Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

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.     

A phylogenetic study ofSaxifraga sect.Saxifraga (Saxifragaceae) based on nrDNA ITS sequences

Parsimony analyses of 54 nrDNA ITS (Internal Transcribed Spacer) sequences ofSaxifraga sect.Saxifraga were performed. In addition to some unresolved clades, there is strong disagreement between the ITS phylogeny and previous classifications based primarily on morphology. The extensive cytological instability of sect.Saxifraga prevents previous cytotaxonomical results from resolving the incongruence between molecular and morphological data. Dissimilar topologies between chloroplast (matK) and nuclear (ITS) trees for eight species of sect.Saxifraga suggest that gene trees and the true species tree are not coincident. Recent and mid-term reticulation is proposed as an explanation for the incongruence between morphological, cytological, organellar, and nuclear data. Homogenization in multigene families, such as the ITS region, via concerted evolution may be the key to the interpretation of results based on ITS sequences within sect.Saxifraga. The use of organellar genes in a larger sample should help to determine whether extensive reticulation occurs in sect.Saxifraga, as has been documented in various genera of Saxifragaceae.     

Xenocylindrocladium guianense andX. subverticillatum, two new species of hyphomycetes from plant debris in the tropics

Two new species of hyphomycetes,Xenocylindrocladium guianense andX. subverticillatum, are described from plant debris collected in French Guiana and Singapore, respectively. The genusXenocylindrocladium has thus far been known from one species,X. serpens, which was described from plant debris collected in Ecuador. The two new taxa are compared with and distinguished fromX. serpens based on morphology, cultural characteristics and phylogenetic analysis of DNA sequence data of the 5.8S rDNA with flanking ITS1 and ITS2 regions and the 5′ end of the β-tubulin gene. These species are also compared with other closely related hypocrealean taxa. Present collection data suggest that species ofXenocylindrocladium could be restricted to the tropics.     

Phylogeny ofCyclamen L. (Primulaceae): Evidence from morphology and sequence data from the internal transcribed spacers of nuclear ribosomal DNA

A molecular phylogenetic study of the genusCyclamen L. (Primulaceae) has been undertaken, based on sequence data from the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. Sequence data from 15 species ofCyclamen and seven outgroup taxa selected from Primulaceae and Myrsinaceae were analyzed phylogenetically. A second analysis based on a combined morphological and molecular dataset was performed to evaluate earlier hypotheses of character evolution in the genus. The results indicate that four monophyletic subgroups may be recognized in the genus, viz.Cyclamen, Psilanthum Schwarz,Eucosme Schwarz andGyrophoebe Schwarz. Each of the four subgenera is diagnosed by distinct basic chromosome number, as well as by morphological and molecular characteristics.     

Phylogenetic analysis of Asian<Emphasis Type="Italic"> Symplocos</Emphasis> (Symplocaceae) based on nuclear and chloroplast DNA sequences

Thirty species and one variety of Symplocos (Symplocaceae), including all taxa distributed in Japan, were phylogenetically analyzed with DNA sequence data. The evolution of morphological characters is discussed on the basis of the phylogenetic relationships obtained. All species were Asian, except one, S. austromexicana, from Mexico. The nuclear ribosomal internal transcribed spacer (ITS) region and two intergenic spacer regions, between trnL and trnF, and between trnH and psbA of chloroplast DNA were used. The topologies of trees obtained from ITS and the chloroplast intergenic spacers are largely congruent. S. sonoharae, the representative of the subgenus Symplocos in Asia, was sister to all other species. The position of the American species, S. austromexicana, which also belongs to the subgenus Symplocos, was not well resolved. The phylogenetic tree based on combined sequence data largely supports the monophyletic origin of the infrageneric sections proposed earlier. However, the phylogenetic relationship between them is not well resolved, probably due to rapid diversification. The section Palura, a deciduous group, is well defined in the DNA analysis, suggesting its independent status in the genus Symplocos. In spite of their morphological divergence, the three endemic species of the Bonin Islands are monophyletic. The occurrence of curved seeds seems to be homoplastic, scattered over the phylogenetic tree without showing a particular infrageneric relationship.     

