Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

Obtention with polymerase chain reaction (PCR) of gene rbcL in the scrophulariaceae holoparasite Lathraea clandestina L. Comparison with autotroph Digitalis purpurea L. and Melampyrum pratense L.]

Amyloplastid DNA was extracted from the Scrophulariaceae holoparasite Lathraea clandestina L. and then purified. An rbcL gene amplification was performed using polymerase chain reaction. Two regions of well conserved sequences in Tobacco, Spinach and Maize rbcL gene have been used as primers. PCR yields a sequence of about 1,230 base pairs, almost nine tenths of the rbcL coding region. Identical results were obtained with plastid DNAs from Tobacco and two other species of Scrophulariaceae: the non-parasitic Digitalis purpurea L. and the hemiparasite Melampyrum pratense L. PCR products were digested with BamHI restriction enzyme and no changes were shown in the localization of the restriction site whatever the species assayed. Identical restriction patterns were also observed with Tobacco and Digitalis PCR products restricted with PstI and KpnI, whereas Melampyrum and Lathraea exhibited a different restriction pattern with PstI. So, despite slight differences, some analogies between Lathraea and Digitalis or Tobacco gene were evidenced. Cloning and sequencing of these PCR products could give a more accurate response to the following question: to what extent have changes occurred in the rbcL gene in a plant which lacks chlorophyll?     

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.     

Genome fragmentation is not confined to the peridinin plastid in dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3'-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates.     

A multigene phylogeny of the Gelidiales including nuclear large-subunit rRNA sequence data

Partial sequences (1032 bp) of the nuclear-encoded large ribosomal RNA gene (LSU) were determined for 16 gelidialean species, and analyzed separately and in combination with plastid rbcL and nuclear SSU gene sequences. The number of informative characters and levels of sequence divergence among taxa are intermediate in LSU sequences as compared to that for rbcL and SSU. Analyses of the separate LSU, and a combined LSU, SSU, and rbcL data sets have identified early-diverging lineages within the Gelidiales including Gelidiella, Pterocladia, Pterocladiella, and a lineage including Gelidium and species classified in other genera. The relationships among most gelidialean taxa are well-resolved and well-supported by analyses of the combined data; however, the relationships of Ptilophora and Capreolia remain unclear. It is speculated that these two lineages have diverged from a common ancestor over an evolutionarily short period of time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prospects for discriminating Zingiberaceae species in India using DNA barcodes

We evaluated nine plastid(matK, rbcL, rpoC1, rpoB,rpl36-rps8, ndhJ, trnL-F, trnH-psbA, accD) and two nuclear(ITS and ITS2) barcode loci in family Zingiberaceae by analyzing 60 accessions of 20 species belonging to seven genera from India.Bidirectional sequences were recovered for every plastid locus by direct sequencing of polymerase chain reaction(PCR)amplicons in all the accessions tested. However, only 35(58%)and 40 accessions(66%) yielded ITS and ITS2 sequences,respectively, by direct sequencing. In different bioinformatics analyses, matK and rbcL consistently resolved 15 species(75%)into monophyletic groups and five species into two paraphyletic groups. The 173 ITS sequences, including 138 cloned sequences from 23 accessions, discriminated only 12 species(60%), and the remaining species were entered into three paraphyletic groups. Phylogenetic and genealogic analyses of plastid and ITS sequences imply the possible occurrence of natural hybridizations in the evolutionary past in giving rise to species paraphyly and intragenomic ITS heterogeneity in the species tested. The results support using matK and rbcL loci for barcoding Zingiberaceae members and highlight the poor utility of ITS and the highly regarded ITS2 in barcoding this family, and also caution against proposing ITS loci for barcoding taxa based on limited sampling.     

Natural Hybrids between Ligularia vellerea and L.subspicata (Asteraceae: Senecioneae)

Three types of intermediate morphological individuals were found in the sympatric distribution areas of Ligularia vellerea and Lsubspicata. These intermediate individuals were hypothesized to be the hybrids of these two species based on the detailed comparison of their morphological characters. To test this hypothesis, we compared the DNA sequence of Lvellerea, Lsubspicata and the intermediate individuals by using the internal transcribed spacer region of nuclear ribosomal and atpB rbcL intergenic spacer region of the chloroplast. Direct sequencing of ITS clearly had additives and then cloned sequencing cloned out two kinds of sequences that were identical to those putative parental species, thus these putative hybrids were approved. With different morphological characters among three types, their plastid donors were all Lsubspicata according to the maternal sequences of atpB rbcL.     

