The complete chloroplast genome sequence of Pelargonium x hortorum: organization and evolution of the largest and most highly rearranged chloroplast genome of land plants

The chloroplast genome of Pelargonium x hortorum has been completely sequenced. It maps as a circular molecule of 217,942 bp and is both the largest and most rearranged land plant chloroplast genome yet sequenced. It features 2 copies of a greatly expanded inverted repeat (IR) of 75,741 bp each and, consequently, diminished single-copy regions of 59,710 and 6,750 bp. Despite the increase in size and complexity of the genome, the gene content is similar to that of other angiosperms, with the exceptions of a large number of pseudogenes, the recognition of 2 open reading frames (ORF56 and ORF42) in the trnA intron with similarities to previously identified mitochondrial products (ACRS and pvs-trnA), the losses of accD and trnT-ggu and, in particular, the presence of a highly divergent set of rpoA-like ORFs rather than a single, easily recognized gene for rpoA. The 3-fold expansion of the IR (relative to most angiosperms) accounts for most of the size increase of the genome, but an additional 10% of the size increase is related to the large number of repeats found. The Pelargonium genome contains 35 times as many 31 bp or larger repeats than the unrearranged genome of Spinacia. Most of these repeats occur near the rearrangement hotspots, and 2 different associations of repeats are localized in these regions. These associations are characterized by full or partial duplications of several genes, most of which appear to be nonfunctional copies or pseudogenes. These duplications may also be linked to the disruption of at least 1 but possibly 2 or 3 operons. We propose simple models that account for the major rearrangements with a minimum of 8 IR boundary changes and 12 inversions in addition to several insertions of duplicated sequence.     

Molecular phylogeny of Osmanthus (Oleaceae) based on non‐coding chloroplast and nuclear ribosomal internal transcribed spacer regions

Abstract The phylogenetic relationships of Osmanthus Lour. were investigated using the nuclear ribosomal internal transcribed spacer (ITS) regions and non‐coding chloroplast regions (psbA‐trnH, trnL‐F). The two datasets support the conclusion that Osmanthus is polyphyletic, with some species of the subtribe Oleinae nested within Osmanthus. Osmanthus didymopetalus P. S. Green is nested within the clade formed by species of section Osmanthus in two trees. Osmanthus attenuatus P. S. Green, O. yunnanensis P. S. Green, and O. gracilinervis R. L. Lu of traditional section Osmanthus are clearly divergent from other accessions, and do not form a monophyletic group with other Osmanthus accessions. Osmanthus marginatus Hemsl. is embedded in the clade formed by species of section Osmanthus in the ITS tree. In cpDNA trees all species of section Osmanthus are placed in the large clade and all species of section Leiolea formed a group. The taxonomic incongruence among trees for ITS and cpDNA indicate hybridization, as introgression may have occurred among some species of sections Osmanthus and Leiolea. Phylogeny of Osmanthus is discussed in light of molecular and morphological data, and a revised infrageneric classification with three sections (Leiolea, Siphosmanthu, and Osmanthus) is presented. The section Linocieroides is abandoned and united with section Osmanthus.     

On the systematic position of some Asian enigmatic genera of Asclepiadoideae (Apocynaceae)