Intercontinental dispersal: The origin of the widespread South American plant speciesGilia laciniata (Polemoniaceae) from a rare California and Oregon coastal endemic

Although separated by 7000-km,Gilia millefoliata, a rare annual plant from California and Oregon coastal dunes andG. valdiviensis, a rare Chilean coastal endemic are morphologically and ecologically quite similar. Their disjunct distribution was proposed to result from recent, birdmediated, intercontinental long-distance dispersal. Both species are morphologically similar to the abundant and ecologically diverse South American taxonG. laciniata. The relationship among these three taxa was investigated using DNA sequence from the nuclear ribosomal (ITS) and chloroplasttrnL regions, as well as isozyme and morphological variation to determine the roles of long-distance dispersal and ecological adaptation in the evolution of the group. These data suggest that aG. millefoliata-like ancestor underwent long-distance dispersal to South America, and there gave rise to the narrow endemicG. valdiviensis and the widespreadG. laciniata.     

Molecular phylogeny ofSalicaceae and closely relatedFlacourtiaceae: Evidence from 5.8 S, ITS 1 and ITS 2 of the rDNA

A ribosomal DNA region, including the entire 5.8S RNA gene and the internal transcribed spacers ITS 1 and ITS 2, was used for studying the phylogeny ofSalicaceae and the relationship betweenSalicaceae andFlacourtiaceae. The length of the ITS regions withinSalicaceae andFlacourtiaceae was similar to that found in other angiosperms. The GC content of both ITS regions was high, varying 62.7-72.2%. The most parsimonious tree clusters the wind-pollinatedChosenia bracteosa among theSalix species, suggesting that it should be included in the genusSalix. The grouping withinSalix leaves subg.Salix as paraphyletic, for which reason the subgeneric division is questionable.Populus was monophyletic and formed a sister group toSalix. The interspecific variation of the ITS sequences was very small inSalicaceae, which is in contradiction to the age of the group according to the evidence from fossil data.Idesia polycarpa fromFlacourtiaceae shows great sequence similarity withSalicaceae, but the analysis of 5.8S rDNA supports monophyly of the four species ofFlacourtiaceae sampled for this study.     

Allotetraploid origin ofAllium altyncolicum (Alliaceae, Allium sect.Schoenoprasum) as investigated by karyological and molecular markers

The tetraploidAllium altyncolicum (2n = 4x = 32) is considered to be of hybrid origin, because most of its morphological characters are intermediate between those of its putative parents,A. schoenoprasum andA. ledebourianum. In the present work an attempt has been made to ascertain its parentage by several methods: Giemsa C-banding, genomic in situ hybridization (GISH), PCR-RFLP of cpDNA, restriction enzyme mapping of the rDNA, and RAPDs. C-banding and GISH indicates clearly thatA. altyncolicum is a segmental allopolyploid.Allium schoenoprasum andA. ledebourianum are the most likely the parental species and the larger part of the genome ofA. altyncolicum (26 chromosomes) is derived fromA. schoenoprasum. The low genetic divergence between these three species was confirmed by the lack of sequence variation in the ITS sequences of nuclear rRNA genes and of the plastid rbcL-atpB intergenic spacer. Both parental species andA. altyncolicum could be distinguished by RFLP of the rDNA repeats. The geographic origin of the putative parental species was investigated using RAPDs.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

The taxonomic relationships within the genus Excoecaria L. (Euphorbiaceae) based on leaf morphology and rDNA sequence data

