Sources of error and confidence intervals in estimating the age of angiosperms from rbcL and 18S rDNA data

Molecular estimates of the age of angiosperms have varied widely, and many greatly predate the Early Cretaceous appearance of angiosperms in the fossil record, but there have been few attempts to assess confidence limits on ages. Experiments with rbcL and 18S data using maximum likelihood suggest that previous angiosperm age estimates were too old because they assumed equal rates across sites-use of a gamma distribution of rates to correct for site-to-site variation gives 10-30 my (million years) younger ages-and relied on herbaceous angiosperm taxa with high rates of molecular evolution. Ages based on first and second codon positions of rbcL are markedly older than those based on third positions, which conflict with the fossil record in being too young, but all examined data partitions of rbcL and 18S depart substantially from a molecular clock. Age estimates are surprisingly insensitive to different views on seed-plant relationships. Randomization schemes were used to quantify confidence intervals due to phylogenetic uncertainty, substitutional noise, and lineage effects (deviations from a molecular clock). Estimates of the age of crown-group angiosperms range from 68 to 281 mya (million years ago), depending on data, tree, and assumptions, with most ～140-190 mya (Early Jurassic-earliest Cretaceous). Approximate 95% confidence intervals on ages are wider for rbcL than 18S, ranging up to 160 my for phylogenetic uncertainty, 90 my for substitutional noise, and 70 my for lineage effects. These intervals overlap the oldest occurrences of angiosperms in the fossil record, as well as some estimates from previous molecular studies.     

18S rDNA sequences and the holometabolous insects

The Holometabola (insects with complete metamorphosis: beetles, wasps, flies, fleas, butterflies, lacewings, and others) is a monophyletic group that includes the majority of the world's animal species. Holometabolous orders are well defined by morphological characters, but relationships among orders are unclear. In a search for a region of DNA that will clarify the interordinal relationships we sequenced approximately 1080 nucleotides of the 5' end of the 18S ribosomal RNA gene from representatives of 14 families of insects in the orders Hymenoptera (sawflies and wasps), Neuroptera (lacewing and antlion), Siphonaptera (flea), and Mecoptera (scorpionfly). We aligned the sequences with the published sequences of insects from the orders Coleoptera (beetle) and Diptera (mosquito and Drosophila), and the outgroups aphid, shrimp, and spider. Unlike the other insects examined in this study, the neuropterans have A-T rich insertions or expansion regions: one in the antlion was approximately 260 bp long. The dipteran 18S rDNA evolved rapidly, with over 3 times as many substitutions among the aligned sequences, and 2-3 times more unalignable nucleotides than other Holometabola, in violation of an insect-wide molecular clock. When we excluded the long-branched taxa (Diptera, shrimp, and spider) from the analysis, the most parsimonious (minimum-length) trees placed the beetle basal to other holometabolous orders, and supported a morphologically monophyletic clade including the fleas+scorpionflies (96% bootstrap support). However, most interordinal relationships were not significantly supported when tested by maximum likelihood or bootstrapping and were sensitive to the taxa included in the analysis. The most parsimonious and maximum-likelihood trees both separated the Coleoptera and Neuroptera, but this separation was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

High rates of nucleotide substitution in nuclear small-subunit (18S) rDNA from holoparasitic flowering plants

Relative rate tests, using Gnetum as a reference taxon, were conducted on nuclear 18S rRNA sequences from 10 angiosperms including autotrophic nonparasites (Arabidopsis, Asarum, Glycine, Malpighia, and Zea), a chlorophyllous hemiparasite (Arceuthobium—Viscaceae), and achlorophyllous holoparasites (Balanophora—Balanophoraceae, Prosopanche—Hydnoraceae, and Rafflesia and Rhizanthes—Rafflesiaceae). Compared with Glycine, the mean number of substitutions per site (K) for five autotrophic angiosperms is 0.036 whereas for the holoparasites K = 0.126, i.e., 3.5 times higher. Comparisons of autotrophic species with short and long generation times showed no differences in K; hence, divergent rRNA sequences in the holoparasites are likely attributable to other mechanisms. These might include genetic bottlenecks, effective population size, and/or molecular drive. High substitution rates appear to be associated only with those parasitic angiosperms that have developed a highly modified haustorial system and extreme nutritional dependence upon the host. At present, high substitution rates in these parasites confound attempts to determine their phylogenetic position relative to other angiosperms. Correspondence to: D.L. Nickrent     

