Comprehensive molecular phylogeny of the sub-family Dipterocarpoideae (Dipterocarpaceae) based on chloroplast DNA sequences

Dipterocarpoideae, the largest sub-family of well-known plant family Dipterocarpaceae, dominates in South Asian rain forests. Although several previous studies addressed the phylogeny of the Dipterocarpaceae family, relationships among many of its genera from the Dipterocarpoideae sub-family are still not well understood. In particular, little is known about the relationships of the genera Vateriopsis, Stemonoporus, Vateria and inconsistence remains between phylogenetic results and taxonomic classifications of Shorea and Hopea species. We studied molecular phylogeny of the sub-family Dipterocarpoideae using the trnL-trnF spacer, trnL intron and the matK gene sequences of chloroplast DNA (cpDNA). This study is the first comprehensive phylogeny reconstruction for the sub-family Dipterocarpoideae based on cpDNA, as it includes most genera (14) and a large number of species (79) with most species endemic to Sri Lanka, as well as one species from Seychelles and one species from the genus Monotes from Madagascar. Phylogenetic trees were constructed using the Neighbor Joining (NJ) and Maximum Likelihood (ML) methods using combined set of sequences including all three cpDNA regions. The topologies of the NJ and ML trees were to a certain extent, consistent with the current taxonomy of Dipterocarpoideae based on morphology and with previous molecular phylogenies based on cpDNA. Furthermore, our results provided new evidence regarding the relationships of the following genera: Vateriopsis and Stemonoporus and about the validity of the previous morphology based classifications of Shorea species. In addition, the topology of our trees was consistent with the classification of Shorea species proposed by Maury (1978), Maury-Lechon (1979) and Symington (1943). Finally, our results provided evidence for the affinity of the genus Monotes to Asian Dipterocarpoideae rather than to Tiliaceae and indicated that it is a good candidate for outgroup species for future studies of the former sub-family.     

Molecular phylogeny of Dipterocarpaceae in Southeast Asia using RFLP of PCR-amplified chloroplast genes

Dipterocarpaceae is the dominant family of Southeast Asia's climax tropical rain forest region, and it contains the region's most important commercial timber species. A molecular phylogeny of the Dipterocarpaceae subfamily Dipterocapoideae was constructed using restriction fragment length polymorphisms of polymerase chain reaction-amplified specific genes in chloroplast DNA. A total of 141 site changes were detected among ten genera and 30 species in 11 different genes: rbcL, psbA, psbD, rpoB, rpoC, petB, atpH, 16S, psaA, petA and trnK. Phylogenetic trees constructed by Wanger parsimony and neighbor-joining methods, using Upuna as the outgroup, displayed five monophytelic groups that included Upuna: HopeaShorea-Parashorea-Neobalanocarpus; Dryobalanops; Dipterocarpus; Anisoptera-Vatica-Cotylelobium; and Upuna. The phylogenetic trees clearly separate species with two different base chromosome numbers: the first group is x=7, and the other is x=11. The x=7 group is thought to be in a synapomorphic character state. Parashorea lucida is a sister to most Shorea species. Neobalanocarpus heimii and Hopea from a clade of a sister to two Shorea species, and Cotylelobium and Vatica are closely related species. Our conclusions agree with a phylogeny derived from wood anatomy data analysis, and with Symington's and Ashton's taxonomic classifications.The raw data of the PCR-RFLP analysis can be obtained from the authors     

Molecular phylogeny ofForsythia (Oleaceae) based on chloroplast DNA variation

Phylogenetic relationships of ten wild species and several cultivars ofForsythia were reconstructed based on the chloroplast (cp) DNA variation. A total of 216 cpDNA variants, 44 of which were potentially phylogenetically informative, was detected using 24 restriction endonucleases. Phylogenetic analysis usingFontanesia andAbeliophyllum as outgroups revealed four well defined species groups in the genus: 1)F. suspensa, 2)F. europaea — F. giraldiana, 3)F. ovata — F. japonica — F. viridissima, and 4)F. koreana — F. manshurica — F. saxatilis. The amount of support for each monophyletic group was evaluated by various methods including character number, decay analysis, parsimony bootstrapping, Neighbour-Joining (NJ) — bootstrapping, NJ-jackknifing, and the topology-dependent permutation tail probability (T-PTP) test. The data do not support the hybrid origin ofF. intermedia fromF. suspensa andF. viridissima. The disjunctly distributed European species,F. europaea, was identified as a sister species of the ChineseF. giraldiana and it was probably derived through recent long distance dispersal.     

