Dressier, andS. emarginata (Garay) Dressler.     

Molecular phylogenetics of Hippophae L. (Elaeagnaceae) based on the internal transcribed spacer (ITS) sequences of nrDNA

 The genus Hippophae comprises 7 species and 8 subspecies according to the latest classification, and has shown enormous ecological, nutrient and medicinal values. Here we analyzed the phylogenetic relationships among 15 taxa of the genus by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). ITS sequences in Hippophae varied in length from 651 bp to 666 bp. The aligned sequences were 690 bp in length and 269 (39.0%) were variable sites with 150 being parsimony-informative. The amount of polymorphism observed within a taxon was extremely low in most taxa except for two putative hybrid species. The aligned sequences were analyzed by maximum parsimony (MP) and neighbor-joining (NJ) methods. In the strict consensus trees of parsimony analysis, the monophyly of Hippophae was supported by 100% bootstrap value. H. tibetana was at the basal position of the genus, and the remaining taxa formed two clades with high bootstrap support. The first clade included subspecies of H.␣rhamnoides and the other one consisted of remaining species. Parsimony analysis also suggested that the species H. tibetana, H. neurocarpa and H.␣salicifolia were all distinct. Although the sequence divergence among subspecies of H. rhamnoides was also remarkably high, the molecular data supported the monophyly of H. rhamnoides when H. rhamnoides subsp. gyantsensis Rousi was excxluded. The NJ trees showed essentially the same topology. The taxonomical arrangement that divided the genus into two sections was not supported based on the ITS sequences. However, the hybrid origin of H. goniocarpa and H. litangensis proposed previously was supported by the present ITS data. Received January 7, 2002; accepted May 10, 2002 Published online: November 22, 2002 Addresses of the authors: Kun Sun, Xuelin Chen, Ruijun Ma, Qin Wang, Institute of Botany, Northwest Normal University, Lanzhou 730070, China. Changbao Li, Song Ge (e-mail: gesong@ns.ibcas.ac.cn or song_ge@hotmail.com), Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.     

Phylogenetic analysis of Eremurus,Asphodelus, and Asphodeline (Xanthorrhoeaceae-Asphodeloideae) inferred from plastid trnL-F and nrDNA ITS sequences

The current study represents phylogenetic analyses of Eremurus, Asphodelus and Asphodeline (Xanthorrhoeaceae-Asphodeloideae) using both plastids genome (trnL-F) and the nuclear ribosomal internal transcribed spacer (nrDNA ITS) sequence data. The analyses revealed that each of the investigated genera is monophyletic. Eremurus subgenus Eremurus is monophyletic, whereas the E. subgenus Henningia is paraphyletic. Trachyandra is the closest relative of Eremurus. Bulbinella and Kniphofia are subsequent sisters of Eremurus and Trachyandra. Aloe, Haworthia and Bulbine were nested in a single clade, sister to the last four genera. Asphodeline section Asphodeline appeared to be non-monophyletic, because of the inclusion of A. damascena. All species of Asphodelus analyzed herein, formed a well-supported clade that it is sister to the clade of Asphodeline species.     

Heterogeneity of the internal transcribed spacer 1 (ITS1) inTulipa (Liliaceae)

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana Christmas Marvel to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.     

Phylogenetic relationships withinPelargonium sect.Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons

Phylogenetic analysis of nrDNA ITS and trnL (UAA) 5 exon-trnF (GAA) chloroplast DNA sequences from 17 species ofPelargonium sect.Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjunct South African and Australian species of sect.Peristera. Representatives fromPelargonium sectt.Reniformia, Ligularia s. l. andIsopetalum (the St. Helena endemicP. cotyledonis) appear to be nested within thePeristera clade. The close relationship between the South African and AustralianPeristera is interpreted as being caused by long-range dispersal to Australia, probably as recent as the late Pliocene.