Flatworm phylogeny from partial 18S rDNA sequences

Partial 18S rDNA sequences from 29 flatworms and 2 outgroup taxa were used in a cladistic analysis of the Platyhelminthes. Support for the clades in the resulting single most parsimonious tree was estimated through bootstrap analysis, jack-knife analysis and decay indices. The Acoelomorpha (Acoela and Nemertodermatida) were absent from the most parsimonious tree. The Acoela and the Fecampiidae form a strongly supported clade, the sister group of which may be the Tricladida. There is some support for monophyly of the rhabdocoel taxon Dalyellioida, previously regarded as paraphyletic. The sister group of the Neodermata remains unresolved. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogenetic relationships among rodent Eimeria species determined by plastid ORF470 and nuclear 18S rDNA sequences

Phylogenetic analyses for 10 rodent Eimeria species from different host genera based on plastid ORF470 and nuclear 18S rDNA sequences were done to infer the evolutionary relationships of these rodent Eimeria species and their correlation to morphology and host specificity. The phylogenies based on both data sets clearly grouped the 10 rodent Eimeria species into two major lineages, which reflect more their morphological differences than host specificity. Species in lineage A have spheroidal to subspheroidal sporulated oocysts, are similar in size (18-29 x 17-23; xbar = 22 x 20 microm), have an oocyst residuum and one-two polar granules; these include Eimeria albigulae (Neotoma), Eimeria arizonensis (Peromyscus, Reithrodontomys), Eimeria onychomysis (Onychomys) and Eimeria reedi (Perognathus). Species in lineage B, including Eimeria falciformis (Mus), Eimeria langebarteli (Reithrodontomys), Eimeria nieschulzi (Rattus), Eimeria papillata (Mus), Eimeria separata (Rattus) and Eimeria sevilletensis (Onychomys) have different shapes (ovoid, ellipsoid, elongated ellipsoid, etc.), differ greatly in size (10-27 x 9-24; xbar = 19 x 16 microm) and all lack an oocyst residuum. Thus, The oocyst residuum was the most determinant feature that differentiated the two lineages. The accession numbers of ORF470 of E. albigulae, E. arizonensis, E. falciformis, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis, E. langebarteli are AF311630-AF311639 and 18S rDNA of E. langebarteli, E. papillata, E. reedi, E. separata, E. sevilletensis are AF311640-AF311644.     

Phylogeny of Nematoda and Cephalorhyncha Derived from 18S rDNA

Phylogenetic relationships of nematodes, nematomorphs, kinorhynchs, priapulids, and some other major groups of invertebrates were studied by 18S rRNA gene sequencing. Kinorhynchs and priapulids form the monophyletic Cephalorhyncha clade that is the closest to the coelomate animals. When phylogenetic trees were generated by different methods, the position of nematomorphs appeared to be unstable. Inclusion of Enoplus brevis, a representative of a slowly evolving nematode lineage, in the set of analyzed species refutes the tree patterns, previously derived from molecular data, where the nematodes appear as a basal bilateral lineage. The nematodes seem to be closer to the coelomate animals than was speculated earlier. According to the results obtained, nematodes, nematomorphs, tardigrades, arthropods, and cephalorhynchs are a paraphyletic association of closely related taxa. Received: 1 December 1997 / Accepted: 9 April 1998     

Phylogeny of Hesionidae (Aciculata, Polychaeta), assessed from morphology, 18S rDNA, 28S rDNA, 16S rDNA and COI

We assess phylogenetic relationships within the polychaete family Hesionidae from morphological data combined with nucleotide data from 18S rDNA, 28S rDNA, 16S rDNA and COI. Parsimony and Bayesian analyses were performed on two data sets; the first was based on a more restricted set of terminals with both morphological and molecular data (17 ingroup terminals), while the second included additional taxa with morphological data only (25 ingroup terminals). The different sets of terminals yielded fully congruent results, as did the parsimony and the Bayesian analyses. Our results indicate high levels of homoplasy in traditionally used morphological characters in the group, and that Hesioninae, Gyptini and Gyptis are nonmonophyletic. Hesionini (mainly Hesione and Leocrates ), Psamathini (mainly Hesiospina , Micropodarke , Nereimyra , Psamathe and Syllidia ), Ophiodrominae (Gyptini and Ophiodromini) and Ophiodromini (mainly Heteropodarke , Ophiodromus and Podarkeopsis ) are monophyletic and agree with previous classifications, and Hesionini is probably the sister to all other hesionids. The placements of the small hesionids capricornia and Lizardia , the hydrothermal vent taxa Hesiodeira and Hesiolyra , and the newly described Hesiobranchia , remain uncertain.     