Molecular phylogeny of chloroplast DNA of Nicotiana species

Amplified fragment length polymorphism (AFLP) analysis of chloroplast DNA was used to study the relationships within the genus Nicotiana. Resulting phylogenetic tree, reconstructed using the UPGMA method, generally agreed with the existing taxonomic classification based on morphological and cytogenetic data, as well as sequence comparison of the internal transcribed spacer of the nuclear ribosomal DNA.     

Molecular phylogeny ofNothofagus (Nothofagaceae) based on theatpB-rbcL intergenic spacer of the chloroplast DNA

The genusNothofagus is distributed in the Southern Hemisphere from South America to Oceania, and its distribution has been assumed to be formed by continental drift by means of Gondwana break-up during the Mesozoic era. The phylogeny of the genus was elucidated by the sequences ofatpB-rbcL intergenic spacer of cpDNA for the better understanding of its evolution and biogeography. The phylogeny ofNothofagus corresponded completely to the pollen morphology which recognizes four pollen types in extant species, and agrees well with the taxonomic system of Hill and Read (1991) although there, the subgenusNothofagus showed in unresolved polytomy. The topology of the phylogenetic tree reveals that subgenusLophozonia was derived first, and thenFuscospora, Nothofagus andBrassospora. Species from South America and New Zealand were assigned to each cluster according to their pollen morphology. Therefore, diversification ofNothofagus should have already proceeded at the subgenus level before the completion of Gondwana break-up Tropical species distributed in New Guinea and New Caledonia whose evolutionary history has been controversial were revealed to be a derived group. All five New Caledonian species formed a monophyletic group with very few sequence divergences in the intergenic spacer of cpDNA, thus showing rapid adaptive radiation in the island. Evolutionary trends of several morphological traits ofNothofagus are discussed. The evolution of valve number of cupules, number of nuts per cupule, and habit of leaf-fall (evergreen or deciduous) which are diversified in the genus, were revealed as having occurred several times as the result of convergence.     

Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA

With the completion of the first gymnosperm mitochondrial genome (mtDNA) from Cycas taitungensis and the availability of more mtDNA taxa in the past 5 years, we have conducted a systematic analysis of DNA transfer from chloroplast genomes (cpDNAs) to mtDNAs (mtpts) in 11 plants, including 2 algae, 1 liverwort, 1 moss, 1 gymnosperm, 3 monocots, and 3 eudicots. By using shared gene order and boundaries between different mtpts as the criterion, the timing of cpDNA transfer during plant evolution was estimated from the phylogenetic tree reconstructed independently from concatenated protein-coding genes of 11 available mtDNAs. Several interesting findings emerged. First, frequent DNA transfer from cpDNA to mtDNA occurred at least as far back as the common ancestor of extant gymnosperms and angiosperms, about 300 MYA. The oldest mtpt is trnV(uac)-trnM(cau)-atpE-atpB-rbcL. Three other mtpts--psaA-psaB, rps19-trnH(gug)-rpl2-rpl23, and psbE-psbF--were dated to the common ancestor of extant angiosperms, at least 150 MYA. However, all protein-coding genes of mtpts have degenerated since their first transfer. Therefore, mtpts contribute nothing to the functioning of mtDNA but junk sequences. We discovered that the cpDNA transfers have occurred randomly at any positions of the cpDNAs. We provide strong evidence that the cp-derived tRNA-trnM(cau) is the only mtpt (1 out of 3 cp-derived tRNA shared by seed plants) truly transferred from cpDNA to mtDNA since the time of the common ancestor of extant gymnosperms and angiosperms. Our observations support the proposition of Richly and Leister (2004) that "primary insertions of organellar DNAs are large and then diverge and fragment over evolutionary time."     

Molecular phylogeny ofIva (Asteraceae,Heliantheae) based on chloroplast DNA restriction site variation

Iva s.str. (comprising ten species) was examined by cpDNA restriction site variation to determine phyletic relationships within the group. The results were compared with relationships proposed from other data. A total of 86 restriction site mutations was detected, 47 of which proved phylogenetically informative. A single most parsimonious tree was obtained using both Wagner and Dollo parsimony. The tree revealed three main lineages that are congruent with the three chromosome lineages (base numbers of x = 16, 17, 18). The monophyly of the x = 16 and 18 groups was supported strongly by molecular data, while the monophyly of x = 17 lineage was only supported moderately. Relationships among the three lineages indicate that the sect.Iva is paraphyletic because sect.Linearbractea is nested within it. Both morphological data and the secondary chemical data are in agreement with the proposed cpDNA phylogeny. Because of this agreement, sect.Iva is revised such that,I. axillaris was excluded and positioned within the newly proposed sect.Rhizoma. Patterns and rates of cpDNA evolution were also examined. The results indicated an uneven evolution in the chloroplast genome with different rates of cpDNA evolution in at least a few species ofIva. However, the evolutionary clock hypothesis can not be rejected within most of the lineages inIva.     

