The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Principal features of the cpDNA evolution in Taraxacum (Asteraceae, Lactuceae): a conflict with taxonomy

Phylogenetic relationships based on the chloroplast genome of Taraxacum were studied. Representative samples of 44 sections or species groups and a number of isolated species were analyzed. On the basis of the sequence variation in psbA–trnH and in trnL–trnF, mutations associated with RFLPs were monitored. Five RFLPs without homoplasy were recognized and used to reconstruct four main cpDNA groups (haplotypes); Group I is ancestral and, contrary to the information in the primary sequences, the RFLPs were not distinct from those of the outgroup species of Agoseris and Prenanthes. This group corresponds to dandelions believed to be ancestral on the basis of morphological data and previous studies of the chloroplast genome. A comparison of parsimony analysis of morphological and chloroplast data showed an overall lack of congruence. The conflict can most probably be accounted for as a consequence of reticulation.     

DIR: a novel DNA rearrangement associated with inverted repeats.

A novel DNA rearrangement has been characterised that is both a direct and inverted repeat.This rearrangement involves the 2-fold duplication of a plasmid sequence adjacent to the site of insertion of a long palindrome.The sequence of this rearrangement suggests that it has arisen by strand slippage from the leading to the lagging strand of the replication fork as a consequence of the presence of the long palindrome.     

Comparison of chloroplast and nuclear phylogeny in the autogamous annualMicroseris douglasii (Asteraceae: Lactuceae)

Morphology suggests that the Californian annualMicroseris douglasii is a monophyletic sister group to the other three diploid annuals ofMicroseris. Phylogenetic analysis of 44 inbred strains ofM. douglasii derived from 23 populations with 72 RAPD markers in the nuclear DNA strongly supports this phylogeny. However, 13 chloroplast RFLPs divideM. douglasii into four distinct groups. Two of these each share one or more cpRFLPs withM. bigelovii andM. pygmaea. Several hypotheses can explain the incongruence between nuclear and chloroplast phylogeny: (1) random sorting out of chloroplasts during phylogeny from a polymorphic pool, (2) cytoplasmic introgression from the related annualM. bigelovii intoM. douglasii after hybridization followed by elimination of theM. bigelovii nuclear genome. We suggest cytoplasmic introgression as the most likely origin. Possible remnants of nuclear introgression have been found in two populations ofM. douglasii that are polymorphic for chloroplast types. In these populationsM. bigelovii type chloroplast DNA seems to be accompanied by nuclear genes for flower color and leaf shape.     

Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.     

Identification of genes that are associated with DNA repeats in prokaryotes

Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.     

Evolution of microsporangium numbers in Microseris (Asteraceae: Lactuceae)

The genus Microseris contains species with disporangiate stamens and species with tetrasporangiate stamens. We determined the number of microsporangia per stamen in serial sections of heads from all 13 species of Microseris, its close relative Uropappus lindleyi, and the two allopolyploid species of Slebbinsoseris. Four Microseris species, three diploid and one tetraploid, have two microsporangia per stamen; all other species investigated have four. The most parsimonious assumption is that the disporangiate condition is derived and arose once in the evolution of Microseris. The inheritance of the number of microsporangia per stamen in crosses between M. bigelovii (disporangiate) and M. douglasii (tetrasporangiate) was determined. Segregation of microsporangium number per stamen in F2s derived from these crosses is quantitative rather than Mendelian. The average number of microsporangia per stamen in the F2 plants ranges from 2.0 to 4.0. There is a predominance of tetrasporangiate stamens in the F1 and in most F2 plants. The observed pattern of inheritance suggests a major gene with dominance and quantitative modifiers.     

Chloroplast DNA evidence for the roles of island colonization and extinction in Tolpis (Asteraceae: Lactuceae)

Tolpis consists of ～13 species native to Africa, Europe, and Macaronesia, with at least one species endemic to each of the four major archipelagos of the Azores, Madeira Islands, Canary Islands, and Cape Verde Islands. All but two of these species develop woody stems by maturity. Chloroplast DNA restriction site variation was analyzed for all species of Tolpis and four outgroups in order to understand the patterns of island colonization and evolution of woodiness in this genus. Parsimony analyses revealed a strongly supported monophyletic Tolpis. Within the genus, the following three well-supported groups were detected: all species from the Canary Islands and Cape Verde Islands, both Azorean species, and both continental species. The Canary Island/Cape Verde clade was sister to the two continental species, and the Azorean clade was sister to this group. The two Madeiran species of Tolpis occupied the basalmost positions within the genus. When biogeography was mapped onto this phylogeny, nine equally parsimonious reconstructions (five steps each) of dispersal history were detected, which fell into two groups: eight reconstructions implied that Tolpis colonized Madeira from the continent, followed by continental extinction and subsequent continental recolonization, while one reconstruction implied that Tolpis colonized Macaronesia four times. Two of the reconstructions involving continental extinction required the least amount of overall dispersal distance. The cpDNA phylogeny also suggests that woodiness arose in the common ancestor of all extant Tolpis, followed by two independent reversals to an herbaceous habit. Assuming that one of the eight reconstructions favoring continental extinction and recolonization is true, our results suggest that Tolpis may represent the first documented example of a woody plant group in Macaronesia that has recolonized the mainland in herbaceous form.     