Variability of nuclear 18S-25S rDNA of Gentiana lutea L. in nature and in tissue culture in vitro

18S-25S rDNA sequence in genomes of G. lutea plants from different natural populations and from tissue culture has been studied with blot-hybridization method. It was shown that ribosomal repeats are represented by the variants which differ for their size and for the presence of additional HindIII restriction site. Genome of individual plant usually possesses several variants of DNA repeats. Interpopulation variability according to their quantitative ratio and to the presence of some of them has been shown. Modifications of the range of rDNA repeats not exceeding intraspecific variability were observed in callus tissues in comparison with the plants of initial population. Non-randomness of genome modifications in the course of cell adaptation to in vitro conditions makes it possible to some extent to forecast these modifications in tissue culture.     

Graniferous tracheary elements in haustoria of root parasitic angiosperms

Graniferous tracheary elements are unusual xylem conducting cells, characterized by having structural material in the lumen. They are known particularly from certain root parasitic angiosperms. The included material is usually granular but may also be amorphous or fibrillar, all having the same origin during differentiation of the tracheary element. Vessels and tracheids with such inclusions were first reported in 1895 by Heinricher inLathraea (Scrophulariaceae). During the early decades of this century graniferous tracheary elements were noted in a few other taxa by different workers but were largely forgotten until the early 1960’s. This paper reviews the early literature and the research carried out during the past twenty-five years on these peculiar cells. Graniferous tracheary elements are found typically in the body of the haustorium of the root parasite, especially in the expanded xylem tissue or “vascular core.” The cells are most widely documented for the hemi-parasitic Santalaceae and were first recorded there in 1910 by Benson. She named the cells “phloeotracheides,” believing they combined the functions of phloem and xylem conducting elements. Heinricher and Benson both considered the granules to be composed of amylodextrin starch and Benson also believed the cells contained an enucleated protoplast. Our work has demonstrated that the granules in the Santalaceae are proteinaceous and that the cells are dead at maturity. In 1978 we therefore renamed them “graniferous tracheary elements.” They occur in all species of the Santalaceae so far investigated and inAtkinsonia ligustrina andNuytsia floribunda of root parasitic Loranthaceae. In these two families graniferous tracheary elements have the same organization. Their occurrence in haustoria of root parasites from other families is also reviewed. Although few observations are yet available in the Olacaceae, the granules inXimenia americana are found to be starch grains, like those inLathraea, whereas those inOlax phyllanthi are protein. Such fundamentally different material in haustorial tracheary elements within the same taxonomic group naturally raises the question of relationships within the family. The function of graniferous tracheary elements has not been experimentally investigated but we have suggested that for some Santalaceae they might serve as a device for regulating the flow of xylem sap through the haustorium.     

Phylogenetic relationships within the class Anthozoa (phylum Cnidaria) based on nuclear 18S rDNA sequences

Taxonomic relationships within the corals and anemones (Phylum Cnidaria: Class Anthozoa) are based upon few morphological characters. The significance of any given character is debatable, and there is little fossil record available for deriving evolutionary relationships. We analyzed complete 18S ribosomal sequences to examine subclass-level and ordinal-level organization within the Anthozoa. We suggest that the Subclass Ceriantipatharia is not an evolutionarily relevant grouping. The Order Corallimorpharia appears paraphyletic and closely related to the Order Scleractinia. The 18S rRNA gene may be insufficient for establishing robust phylogenetic hypotheses concerning the specific relationships of the Corallimorpharia and the Ceriantharia and the branching sequence for the orders within the Hexacorallia. The 18S rRNA gene has sufficient phylogenetic signal, however, to distinguish among the major groupings within the Class Anthozoa, and we use this information to suggest relationships for the enigmatic taxa Dactylanthus and Dendrobrachia.     