The complete chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: organelle genomes of the smallest eukaryote are examples of compaction

The complete nucleotide sequence of the mt (mitochondrial) and cp (chloroplast) genomes of the unicellular green alga Ostreococcus tauri has been determined. The mt genome assembles as a circle of 44,237 bp and contains 65 genes. With an overall average length of only 42 bp for the intergenic regions, this is the most gene-dense mt genome of all Chlorophyta. Furthermore, it is characterized by a unique segmental duplication, encompassing 22 genes and covering 44% of the genome. Such a duplication has not been observed before in green algae, although it is also present in the mt genomes of higher plants. The quadripartite cp genome forms a circle of 71,666 bp, containing 86 genes divided over a larger and a smaller single-copy region, separated by 2 inverted repeat sequences. Based on genome size and number of genes, the Ostreococcus cp genome is the smallest known among the green algae. Phylogenetic analyses based on a concatenated alignment of cp, mt, and nuclear genes confirm the position of O. tauri within the Prasinophyceae, an early branch of the Chlorophyta.     

The value of repetitive sequences in chloroplast DNA for phylogeographic inference: a comment on Vachon & Freeland 2011

In a recent Technical Advance article, Vachon and Freeland (2011, Molecular Ecology Resources, 11, 279-285.) evaluate the utility of repetitive and non-repetitive variation in the chloroplast genome for phylogeographic inference, using variation in Phragmites australis as an example. While we agree that repetitive and nonrepetitive regions evolve at different rates and homoplasy can impact results, we disagree with the conclusion that repetitive regions are inappropriate for large-scale phylogeographic studies. Here we describe limitations to the study dataset and analysis, and provide an alternative viewpoint on the utility of repetitive regions for phylogeographic studies.     

Phylogeographic inferences from chloroplast DNA: quantifying the effects of mutations in repetitive and non-repetitive sequences

Phylogeographic inference can be a powerful tool in reconstructing species’ evolutionary histories; however, although inferred phylogeographic patterns should depend in part on the underlying types and rates of mutations, the effects of different types of mutations have seldom been quantified. In this study we identified two chloroplast minisatellites in the common reed Phragmites australis, and showed that these are more variable than chloroplast microsatellites. We then recreated parsimony networks of the global phylogeography of P. australis based on data that either included or excluded repetitive sequences (minisatellites and microsatellites), thereby illustrating the influence that these repetitive sequences can have on large‐scale phylogeographic inference. The resulting networks differed in the numbers of mutational steps, degrees of uncertainty, and total numbers of haplotypes. In addition, the suggested ancestor‐descendant relationships among lineages changed substantially depending on whether repetitive sequences were included. We therefore caution against the inclusion of repetitive sequences in large‐scale networks because of their high potential for homoplasy. Nevertheless, we advocate the inclusion of repetitive sequences in other analyses: specifically, we show that the ratio of mutations in repetitive vs. non‐repetitive regions can provide insight into the relative ages of lineages.     

Single nucleotide sequence analysis: a cost- and time-effective protocol for the analysis of microsatellite- and indel-rich chloroplast DNA regions

We present a simple method to screen for DNA sequence variation in microsatellite- and indel-rich regions of the chloroplast genome. The single nucleotide sequence (SNS) analysis provides a trade-off between the time- and cost-effective, but less informative and homoplasy-sensitive electrophoretic detection of microsatellite and indel size variation on the one hand, and more costly, but also more accurate methods like DNA sequencing on the other. The principle of the SNS method is to sequence one instead of all four nucleotides of a target region amplified by polymerase chain reaction. By careful selection of the respective nucleotide, almost the same amount of information can be retrieved from these partial sequences as could be from complete sequences; however, only a third to a fourth of the money and time resources are needed.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Heteroplasmy of chloroplast DNA in Medicago

Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.Contribution No 88-547-J from the Kansas Agricultural Experiment Station, Manhattan.     