Elatinaceae are sister to Malpighiaceae; Peridiscaceae belong to Saxifragales

Phylogenetic data from plastid (ndhF and rbcL) and nuclear (PHYC) genes indicate that, within the order Malpighiales, Elatinaceae are strongly supported as sister to Malpighiaceae. There are several putative morphological synapomorphies for this clade; most notably, they both have a base chromosome number of X = 6 (or some multiple of three or six), opposite or whorled leaves with stipules, unicellular hairs (also uniseriate in some Elatinaceae), multicellular glands on the leaves, and resin (Elatinacae) or latex (Malpighiaceae). Further study is needed to determine if these features are synapomorphic within the order. Malpighiaceae have previously been inferred as sister to Peridiscaceae based on rbcL sequence data, but the rbcL sequence of Whittonia is a chimera of two sequences, neither of which appears to be Whittonia. Our data from plastid (atpB, rbcL) and nuclear (18S rDNA) genes instead place Peridiscaeace as a member of the Saxifragales.     

rbcL Plastid Pseudogene as a Tool for Orobanche (Subsection Minores) Identification

Abstract: Among members of the Orobanchaceae, parasitism has led to a drastic reduction in the morphological characters useful in identification. This is especially the case of species belonging to the subsection Minores (section Orobanche ), which is a real tangle for agronomists and botanists. In this study, more than 120 specimens collected in the west of France were studied for their morphological characters and molecular markers. Their rbcL plastid gene was analyzed by RFLP and nucleotide sequencing. Because of several substitutions, insertions and deletions in the coding sequence, leading to stop codons and frameshift, this rbcL gene could be considered as a pseudogene. Variations in both 5' upstream and coding regions allowed us to distinguish among specimens inside the Minores subsection. It was then possible to point out morphological characters, not used in floras, that fit with molecular analysis and confirm the existence of four taxa. Consequently, a new key to species determination using molecular analysis or morphological characters or a combination of both is proposed for O. hederae, O. amethystea, O. minor and O. loricata. Finally, the value of plastid pseudogenes in taxonomy is discussed.     

Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae

DNA sequences of the plastid gene psaB were completed for 182 species of Orchidaceae (representing 150 different genera) and outgroup families in Asparagales. These data were analyzed using parsimony, and resulting trees were compared to a rbcL phylogeny of Orchidaceae for the same set of taxa after an additional 30 new rbcL sequences were added to a previously published matrix. The psaB tree topology is similar to the rbcL tree, although the psaB data contain less homoplasy and provide greater bootstrap support than rbcL alone. In combination, the two-gene tree recovers the five monophyletic subfamilial clades currently recognized in Orchidaceae, but fails to resolve the positions of Cypripedioideae and Vanilloideae. These new topologies help to clarify some of the anomalous results recovered when rbcL is analyzed alone. Both genes appear to be absent from the plastid genome of several achlorophyllous orchids, but are present in the form of presumably non-functional pseudogenes in Cyrtosia. This study is the first to document the utility of psaB sequences for phylogenetic studies of plants below the family level.     

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae)

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.     

Phylogeny of epiphytic Huperzia (Lycopodiaceae): paleotropical and neotropical clades corroborated by rbcL sequences

The phylogenetic relationships of Lycopodiaceae, and specifically the relationships among epiphytic Huperzia , were investigated by means of a cladistic analysis using plastid rbcL gene sequences. Huperzia species are partitioned into neotropical and paleotropical clades, a result that is incongruent with traditional morphology based taxonomy. The rbcL data corroborate an earlier study based on tmL intron and tmL-trnF spacer sequences. Together, these two molecular studies provide strong evidence that the diversification of epiphytic Huperzia is a comparatively recent phenomenon (Late Cretaceous-Tertiary), and that there has been widespread morphological convergence in epiphytes throughout the tropics.     

Phylogenetic studies of Ophioglossaceae: evidence from rbcL and trnL-F plastid DNA sequences and morphology

Ophioglossaceae are a putatively ancient lineage of ferns in which the aerial portion of the plant is composed of a single leaf. The simplicity of foliar morphology has limited the number of characters available for constructing classifications and contributed to taxonomic difficulties at nearly every level of classification within the family. Analysis of plastid DNA rbcL sequences from 36 species representing the diversity of Ophioglossaceae supported the monophyly of the family. Intrafamilial relationships were examined using rbcL and trnL-F plastid DNA sequences and morphological data. Individual and combined analyses of the three data sets revealed two main clades within the family, here termed ophioglossoid and botrychioid. In the botrychioid clade, Helminthostachys was sister to a broadly defined Botrychium, within which Botrychium in the narrow sense of some authors and Sceptridium were sister. Botrypus was paraphyletic, with Botrypus virginianus sister to Botrychium plus Sceptridium, and with Botrypus strictus sister to all other botrychioid species except Helminthostachys. In the ophioglossoid clade, Ophioglossum in the narrow sense was sister to Cheiroglossa plus Ophioderma, but relationships within Ophioglossum were not well supported.     