Radiolaria divided into Polycystina and Spasmaria in combined 18S and 28S rDNA phylogeny

Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis.     

Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences

To study the inter- and infrafamilial phylogenetic relationships in the order Caryophyllales sensu lato (s.l.), ～930 base pairs of the matK plastid gene have been sequenced and analyzed for 127 taxa. In addition, these sequences have been combined with the rbcL plastid gene for 53 taxa and with the rbcL and atpB plastid genes as well as the nuclear 18S rDNA for 26 taxa to provide increased support for deeper branches. The red pigments of Corbichonia, Lophiocarpus, and Sarcobatus have been tested and shown to belong to the betacyanin class of compounds. Most taxa of the order are clearly grouped into two main clades (i.e., "core" and "noncore" Caryophyllales) which are, in turn, divided into well-defined subunits. Phytolaccaceae and Molluginaceae are polyphyletic, and Portulacaceae are paraphyletic, whereas Agdestidaceae, Barbeuiaceae, Petiveriaceae, and Sarcobataceae should be given familial recognition. Two additional lineages are potentially appropriate to be elevated to the family level in the future: the genera Lophiocarpus and Corbichonia form a well-supported clade on the basis of molecular and chemical evidence, and Limeum appears to be separated from other Molluginaceae based on both molecular and ultrastructural data.     

Plant eats plant: sap-feeding witchweeds and other parasitic angiosperms

About one in every hundred species of flowering plant is parasitic and obtain some or all of their carbon, nutrients and water from the sap of their hosts. They possess unique morphological and metabolic adaptations but are more than just botanical curiosities.     

Reproductive development and nuclear DNA content in angiosperms

Correlates of nuclear DNA content in angiosperms have been noted previously for a range of features, cellular to geographic. A new hypothesis, the correlation between nuclear DNA content and reproductive developmental features (after Cavalier-Smith, Journal of Cell Science 34, 247–268, 1978) is posed and tested here. Of three features tested (megasporogenesis, microsporogenesis, and endosperm development), megasporogenesis alone was shown to be correlated with nuclear DNA amount. The hypothesis was examined in 107 families of angiosperms using nonparametric statistics, and in 53 families of monocotyledons and outgroups using a phylogenetic test of association. A correlation was found between large genomes and successive microsporogenesis for all angiosperms, but not for monocots and dicots analyzed separately, thus underlining the importance of taking into account phylogenetic relationships in such studies. A correlation between cellular endosperm and large genomes in dicotyledons needs to be confirmed in a phylogenetic context. A tendency for deviations from monosporic megasporogenesis to occur in taxa that have a nuclear DNA content of over 9.0 pg/C was demonstrated using both phylogenetic and nonphylogenetic tests. It is hypothesized that cytoskeleton dynamics are affected in reproductive cells, enabling decoupling between nuclear and cytoplasmic cell cycles and leading to variation in reproductive development.     

Paraphyly of Homoptera and Auchenorrhyncha inferred from 18S rDNA nucleotide sequences