The tropical Indo-Pacific genus Excoecaria L. (Euphorbiaceae) has several closely related species in Australia whose taxonomic relationships are unclear. The most widely reported species in Australia is the mangrove species Excoecaria agallocha L. (type species), whose taxonomic and geographic limits are difficult to define from its closely related species or sub-species. Two additional taxa have also been described but not clearly differentiated from the type species: Excoecaria dallachyana Baillon and Excoecaria ovalis Endl. This project aimed to determine the taxonomic relationships of the Australian Excoecaria species using both leaf morphological data and DNA sequence data from the internal transcribed spacer (ITS) region of ribosomal genes. The nucleotide differences in the examined ITS1 region show that E. agallocha from eastern Australia and E. ovalis from Western Australia respectively, are genetically uniform within species but differ from each other consistently, thus supporting species status. The leaf morphological data also support this view: single factor analysis of variance consistently separated E. ovalis from E. agallocha on the basis of leaf width, leaf length and length of petiole. In contrast, E. ovalis from the Gulf of Carpentaria differs only slightly from E. ovalis in Western Australia, but no evidence was found to suggest any leaf morphological differentiation within this species. The analysis also suggests that E. dallachyana is not closely related to either mangrove species E. agallocha or E. ovalis, despite superficial morphological similarities.     

Relationships between three species ofFestuca sect.Bovinae (Poaceae)

Seed protein electrophoresis confirms the existence of polymorphism among hexaploid populations ofFestuca arundinacea. Both protein and morphological results suggest thatF. pratensis andF. arundinacea should retain independent specific status. High protein homology of these two species withF. gigantea points towards phylogenetic links between these taxa.     

ITS sequences from nuclear rDNA suggest unexpected phylogenetic relationships between Euro-Mediterranean, East Asiatic and North American taxa ofQuercus (Fagaceae)

Nucleotide sequences from the internal transcribed spacer (ITS) regions of the 18S–26S nuclear ribosomal DNA have been studied from ten species ofQuercus (representing four subgenera),Castanea sativa andFagus sylvatica, as a preliminary molecular contribution to the still poorly understood systematics and evolution ofFagaceae. The resulting matrix has been used to calculate pair-wise sequence divergence indices and to construct a maximum parsimony tree forQuercus coding indels as a fifth state. Divergence is greater forQuercus vs.Fagus than forQuercus vs.Castanea. The tree for theQuercus taxa studied reveals two clearly divergent clades. In clade I the evergreen W MediterraneanQ. suber appears in a basal position as sister to more distal deciduous taxa, i.e. the E MediterraneanQ. macrolepis and the E AsiaticQ. acutissima (all formerly united as different sections under the apparently polyphyletic subg.Cerris), andQ. rubra (a representative of the N American subg.Erythrobalanus), forming a pair withQ. acutissima. In clade II the evergreen southeastern N AmericanQ. virginiana is basal and sister to the remaining three branches, i.e. a pair of evergreen Mediterranean taxa withQ. ilex andQ. coccifera (subg.Sclerophyllodrys), the deciduous but otherwise plesiomorphic SE European/SW AsiaticQ. cerris (type species of subg.Cerris), and the related but more apomorphic European pairQ. petraea andQ. robur (subg.Quercus). These results partly conflict with current taxonomic classification but are supported by some anatomical and morphological characters. They document polyphyletic lines from evergreen to deciduous taxa and suggest Tertiary transcontinental connections within the genus.     

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.     

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.     

A new genus and two new species of gobiid fishes (Perciformes) from the Chagos Archipelago,Central Indian Ocean

Synopsis A recent (1979) expedition to the Chagos Archipelago resulted in the collection of about 40 new taxa of fishes. A new genus,Trimmatom, and two new species,T. nanus andT. offucius, are described here. The new genus is characterized by having all pelvic-fin rays simple (unbranched), a scaleless body, no head pores, a wide gill opening extending anteroventrally to below the eye, and hypurals 1 and 2 fused to the complex formed by the fusion of the ural centrum and hypurals 3 and 4.T. nanuss andT. offucius are differentiated on the basis of fin ray counts and colour pattern.T. nanus is the smallest vertebrate yet to be described. Mature females with ovaries full of eggs are 8–10 mm in standard length.     