The phylogenetic structure of Asclepiadoideae (Apocynaceae) has been elucidated at the tribal and subtribal levels in the last two decades. However, to date, the systematic positions of seven Asian genera, Cosmostigma, Graphistemma, Holostemma, Pentasachme, Raphistemma, Seshagiria and Treutlera, have not been investigated. In this study, we examine the evolutionary relationships among these seven small enigmatic Asian genera and clarify their positions in Asclepiadoideae, using a combination of plastid sequences of rbcL, rps16, trnL and trnL‐ F regions. Cosmostigma and Treutlera are resolved as members of the non‐Hoya clade of Marsdenieae with strong support (maximum parsimony bootstrap support value BS_MP = 96, maximum likelihood bootstrap support value BS_ML = 98, Bayesian‐inferred posterior probability PP = 1.0). Pentasachme is resolved as sister of Stapeliinae to Ceropegieae with moderate support (BS_MP = 64, BS_ML = 66, PP = 0.94). Graphistemma, Holostemma, Raphistemma and Seshagiria are all nested in the Asclepiadeae–Cynanchinae clade (BS_MP = 97, BS_ML = 100, PP = 1.0). The study confirms the generally accepted tribal and subtribal structure of the subfamily. One exception is Eustegia minuta, which is placed here as sister to all Asclepiadeae (BS_MP = 58, BS_ML = 76, PP = 0.99) and not as sister to the Marsdenieae + Ceropegieae clade. The weak support and conflicting position indicate the need for a placement of Eustegia as an independent tribe. In Asclepiadeae, a sister group position of Cynanchinae to the Asclepiadinae + Tylophorinae clade is favoured (BS_MP = 84, BS_ML = 88, PP = 1.0), whereas Schizostephanus is retrieved as unresolved. Oxystelma appears as an early‐branching member of Asclepiadinae with weak support (BS_MP = 52, BS_ML = 74, PP = 0.69). Calciphila and Solenostemma are also associated with Asclepiadinae with weak support (BS_MP = 37, BS_ML = 45, PP = 0.79), but all alternative positions are essentially without support. The position of Indian Asclepiadoideae in the family phylogeny is discussed. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 601–619.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

Chloroplast microsatellites and mitochondrial nad1 intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila)

We studied the phylogenetic relationships among the three stone pine species, Pinus cembra, P. sibirica, and P. pumila, using chloroplast microsatellites and mitochondrial nad1 intron 2 sequences. The three chloroplast microsatellite loci combined into a total of 18 haplotypes. Fourteen haplotypes were detected in 15 populations of P. cembra and one population of P. sibirica, five of which were shared between the two species, and the two populations of P. pumila comprised four species-specific haplotypes. Mitochondrial intron sequences confirmed this grouping of species. Sequences of P. cembra and P. sibirica were identical, but P. pumila differed by several nucleotide substitutions and insertions/deletions. A repeat region found in the former two species showed no intraspecific variation. These results indicate a relatively recent evolutionary separation of P. cembra and P. sibirica, despite their currently disjunct distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help to trace the hybridization in their overlapping distribution area and to identify fossil remains with respect to the still unresolved postglacial re-colonization history of these two species.     

Ribes (Grossulariaceae) phylogeny as indicated by restriction-site polymorphisms of PCR-amplified chloroplast DNA

We surveyed exemplars from all 12 infrageneric taxa ofRibes (Grossulariaceae) for restriction site variation in two cpDNA regions, fromrbcL toaccD and fromrpoC1 torpoC2, in order to develop an explicit phylogenetic hypothesis and to assess the validity of infrageneric classifications. Maximum parsimony analysis resolves sect.Ribes (red currants), sect.Berisia (European alpine currants), sect.Symphocalyx (golden currants), sect.Grossularia plus sect.Grossularioides (true gooseberries and spiny currants), andHesperia, Lobbia, and probably sect.Robsonia (west North American gooseberries) as well-supported monophyletic groups. The clade of sectionsGrossularioides andGrossularia is unexpected, and suggests that subgenusGrossularia is not monophyletic. Alternatively, sect.Grossularioides may have acquired its cpDNA via hybridization and introgression. SectionsCoreosma (black currants) andHeritiera (dwarf currants) are apparently non monophyletic. Relationships among the well-supported lineages and the other sampled taxa remain unresolved. Maximum likelihood analysis is consistent with the parsimony results.     