Evolutionary affiliations of eighteen families of Hemiptera (s.l.) are inferred using molecular phylogenetic analysis of nucleotide (nt) sequences of 18S rDNAs. Exemplar taxa include: Archaeorrhyncha (=Fulgoromorpha): flatid, issid, dictyopharid, cixiid and delphacid; Prosorrhyncha (=Heteropterodea): Peloridiomorpha (=Coleorhyncha) -peloridiid, Heteroptera gerrid, lygaeid and mirid; Clypeorrhyncha [=extant (monophyletic) cicadomorphs]: cicadid, cercopoids (cercopid, aphrophorid), membracid and cicadellids (deltocephaline and cicadelline); and Sternorrhyncha: psyllid, aleyrodid, diaspidid and aphid. Analysed sequences encompass a region beginning ?550 nucleotides (nts) from the 5'-end to ?200 nts upstream from the 3'-end of the gene [?1150 base pairs (bp) in euhemipteran to >1400 bp in sternorrhynchan taxa]. Maximum parsimony and bootstrap analyses (PAUP) identify four principal hemipteran clades, Stenorrhyncha, Clypeorrhyncha, Archaeorrhyncha and Prosorrhyncha. These lineages are identified by synapomorphies distributed throughout the gene. Sternorrhyncha is a sister group to all other Hemiptera (i.e. Euhemiptera sensu Zrzavy), rendering Homoptera paraphyletic. Within Euhemiptera, clades Clypeorrhyncha, Archaeorrhyncha, Prosorrhyncha and Heteroptera are supported by one, three, two and three synapomorphic sites, respectively. There is equitable parsimonious inference for Archaeorrhyncha as the sister group to Prosorrhyncha (Neoherriiptera sensu Sorensen et al.) or Clypeorrhyncha, in either case rendering Auchenorrhyncha paraphyletic. Neohemiptera is supported by one synapomorphy. Within Clypeorrhyncha, clade cicada + cercopoids is the sister group of the clade cicadellids + membracid (Membracoidea sensu Dietrich & Deitz). Among archaeorrhynchans, clade delphacid + cixiid is the sister group of the clade dictyopharid + flatid + issid. Within Prosorrhyncha, the peloridiid is sister to the Heteroptera. Within Heteroptera, gerrid is the sister group of the clade mirid + lygaeid (Panheteroptera sensu Schuh). Based on secondary structure of synonymous 18S rRNA, two synapomorphies each of Sternorrhyncha, Prosorrhyncha and Heteroptera are compensatory substitutions on stem substructures. All other synapomorphies identifying major lineages of Hemiptera are noncompensatory substitutions on either bulges or stems. Short basal internodal distances suggest radiation of hemipteran lineages at the suborder level occurred rapidly. Morphological, palaeoentomological and eco-evolutionary factors supporting the 18S rDNA-based phylogenetic tree are discussed.     

Internal phylogeny of the Chilopoda (Myriapoda, Arthropoda) using complete 18S rDNA and partial 28S rDNA sequences

The internal phylogeny of the 'myriapod' class Chilopoda is evaluated for 12 species belonging to the five extant centipede orders, using 18S rDNA complete gene sequence and 28S rDNA partial gene sequence data. Equally and differentially weighted parsimony, neighbour-joining and maximum-likelihood were used for phylogenetic reconstruction, and bootstrapping and branch support analyses were performed to evaluate tree topology stability. The results show that the Chilopoda constitute a monophyletic group that is divided into two lines, Notostigmophora (= Scutigeromorpha) and Pleurostigmophora, as found in previous morphological analyses. The Notostigmophora are markedly modified for their epigenic mode of life. The first offshoot of the Pleurostigmophora are the Lithobiomorpha, followed by the Craterostigmomorpha and by the Epimorpha s. str. (= Scolopendromorpha + Geophilomorpha), although strong support for the monophyly of the Epimorpha s. lat. (= Craterostigmomorpha + Epimorpha s. str.) is only found in the differentially weighted parsimony analysis.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。     

A preliminary phylogeny of the Pentatomomorpha (Hemiptera: Heteroptera) based on nuclear 18S rDNA and mitochondrial DNA sequences

Pentatomomorpha is the second suborder in size only to Cimicomorpha in Heteroptera. However, the phylogenetic relationships among members of the suborder are not well established. Sequences from partial nuclear ribosomal 18S gene and mitochondrial COX1 gene were analyzed separately and in combination to generate a preliminary molecular phylogeny of Pentatomomorpha based on 40 species representing 17 putative families. Analyses of the combined sequence data provided a better-resolved and more robust hypothesis of Pentatomomorpha phylogeny than did separate analyses of the individual genes. The phylogenies were mostly congruent with morphological studies. Results strongly supported the monophyly of the infraorder Pentatomomorpha, and the placement of Aradoidea as sister to Trichophora. The monophyletic Trichophora was grouped into two major lineages, one being the superfamily Pentatomoidea, and the other comprising Lygaeoidea, Coreoidea, and Pyrrhocoroidea. The analysis of the ML and ME trees of combined dataset supported the monophyletic Pentatomoidea. In all analysis the Pyrrhocoroidea was polyphyletic; the monophyletic Lygaeoidea was supported only in the analysis of ME tree, and Coreoidea was polyphyletic except in the MP tree of combined dataset. The molecular and morphylogical data both indicated that the family Coreoidae should be revised subsequently. Our phylogenetic results suggested that the COX1 segment alone might not be an optimal molecular marker for the phylogeny of Pentatomomorpha.