Morphological and ITS sequence divergence between taxa of Cuminia (Lamiaceae), an endemic genus of the Juan Fernandez Islands, Chile

Morphological and ITS sequence divergence were assessed between the two presently recognized taxa of the endemic genusCuminia (Lamiaceae), on Masatierra of the Juan Fernandez Islands. Morphological studies were based on leaf morphology of 51 individuals. Individuals ofC. fernandezia have narrow and lanceolate leaves with cuneate to acute bases and apices, whereas individuals ofC. eriantha have broadly ovate leaves with rounded bases and acute to obtuse apices. The two taxa can also be distinguished by the presence of pubescence.Cuminia eriantha has hairs on the leaves, young stems, floral peduncles, calyx, and corolla. Alternatively,C. fernandezia is glabrous except for tuft hairs on the nodes, and hairs on calyx teeth and corolla tubes. The ITS 1 and 2 regions of the five plants ofCuminia sequenced are a total of 451 bp long. All plants have identical ITS-1 but pubescent and glabrous plants are consistently different in ITS-2 sequence, revealing 1.3% total sequence divergence between the species. Both morphological and molecular data support the taxonomic recognition of two taxa, and the small but consistent differences appear to justify species status for the two entities. The pubescent populations representC. eriantha and the glabrous ones areC. fernandezia. It is hypothesized that the species diverged from a common ancestral immigrant to the islands, when Masatierra was much larger and more ecologically diverse than it now is. The two taxa seem to maintain their identities with no evidence of hybridization, even though they often grow in close proximity to one other on the island. Each population consists only of glabrous or pubescent plants, with no mixed population detected.     

Phylogenetic analysis of Sesuvioideae (Aizoaceae) inferred from nrDNA internal transcribed spacer (ITS) sequences and morphological data

The phylogeny of the four genera of Aizoaceae subfamily Sesuvioideae (Sesuvium, Cypselea, Trianthema and Zaleya) is elucidated employing internal transcribed spacer (ITS) sequences and 23 morphological characters. Phylogenetic analysis based on ITS sequences and a combined molecular-morphological analysis provide largely congruent results. The monophyly of Sesuvioideae and its close relationship to Aizooideae s.l. and Mesembryanthemoideae is confirmed. Zaleya is placed within Trianthema and Cypselea within Sesuvium by ITS analysis, but in the combined analysis, Zaleya forms an unresolved polytomy with the two Trianthema clades and Cypselea, as well as two Sesuvium species, remain unresolved. Sesuvium sesuvioides and S. hydaspicum, previously treated as synonyms, are closely related, but molecular data do not support conspecifity. The Trianthema triquetra complex needs further intensive study because the African T. triquetra sample is closer to the NE African-Arabian T. sheilae than to Australian samples of T. triquetra. The close relationship of four species of Trianthema (T. patellitecta, T. rhynchocalyptra, T. megasperma, and T. pilosa) from Australia based on molecular data is supported morphologically by the exclusive possession of a well-developed indumentum in these taxa.     

Phylogeny of Bonatea (Orchidaceae: Habenariinae) based on molecular and morphological data

The genus Bonatea is widely distributed throughout southern and eastern Africa. Considerable debate surrounds the generic status of Bonatea, but there have been neither previous studies of evolutionary relationships among Bonatea species, nor any tests of the monophyly of the genus in relation to its close relative Habenaria. We investigated phylogenetic relationships between Bonatea and selected Habenaria species using morphology, as well as sequences of the chloroplast gene matK and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. A fully resolved cladogram was obtained using morphological data, but neither the ITS, matK, nor combined data sets yielded well-resolved and well-supported phylogenetic structure for Bonatea. There is poor congruence between ITS and matK data for interspecific relationships in Bonatea, whilst the morphological results are largely congruent with the ITS analysis. Relative to Habenaria, there is little sequence variation between Bonatea species, which could indicate a recent and rapid radiation of Bonatea. Although the sampled Bonatea species form a distinct clade, more extensive sampling of Habenaria would be required to establish unambiguously whether or not Bonatea is monophyletic.     

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.