Molecular phylogeny of the genus Hordeum using three chloroplast DNA sequences.

The genus Hordeum consists of three cytotypes (2x, 4x, and 6x). Its reproductive isolation has been incomplete between closely related species and hence the genetic relationship is reticulate and complex. We used 32 taxa of Hordeum and three chloroplast DNA sequences, matK, atpB-rbcL, and trnL-trnF in the current study. Molecular phylogenetic analysis based on sequence data of the three chloroplast DNA regions clearly demonstrated genetic relationships among taxa and origin of polypoids. The formation of H. secalinum likely involved hybridization between Hordeum marinum subsp. marinum and a Eurasian diploid possessing the H genome. The formation of hexaploid Hordeum brachyantherum involved hybridization between tetraploid H. brachyantherum and diploid H. marinum subsp. gussoneanum. The formation of three tetraploids, H. brachyantherum, Hordeum jubatum, and Hordeum guatemalense, probably involved hybridization between H. brachyantherum subsp. californicum and an altered H genome diploid. The formation of Hordeum arizonicum involved the two taxa Hordeum pusillum and H. jubatum.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.     

Molecular phylogeny of Oryzeae (Poaceae) based on DNA sequences from chloroplast, mitochondrial, and nuclear genomes

The phylogeny and evolutionary history of the rice tribe (Oryzeae) were explored using sequences of five DNA fragments (matK, trnL, nad1, Adh2, and GPA1) from chloroplast, mitochondrial, and nuclear genomes. Results indicate that (1) Oryzeae is monophyletic and falls into two main clades corresponding to the traditionally recognized subtribes; (2) previous recognition of three monotypic genera (Hydrochloa, Porteresia, and Prosphytochloa) is not justified; and (3) close affinities of the monoecious genera are not supported, suggesting the possibility of multiple origins of unisexual florets. Based on the magnitude of matK and GPA1 sequence divergence, we suggest that Oryza and Leersia branched off from the remaining genera of Oryzeae ～20 million years ago (mya), and separated from each other ～14 mya. A divergence time of ～9 mya is obtained for the most basal split within Oryza. These estimates suggest that Oryzeae diverged during the Miocene, and thus imply that long-distance dispersal appears to be one of the important factors in the diversification of the tribe.     

Origin and phylogeny of chloroplasts revealed by a simple correlation analysis of complete genomes

The complete sequenced genomes of chloroplast have provided much information on the origin and evolution of this organelle. In this paper we attempt to use these sequences to test a novel approach for phylogenetic analysis of complete genomes based on correlation analysis of compositional vectors. All protein sequences from 21 complete chloroplast genomes are analyzed in comparison with selected archaea, eubacteria, and eukaryotes. The distance-based analysis shows that the chloroplast genomes are most closely related to cyanobacteria, consistent with the endosymbiotic origin of chloroplasts. The chloroplast genomes are separated to two major clades corresponding to chlorophytes (green plants) s.l. and rhodophytes (red algae) s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution. For instance, the analysis places the chloroplasts of two chromophytes (Guillardia and Odontella) within the rhodophyte lineage, supporting secondary endosymbiosis as the source of these chloroplasts. The relationships among the green algae and land plants in our tree also agree with results from traditional phylogenetic analyses. Thus, this study establishes the value of our simple correlation analysis in elucidating the evolutionary relationships among genomes. It is hoped that this approach will provide insights on comparative genome analysis.     

Major lineages within Apiaceae subfamily Apioideae: a comparison of chloroplast restriction site and DNA sequence data

Traditional sources of taxonomic characters in the large and taxonomically complex subfamily Apioideae (Apiaceae) have been confounding and no classification system of the subfamily has been widely accepted. A restriction site analysis of the chloroplast genome from 78 representatives of Apioideae and related groups provided a data matrix of 990 variable characters (750 of which were potentially parsimony-informative). A comparison of these data to that of three recent DNA sequencing studies of Apioideae (based on ITS, rpoCl intron, and matK sequences) shows that the restriction site analysis provides 2.6–3.6 times more variable characters for a comparable group of taxa. Moreover, levels of divergence appear to be well suited to studies at the subfamilial and tribal levels of Apiaceae. Cladistic and phenetic analyses of the restriction site data yielded trees that are visually congruent to those derived from the other recent molecular studies. On the basis of these comparisons, six lineages and one paraphyletic grade are provisionally recognized as informal groups. These groups can serve as the starting point for future, more intensive studies of the subfamily.