Phylogenetic analysis of Asplenium subgenus Ceterach (Pteridophyta: Aspleniaceae) based on plastid and nuclear ribosomal ITS DNA sequences

Phylogenetic relationships among 20 taxa of the fern genus Asplenium subgenus Ceterach (Filicopsida, represented by 73 accessions) were investigated using DNA sequence data from the nuclear ribosomal internal transcribed spacers (ITS nDNA) and plastid trnL-F intergenic spacer. In addition, a single sample per taxon was used in an analysis of the plastid rbcL gene. Chromosome counts were determined for all the samples, and these demonstrated a range from diploid to octoploid. Analyses of the DNA sequence data indicated that Asplenium subgenus Ceterach is polyphyletic, implicating homoplasy in the characters previously used to circumscribe this taxon. Plastid trnL-F and rbcL analyses resulted in identical tree topologies. The trees produced from the separate plastid and nuclear matrices agree in (1) the recognition of identical groups of accessions corresponding to A. dalhousiae, A. ceterach, A. aureum, A. cordatum, A. phillipsianum, and A. haughtonii; (2) the division of A. subg. Ceterach into two subclades, a Eurasian-Macaronesian and a strictly African alliance; (3) the position of A. dalhousiae as a member of the former subclade; (4) the lack of genetic variation in A. cordatum despite its morphological variability; and (5) the clustering of each autopolyploid with their diploid ancestor. However, the plastid and nuclear trees differ in their placement of A. haughtonii and A. dalhousiae, which might be due to different evolutionary histories of nuclear and plastid genomes, and is possibly an indication of ancient hybridization. The analyses confirm the existence of several strictly African taxa. Asplenium phillipsianum and A. cordatum each form species complexes of diploid and autopolyploid taxa, from which a third, morphologically intermediate, allotetraploid species has originated. Asplenium haughtonii is a distinct endemic species from Saint Helena. The maternally inherited plastid sequences support the hypothesis that A. aureum is an ancestor of A. lolegnamense and of A. octoploideum. Because gene conversion did not eliminate divergent ITS alleles in the allopolyploids, their reticulate ancestry could be demonstrated. Biparentally inherited nrITS sequences support the allopolyploid status of A. aureum, A. lolegnamense, and A. punjabense, indicating they share the ancestral A. javorkeanum genome.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.     

Identifying clades in Asian Annonaceae: monophyletic genera in the polyphyletic Miliuseae

The tribe Miliuseae (Annonaceae) comprises six genera distributed in Asia: Alphonsea, Mezzettia, Miliusa, Orophea, Platymitra, and Phoenicanthus. A phylogenetic study to investigate the putative monophyly of the tribe and the intergeneric relationships is presented here. Nucleotide sequences of the plastid gene rbcL, trnL intron, and trnL-trnF intergenic spacer were analyzed from 114 Annonaceae taxa, including 24 Miliuseae species and two outgroups using maximum parsimony and Bayesian inference. The two data sets (rbcL and the trnL-trnF regions) were analyzed separately and in combination. Miliuseae were found to be polyphyletic due to the position of Mezzettia and are part of a large, predominantly Asian and Central-American clade (miliusoid clade). Although intergeneric relationships were poorly resolved, all genera, except Polyalthia, were monophyletic, supporting previous generic delimitation based on morphology. A group of three Polyalthia species seems the most likely sister group of Miliusa. Several infrageneric groups of Miliusa, Orophea, and Polyalthia are supported by both molecular and morphological data. No morphological synapomorphies have yet been found for the miliusoid clade. Molecular clades within the miliusoid clade, however, can be characterized by size and the shape of the outer petals, number of ovules per carpel, and the size of the fruits.     

Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts

The objective of this study was to determine if mRNA sequences downstream of the translation initiation codon are important for translation of plastid mRNAs. We have employed a transgenic approach, measuring accumulation of the neomycin phosphotransferase (NPTII) reporter enzyme translationally fused with 14 N-terminal amino acids encoded in the rbcL or atpB plastid genes. NPTII accumulation from wild-type and mutant rbcL and atpB segments was compared. We report that silent mutations in the rbcL segment reduced NPTII accumulation 35-fold. In contrast, mutations in the atpB mRNA reduced NPTII accumulation only moderately from approximately 7% (w/w) to approximately 4% (w/w) of the total soluble cellular protein, indicating that the importance of sequences downstream of the translation initiation codon are dependent on the individual mRNA. Information provided here will facilitate transgene design for high-level expression of recombinant proteins in chloroplasts by translational fusion with the N-terminal segment of highly expressed plastid genes or by introduction of silent mutations in the N-terminal part of the coding region.