Cladistic biogeography of Gleditsia (Leguminosae) based on ndhF and rpl16 chloroplast gene sequences

We used cladistic analysis of chloroplast gene sequences (ndhF and rpl16) to test biogeographic hypotheses in the woody genus Gleditsia. Previous morphological comparisons suggested the presence of two eastern Asian-eastern North American species pairs among the 13 known species, as well as other intra- and inter-continental disjunctions. Results from phylogenetic analyses, interpreted in light of the amount of sequence divergence observed, led to the following conclusions. First, there is a fundamental division of the genus into three clades, only one of which contains both Asian and North American species. Second, the widespread and polymorphic Asian species, G. japonica, is sister to the two North American species, G. triacanthos and G. aquatica, which themselves are closely related inter se, but are both polymorphic and paraphyletic. Third, the lone South American Gleditsia species, G. amorphoides, forms a clade with two eastern Asian species. Gleditsia thus appears to have only one Asian-North American disjunction and no intercontinental species pairs. Low sequence divergence between G. amorphoides and its closest Asian relatives implicates long-distance dispersal in the origin of this unusual disjunction. Sequence divergence between Asian and North American Gleditsia is much lower than between Asian and North American species of its closest relative, Gymnocladus. Estimates of Asian-North American divergence times for Gymnocladus are in general accordance with fossil data, but estimates for Gleditsia suggest recent divergences that conflict with ages of known North American Gleditsia fossils.     

A phylogeny of the European gentians inferred from chloroplast trnL (UAA) intron sequences

Phylogenetic relationships between the European species of the genus Gentiana L. (Gentianaceae) were inferred from chloroplast trnL , (UAA) intron sequence data. The phylogeny obtained is largely in accordance with the classification of species into sections Gentiana, Megalanthe and Calathianae. Few synapomorphies support the branching of the main lineages and thus could suggest a rapid radiation following the colonization of Europe. Within section Gentiana , our results are highly congruent with the previous distinction of G. montserratii Vivant from G. lutea L. Section Megalanthe is divided into two well separated lineages, both of which comprise calcicole and calcifuge species. The 'star phylogeny' obtained in section Calathianae suggests that most of the taxa speciated almost simultaneously. Relative-rate tests between two lineages suggested that section Chondropliyllae displays higher mutation rates than the rest of the genus Gentiana and that cpDNA can violate assumptions of rate constancy at lower taxonomic level.     

Phylogenetic Relationships of the Macaques (Cercopithecidae: Macaca), Inferred from Mitochondrial DNA Sequences

To study the phylogenetic relationships of the macaques, five gene fragments were sequenced from 40 individuals of eight species: Macaca mulatta, M. cyclopis, M. fascicularis, M. arctoides, M. assamensis, M. thibetana, M. silenus, and M. leonina. In addition, sequences of M. sylvanus were obtained from Genbank. A baboon was used as the outgroup. The phylogenetic trees were constructed using maximum-parsimony and Bayesian methods. Because five gene fragments were from the mitochondrial genome and were inherited as a single entity without recombination, we combined the five genes into a single analysis. The parsimony bootstrap proportions we obtained were higher than those from earlier studies based on the combined mtDNA dataset. Excluding M. arctoides, our results are generally consistent with the classification of Delson (1980). Our phylogenetic analyses agree with earlier studies suggesting that the mitochondrial lineages of M. arctoides share a close evolutionary relationship with the mitochondrial lineages of the fascicularis group of macaques (and M. fascicularis, specifically). M. mulatta (with respect to M. cyclopis), M. assamensis assamensis (with respect to M. thibetana), and M. leonina (with respect to M. silenus) are paraphyletic based on our analysis of mitochondrial genes.     

Mitochondrial genome phylogeny reveals the deep-time origin of Gomphomastacinae (Orthoptera: Eumastacidae) and its alpine genera in China

Gomphomastacinae is a grasshopper subfamily in Eumastacidae, with a morphology and distribution distinct from other subfamilies. The alpine genera of Gomphomastacinae that inhabit the Qinghai–Tibet Plateau in China show unique characteristics adapted to high-altitude life. However, their phylogenetic position and biogeographic history remain controversial. Thus, to determine the diversification history of these alpine genera and the origin of the subfamily, we obtained mitochondrial genome sequences from all seven Gomphomastacinae genera distributed in China. The reconstructed phylogeny was well supported and confirmed the phylogenetic position of Gomphomastacinae within Eumastacidae. Time calibration revealed a deep-time origin of the subfamily dating back to the Cretaceous period, and the diversification among alpine genera was also an ancient pre-Miocene event (30–50 Ma). Based on phylogeny and time estimates, the most likely biogeographic scenario is that Gomphomastacinae originated from an ancestral lineage that lived in East Gondwana and dispersed to Central and Western Asia through India. Subsequently, the alpine genera likely diverged along with the uplift of the Qinghai–Tibet Plateau and survived drastic climate change by in situ adaptation to high-altitude dwellings.     

Chloroplast DNA restriction site variation and phylogeny of the Berberidaceae

Comparative restriction site mapping of the chloroplast genome was performed to examine phylogenetic relationships among 27 species representing 16 genera of the Berberidaceae and two outgroups. Chloroplast genomes of the species included in this study showed no major structural rearrangements (i.e., they are collinear to tobacco cpDNA) except for the extension of the inverted repeat in species of Berberis and Mahonia. Excluding several regions that exhibited severe length variation, a total of 501 phylogenetically informative sites was mapped for ten restriction enzymes. The strict consensus tree of 14 equally parsimonious trees indicated that some berberidaceous genera (Berberis, Mahonia, Diphylleia) are not monophyletic. To explore phylogenetic utility of different parsimony methods phylogenetic trees were generated using Wagner, Dollo, and weighted parsimony for a reduced data set that included 18 species. One of the most significant results was the recognition of the four chromosomal groups, which were strongly supported regardless of the parsimony method used. The most notable difference among the trees produced by the three parsimony methods was the relationships among the four chromosomal groups. The cpDNA trees also strongly supported a close relationship of several generic pairs (e.g., Berberis-Mahonia, Epimedium-Vancouveria, etc.). Maximum likelihood values were computed for the four different tree topologies of the chromosomal groups, two Wagner, one Dollo, and one weighted topology. The results indicate that the weighted tree has the highest likelihood value. The lowest likelihood value was obtained for the Dollo tree, which had the highest bootstrap and decay values. Separate analyses using only the Inverted Repeat (IR) region resulted in a tree that is identical to the weighted tree. Poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae indicate that they may have radiated from an ancestral stock in a relatively short evolutionary time.     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

Mitochondrial control region and 12S rRNA variation in Przewalski's horse (Equus przewalskii)

Variation in the control region and the 12S rRNA gene of all surviving mitochondrial lineages of Przewalski's horse was investigated. Variation is low despite the present day population being descended from 13 individuals probably representing animals from three different regions of its range. Phylogenetic comparison of these sequences, with sequences for the domestic horse, does not resolve the ancestral status of either horse.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.     

鼬科动物线粒体DNA控制区结构分析

利用PCR技术获得紫貂(Martes zibellina)和黄喉貂(Martes flavigula)线粒体DNA控制区全序列,并结合从GenBank中下载的9种鼬科动物相应序列,用ClustalX排序后对控制区结构进行分析,识别出延长终止序列区、中央区和保守序列区3个区域,指出了一个终止相关序列ETAS1及8个保守序列(CSB-F、E、D、C、B、1、2和3),并给出了序列通式,在CSB1和CSB2之间发现不同形式的短重复序列.此外,以狼为外类群,应用邻接法构建鼬科线粒体控制区全序列的系统进化树,结果表明:臭鼬亚科最先从鼬科中分化出来,随后剩余类群分为两大支系,即貂属种类与貂熊聚为一支,并与獾亚科的狗獾形成姐妹群;另一支为水獭亚科的物种与鼬属的林鼬形成姐妹群,再与虎鼬聚在一起,狗獾与貂属的紫貂亲缘关系最近,水獭亚科与鼬属亲缘关系最近.     

线粒体DNA nad1序列在青海云杉青藏高原台面和周边地区种群中的变异

青藏高原是气候变迁的敏感区。但第四纪以来全球气候的反复变化对该地区植物分布格局的影响至今未进行过仔细研究。线粒体DNA在松科植物中是母系遗传,其变异被广泛用于研究该科植物历史上由于气候变迁造成的瓶颈作用以及冰期后扩张中形成的奠基者效应。对青海云杉位于青藏高原核心、其边缘甘肃和宁夏地区共12个种群155个个体的线粒体nad 1片段的全序列进行测序,研究该序列在各种群中的变异状况。通过测序发现该片段在青海云杉中十分保守,在所有个体中仅发现由于重复序列造成的5种单倍型(H ap A,H ap B,H ap C,H ap D和H ap E)。高原核心的所有种群均只有H ap A,而所有的4种单倍型都在高原边缘的甘肃地区存在,但这一地区仍然是H ap A占优势;但是在远离高原的宁夏地区则存在5种单倍性;多样性的变异趋势是宁夏地区高于高原边缘的甘肃地区,而甘肃地区又显著高于高原核心地区。这一结果表明,在过去气候变迁过程中,不同地区的青海云杉种群经历了不同的气候影响,这种影响造成的奠基者效应或者瓶颈作用是造成目前这种遗传多样性分布格局的主要原因。现有的单倍型分布格局,特别是高原上所有间断分布种群拥有相同的单倍型表明,森林回迁中的奠基者效应应是更为重要的因素。     

Hybridization and Rorippa austriaca (Brassicaceae) invasion in Germany

Introgressive hybridization between the invasive Rorippa austriaca and the native R. sylvestris in Germany has been studied using chloroplast DNA (trnL intron) and amplified fragment length polymorphism. Three hybrid zones between the invasive and native species were located in the Ruhr Valley (Mülheim) and at the River Main near Würzburg (Randersacker, Winterhausen). In each hybrid zone hybridization was indicated by additivity of region-specific amplified fragment length polymorphism markers proving independent hybridization events. The hybrids were either morphologically intermediate (R. x armoracioides) or were close to R. sylvestris. The trnL intron of R. austriaca is characterized by a species-specific deletion. This diagnostic chloroplast marker of R. austriaca was detected in three individuals of R. sylvestris providing evidence for introgression of the invasive chloroplast into the native species. Bidirectional introgression of R. austriaca markers into R. sylvestris and of R. sylvestris markers into R. austriaca was detected in the amplified fragment length polymorphism analysis. Some of the invasive R. austriaca populations showed high within-population variation. A possible association among introgression, within-population variation and invasion success is discussed. The morphologically intermediate hybrid R. x armoracioides is currently spreading in northern Germany. It forms large populations without its parent species R. austriaca and R. sylvestris. It is concluded that hybridization between invasive R. austriaca and native R. sylvestris may lead to the evolution of a new invasive species R. x armoracioides.     

Phylogenetic relationships in the Neotropical tribe Hamelieae (Rubiaceae,Cinchonoideae) and comments on its generic limits

Our molecular phylogenetic analyses shed some light on the evolutionary relationships within the Hamelieae tribe. Phylogenetic reconstructions based on Internal Transcribed Spacer and trnL-F sequence data revealed the presence of three distinct evolutionary lineages. The first clade includes Hamelia and Syringantha, the second clade includes Deppea s.l. (including Bellizinca, Csapodya, and Edithea), and the third clade includes Pinarophyllon, Deppeopsis, Hoffmannia,Pseudomiltemia, Plocaniophyllon, Omiltemia, and Renistipula. The phylogenetic analysis re-evaluated some taxonomical combinations. The transfer of Deppeopsistaxa from Deppea s.l. is supported, but however, the monophyly of the genus is not. The transfer of Renistipula from Rondeletieae is also highly supported. BothCsapodya and Edithea species form a well-defined group among Deppea s.l. with high posterior probabilities, allowing to reconsider the exclusion or integration of these taxa to Deppea.