Multiple Events of Allopolyploidy in the Evolution of the Racemose Lineages in Prunus (Rosaceae) Based on Integrated Evidence from Nuclear and Plastid Data.

Prunus is an economically important genus well-known for cherries, plums, almonds, and peaches. The genus can be divided into three major groups based on inflorescence structure and ploidy levels: (1) the diploid solitary-flower group (subg. Prunus, Amygdalus and Emplectocladus); (2) the diploid corymbose group (subg. Cerasus); and (3) the polyploid racemose group (subg. Padus, subg. Laurocerasus, and the Maddenia group). The plastid phylogeny suggests three major clades within Prunus: Prunus-Amygdalus-Emplectocladus, Cerasus, and Laurocerasus-Padus-Maddenia, while nuclear ITS trees resolve Laurocerasus-Padus-Maddenia as a paraphyletic group. In this study, we employed sequences of the nuclear loci At103, ITS and s6pdh to explore the origins and evolution of the racemose group. Two copies of the At103 gene were identified in Prunus. One copy is found in Prunus species with solitary and corymbose inflorescences as well as those with racemose inflorescences, while the second copy (II) is present only in taxa with racemose inflorescences. The copy I sequences suggest that all racemose species form a paraphyletic group composed of four clades, each of which is definable by morphology and geography. The tree from the combined At103 and ITS sequences and the tree based on the single gene s6pdh had similar general topologies to the tree based on the copy I sequences of At103, with the combined At103-ITS tree showing stronger support in most clades. The nuclear At103, ITS and s6pdh data in conjunction with the plastid data are consistent with the hypothesis that multiple independent allopolyploidy events contributed to the origins of the racemose group. A widespread species or lineage may have served as the maternal parent for multiple hybridizations involving several paternal lineages. This hypothesis of the complex evolutionary history of the racemose group in Prunus reflects a major step forward in our understanding of diversification of the genus and has important implications for the interpretation of its phylogeny, evolution, and classification

Complete chloroplast genome of Trachelium caeruleum: extensiverearrangements are associated with repeats and tRNAs

Chloroplast genome structure, gene order and content arehighly conserved in land plants. We sequenced the complete chloroplastgenome sequence of Trachelium caeruleum (Campanulaceae) a member of anangiosperm family known for highly rearranged chloroplast genomes. Thetotal genome size is 162,321 bp with an IR of 27,273 bp, LSC of 100,113bp and SSC of 7,661 bp. The genome encodes 115 unique genes, with 19duplicated in the IR, a tRNA (trnI-CAU) duplicated once in the LSC and aprotein coding gene (psbJ) duplicated twice, for a total of 137 genes.Four genes (ycf15, rpl23, infA and accD) are truncated and likelynonfunctional; three others (clpP, ycf1 and ycf2) are so highly divergedthat they may now be pseudogenes. The most conspicuous feature of theTrachelium genome is the presence of eighteen internally unrearrangedblocks of genes that have been inverted or relocated within the genome,relative to the typical gene order of most angiosperm chloroplastgenomes. Recombination between repeats or tRNAs has been suggested as twomeans of chloroplast genome rearrangements. We compared the relativenumber of repeats in Trachelium to eight other angiosperm chloroplastgenomes, and evaluated the location of repeats and tRNAs in relation torearrangements. Trachelium has the highest number and largest repeats,which are concentrated near inversion endpoints or other rearrangements.tRNAs occur at many but not all inversion endpoints. There is likely nosingle mechanism responsible for the remarkable number of alterations inthis genome, but both repeats and tRNAs are clearly associated with theserearrangements. Land plant chloroplast genomes are highly conserved instructure, gene order and content. The chloroplast genomes of ferns, thegymnosperm Ginkgo, and most angiosperms are nearly collinear, reflectingthe gene order in lineages that diverged from lycopsids and the ancestralchloroplast gene order over 350 million years ago (Raubeson and Jansen,1992). Although earlier mapping studies identified a number of taxa inwhich several rearrangements have occurred (reviewed in Raubeson andJansen, 2005), an extraordinary number of chloroplast genome alterationsare concentrated in several families in the angiosperm order Asterales(sensu APGII, Bremer et al., 2003). Gene mapping studies ofrepresentatives of the Campanulaceae (Cosner, 1993; Cosner et al.,1997,2004) and Lobeliaceae (Knox et al., 1993; Knox and Palmer, 1999)identified large inversions, contraction and expansion of the invertedrepeat regions, and several insertions and deletions in the cpDNAs ofthese closely related taxa. Detailed restriction site and gene mapping ofthe chloroplast genome of Trachelium caeruleum (Campanulaceae) identifiedseven to ten large inversions, families of repeats associated withrearrangements, possible transpositions, and even the disruption ofoperons (Cosner et al., 1997). Seventeen other members of theCampanulaceae were mapped and exhibit many additional rearrangements(Cosner et al., 2004). What happened in this lineage that made itsusceptible to so many chloroplast genome rearrangements? How do normallyvery conserved chloroplast genomes change? The cause of rearrangements inthis group is unclear based on the limited resolution available withmapping techniques. Several mechanisms have been proposed to explain howrearrangements occur: recombination between repeats, transposition, ortemporary instability due to loss of the inverted repeat (Raubeson andJansen, 2005). Sequencing whole chloroplast genomes within theCampanulaceae offers a unique opportunity to examine both the extent andmechanisms of rearrangements within a phylogenetic framework.We reporthere the first complete chloroplast genome sequence of a member of theCampanulaceae, Trachelium caeruleum. This work will serve as a benchmarkfor subsequent, comparative sequencing and analysis of other members ofthis family and close relatives, with the goal of further understandingchloroplast genome evolution. We confirmed features previously identifiedthrough mapping, and discovered many additional structural changes,including several partial to entire gene duplications, deterioration ofat least four normally conserved chloroplast genes into gene fragments,and the nature and position of numerous repeat elements at or nearinversion endpoints. The focus of this paper is on analyses of sequencesat or near these rearrangements in Trachelium caeruleum. Inversions arebelieved to occur due to the presence of repeat elements subject tohomologous recombination (Palmer, 1991; Knox et al., 1993). Repeats mayfacilitate inversions or other genome rearrangements (Achaz et al.,2003), and higher incidences of repeats have been correlated with greaternumbers of rearrangements (Rocha, 2003). Alternatively, repeats mayproliferate within a genome asa result of DNA strand repair mechanismsfollowing a rearrangement event such as an inversion. Gen

Mass Taxon-Sampling as a Strategy towards Illuminating the Natural History of Campanula (Campanuloideae)

Speciose clades usually harbor species with a broad spectrum of adaptive strategies and complex distribution patterns, and thus constitute ideal systems to disentangle biotic and abiotic causes underlying species diversification. The delimitation of such study systems to test evolutionary hypotheses is difficult because they often rely on artificial genus concepts as starting points. One of the most prominent examples is the bellflower genus Campanula with some 420 species, but up to 600 species when including all lineages to which Campanula is paraphyletic. We generated a large alignment of petD group II intron sequences to include more than 70% of described species as a reference. By comparison with partial data sets we could then assess the impact of selective taxon sampling strategies on phylogenetic reconstruction and subsequent evolutionary conclusions

Methods for Obtaining and Analyzing Whole Chloroplast Genome Sequences

During the past decade there has been a rapid increase in our understanding of plastid genome organization and evolution due to the availability of many new completely sequenced genomes. Currently there are 43 complete genomes published and ongoing projects are likely to increase this sampling to nearly 200 genomes during the next five years. Several groups of researchers including ours have been developing new techniques for gathering and analyzing entire plastid genome sequences and details of these developments are summarized in this chapter. The most important recent developments that enhance our ability to generate whole chloroplast genome sequences involve the generation of pure fractions of chloroplast genomes by whole genome amplification using rolling circular amplification, cloning genomes into Fosmid or BAC vectors, and the development of an organellar annotation program (DOGMA). In addition to providing details of these methods, we provide an overview of methods for analyzing complete plastid genome sequences for repeats and gene content, as well as approaches for using gene order and sequence data for phylogeny reconstruction. This explosive increase in the number of sequenced plastid genomes and improved computational tools will provide many insights into the evolution of these genomes and much new data for assessing relationships at deep nodes in plants and other photosynthetic organisms

Phylogeny and comparative chloroplast genomics of the Campanulaceae

The Campanulaceae is a large, nearly cosmopolitan family, chiefly of temperate regions, with a huge range of variation in morphology, breeding systems, ecology, pollen and seed morphology. The monophyly of the family is well-established, but intrafamiliar and generic circumscription remains controversial. I reconstructed a molecular phylogeny of Campanulaceae using the chloroplast genes atpB, matK and rbcL, sampling a wide diversity of the family, and used the phylogeny to clarify major family relationships and address a number of other problems related to taxonomy and biogeography of the family. Phylogenetic analyses showed strong evidence that there are two major clades in the family, the platycodonoids, and a larger clade comprised of the wahlenbergioids and campanuloids. I also found that a number of historical genera are not monophyletic. In addition, phylogenetic evidence suggests that some oceanic island taxa arose from single introductions, and others from multiple introductions. The phylogenetic evidence also suggests that the North American Campanulaceae are non-monophyletic. The Campanulaceae is also known for having highly rearranged chloroplast genomes, based on earlier gene mapping and restriction site studies. Chloroplast genome organization, gene order and content are highly conserved among most land plants. I sequenced the complete chloroplast genome sequences of two members of the Campanulaceae, Trachelium caeruleum L. and Platycodon grandiflorus (Jacq.) A.DC., and Pseudonemacladus oppositifolius (BL Rob.) McVaugh, a member of the closely related Nemacladaceae. These genomes are far more rearranged than originally suggested, with multiple inversions, gene duplications and losses, and possible transpositions. Recombination between repeats or tRNAs has been suggested as two means of chloroplast genome rearrangements. In a comparison with other angiosperms, the Trachelium, Platycodon, and Pseudonemacladus chloroplast genomes have both the highest number and largest repeated intergenic sequences, and these are concentrated near rearrangement endpoints. Genes for tRNAs occur at many but not all inversion endpoints, so it is possible that some combination of repeats and tRNAs may have mediated recombination leading to these rearrangements. This does not preclude the possibility that some other mechanisms of structural evolution may also have participated in the extraordinary chloroplast genome evolution in these taxa.Biological Sciences, School o

Complete chloroplast genome of Trachelium caeruleum: extensive rearrangements are associated with repeats and tRNAs

Abstract Chloroplast genome structure, gene order and content are highly conserved in land plants. We sequenced the complete chloroplast genome sequence of Trachelium caeruleum (Campanulaceae) a member of an angiosperm family known for highly rearranged chloroplast genomes. The total genome size is 162,321 bp with an IR of 27,273 bp, LSC of 100,113 bp and SSC of 7,661 bp. The genome encodes 115 unique genes, with 19 duplicated in the IR, a tRNA (trnI-CAU) duplicated once in the LSC and a protein coding gene (psbJ) duplicated twice, for a total of 137 genes. Four genes (ycf15, rpl23, infA and accD) are truncated and likely nonfunctional; three others (clpP, ycf1 and ycf2) are so highly diverged that they may now be pseudogenes. The most conspicuous feature of the Trachelium genome is the presence of eighteen internally unrearranged blocks of genes that have been inverted or relocated within the genome, relative to the typical gene order of most angiosperm chloroplast genomes. Recombination between repeats or tRNAs has been suggested as two means of chloroplast genome rearrangements. We compared the relative number of repeats in Trachelium to eight other angiosperm chloroplast genomes, and evaluated the location of repeats and tRNAs in relation to rearrangements. Trachelium has the highest number and largest repeats, which are concentrated near inversion endpoints or other rearrangements. tRNAs occur at many but not all inversion endpoints. There is likely no single mechanism responsible for the remarkable number of alterations in this genome, but both repeats and tRNAs are clearly associated with these rearrangements. Land plant chloroplast genomes are highly conserved in structure, gene order and 3 content. The chloroplast genomes of ferns, the gymnosperm Ginkgo, and mos

Complete chloroplast genome of Trachelium caeruleum: extensive rearrangements are associated with repeats and tRNAs

Chloroplast genome structure, gene order and content are highly conserved in land plants. We sequenced the complete chloroplast genome sequence of Trachelium caeruleum (Campanulaceae) a member of an angiosperm family known for highly rearranged chloroplast genomes. The total genome size is 162,321 bp with an IR of 27,273 bp, LSC of 100,113 bp and SSC of 7,661 bp. The genome encodes 115 unique genes, with 19 duplicated in the IR, a tRNA (trnI-CAU) duplicated once in the LSC and a protein coding gene (psbJ) duplicated twice, for a total of 137 genes. Four genes (ycf15, rpl23, infA and accD) are truncated and likely nonfunctional; three others (clpP, ycf1 and ycf2) are so highly diverged that they may now be pseudogenes. The most conspicuous feature of the Trachelium genome is the presence of eighteen internally unrearranged blocks of genes that have been inverted or relocated within the genome, relative to the typical gene order of most angiosperm chloroplast genomes. Recombination between repeats or tRNAs has been suggested as two means of chloroplast genome rearrangements. We compared the relative number of repeats in Trachelium to eight other angiosperm chloroplast genomes, and evaluated the location of repeats and tRNAs in relation to rearrangements. Trachelium has the highest number and largest repeats, which are concentrated near inversion endpoints or other rearrangements. tRNAs occur at many but not all inversion endpoints. There is likely no single mechanism responsible for the remarkable number of alterations in this genome, but both repeats and tRNAs are clearly associated with these rearrangements. Land plant chloroplast genomes are highly conserved in structure, gene order and content. The chloroplast genomes of ferns, the gymnosperm Ginkgo, and most angiosperms are nearly collinear, reflecting the gene order in lineages that diverged from lycopsids and the ancestral chloroplast gene order over 350 million years ago (Raubeson and Jansen, 1992). Although earlier mapping studies identified a number of taxa in which several rearrangements have occurred (reviewed in Raubeson and Jansen, 2005), an extraordinary number of chloroplast genome alterations are concentrated in several families in the angiosperm order Asterales (sensu APGII, Bremer et al., 2003). Gene mapping studies of representatives of the Campanulaceae (Cosner, 1993; Cosner et al.,1997, 2004) and Lobeliaceae (Knox et al., 1993; Knox and Palmer, 1999) identified large inversions, contraction and expansion of the inverted repeat regions, and several insertions and deletions in the cpDNAs of these closely related taxa. Detailed restriction site and gene mapping of the chloroplast genome of Trachelium caeruleum (Campanulaceae) identified seven to ten large inversions, families of repeats associated with rearrangements, possible transpositions, and even the disruption of operons (Cosner et al., 1997). Seventeen other members of the Campanulaceae were mapped and exhibit many additional rearrangements (Cosner et al., 2004). What happened in this lineage that made it susceptible to so many chloroplast genome rearrangements? How do normally very conserved chloroplast genomes change? The cause of rearrangements in this group is unclear based on the limited resolution available with mapping techniques. Several mechanisms have been proposed to explain how rearrangements occur: recombination between repeats, transposition, or temporary instability due to loss of the inverted repeat (Raubeson and Jansen, 2005). Sequencing whole chloroplast genomes within the Campanulaceae offers a unique opportunity to examine both the extent and mechanisms of rearrangements within a phylogenetic framework.We report here the first complete chloroplast genome sequence of a member of the Campanulaceae, Trachelium caeruleum. This work will serve as a benchmark for subsequent, comparative sequencing and analysis of other members of this family and close relatives, with the goal of further understanding chloroplast genome evolution. We confirmed features previously identified through mapping, and discovered many additional structural changes, including several partial to entire gene duplications, deterioration of at least four normally conserved chloroplast genes into gene fragments, and the nature and position of numerous repeat elements at or near inversion endpoints. The focus of this paper is on analyses of sequences at or near these rearrangements in Trachelium caeruleum. Inversions are believed to occur due to the presence of repeat elements subject to homologous recombination (Palmer, 1991; Knox et al., 1993). Repeats may facilitate inversions or other genome rearrangements (Achaz et al., 2003), and higher incidences of repeats have been correlated with greater numbers of rearrangements (Rocha, 2003). Alternatively, repeats may proliferate within a genome as a result of DNA strand repair mechanisms following a rearrangement event such as an inversion. Gene mapping studies previously identified five families of dispersed repeats in Trachelium at or near inversion endpoints (Cosner et al., 1997). Here we examine the sequences of these repeats and identify, map and characterize numerous additional repeats within the genome. We compare the number and size of repeats in typical unrearranged angiosperm chloroplast genomes to what we find in the highly rearranged chloroplast genome of Trachelium. The Trachelium chloroplast genome has the highest number and the largest repeats of diverse origin of any sequenced angiosperm chloroplast genome. These repeats are generally clustered at or near rearrangements and they are of diverse origins: partial or entire chloroplast gene duplications, noncoding chloroplast sequences or novel DNA with no clear sequence identity to any existing chloroplast DNA sequences. The Trachelium chloroplast genome represents the most highly rearranged sequenced genome of land plants and its bizarre organization is clearly associated with the high incidence of dispersed repetitive DNA

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PollenPresent_AntherArrangement_Matrix

PollenPresent_AntherArrangement_Matri