Self-fertility and associated flower head traits in the Iberian taxa ofLactuca and related genera (Asteraceae: Lactuceae)

Sexual reproduction is the main reproduction mechanism among the 14 wild Iberian taxa ofLactuca, Prenanthes, Cicerbita, andMycelis. High levels of self-fertilization occur inLactuca, as well as facultative and obligate xenogamy. Xenogamy is strongly correlated with large capitula having blue or bright yellow colouration, high P/O ratios, and long anthesis, whereas self-fertilization is correlated with smaller capitula having pale yellow colouration, small P/O ratios, and short anthesis. Large variations occur in P/O index among taxa showing similar fertilization mechanism, probably in relation to the number of florets included in flower heads. Interesting differences in reproductive systems have been detected between subspecies ofLactuca viminea.     

Distribution of reproductive systems and taxonomical concepts in the genus Taraxacum F.H. Wigg. (Asteraceae,Lactuceae) in Germany

Reproductive studies of the genus Taraxacum F.H. Wigg. (Asteraceae) were carried out in the Eastern part of Germany (approx. 1,300 individuals). Using the method of variability of pollen diameter we observed almost in all cases only obligately agamospermous plants in the area studied. With regard to Germany, regions with 1. obligately sexual and obligately agamospermous 2. facultatively and obligately agamospermous 3. exclusively obligately agamospermous individuals, could be distinguished. From these facts, it is concluded that the microspecies concept is preferable to the traditional macrospecies concept, especially in regions with exclusively obligately agamospecies. The microspecies concept is also applicable in a differentiated form in the remaining regions.     

Nuclear DNA Polymorphisms Among Strains of Microseris bigelovii (Asteraceae: Lactuceae) Amplified from Arbitrary Primers

The autogamous annual Microseris bigelovii occurs in isolated populations along a narrow strip of land along the Pacific Coast from Southern California to Victoria, B. C. — Thirteen inbred strains were derived from 11 representative populations. Nuclear DNA from these strains was amplified using single arbitrary 10-base-pair primers. 194 amplification products were obtained with 22 primers. Of these, 44 were shared by all strains, 32 were present or absent in only one of the strains. The remaining 118 amplification products were shared by 2 to 11 strains. Attempts to construct a cladogram of the 13 inbred genotypes revealed 5 monophyletic groups. Two of these consist of duplicate isolates from two populations each, two comprise two strains each derived from neighboring populations, and one comprises the three populations from Victoria, B. C., which are postulated to be the result of local differentiation after a single founder event. The Victoria populations share the only known chloroplast mutation in M. bigelovii with a strain from the San Mateo County south of San Francisco. Beyond the local groups of strains we found more or less random allele associations rather than cladistic groupings, with overall genetic distances (UPGMA) more or less related to geographic distance. Achene transport, too rare for direct observationmust be effective to allow gene flow among populationsin M. bigelovii.     

Phenotypic Plasticity of Capitulum Morphogenesis in Microseris pygmaea (Asteraceae: Lactuceae)

Inbred populations of the annual Chilean species Microserispygmaea show phenotypic plasticity in the number of floretsper capitulum. In order to find out how this plastic variationmay arise during development, two inbred lines (A92 and C96)were grown under short day conditions. Groups of plants fromeach strain were transferred to long day conditions at about2 week intervals. In this way we introduced variation for plantsize (number of leaves per plant) at onset of flowering. Thenumber of florets per mature capitulum increased linearly withplant size. After transfer to long day conditions, plants wereharvested daily for light microscopic measurements of meristemwhole mounts. Only the first capitulum of each plant was analysed.All florets were formed after 12 d in strain C96 and after 14d in strain A92. In order to detect the effect of plant sizeon morphogenesis, we performed a multiple regression analysisof developmental parameters on time and number of leaves. Widthand height of the capitulum receptacle increased daily witha growth rate depending on the starting size. Differences inmeristem size were detected already in vegetative plants ofdifferent sizes. In contrast, there was no influence of plantsize on floret primordium size. We combined the multiple regressionmodels in one simple model for prediction of floret numbersfrom numbers of leaves per plant at onset of flowering. Predictionsof this model agree with observed relationships in both inbredlines.Copyright 1994, 1999 Academic Press Asteraceae, capitulum, inflorescence, meristem, meristic character, Microseris pygmaea, morphogenesis, phenotypic plasticity     

Mitochondrial-like DNA sequences flanked by direct and inverted repeats in the nuclear genome of Toxoplasma gondii.

In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.     

Nonautonomous inverted repeat Alien transposable elements are associated with genes of both monocotyledonous and dicotyledonous plants

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5′-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.     

The interactions of enzyme and chemical probes with inverted repeats in supercoiled DNA

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as S1, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endonucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Distributions of dimeric tandem repeats in non-coding and coding DNA sequences

We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast). For coding DNA, we find that all 16 distribution functions are exponential. For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function. We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms. These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations. By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer.