Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

When is enough,enough in phylogenetics? A case in point from Hordeum (Poaceae)

Direct optimization was used to reconstruct the phylogeny of the 26 diploid taxa included in the genus Hordeum. The total data set was composed of 16 nucleotide sequence regions from the nuclear as well as the plastid genome. The nine nuclear regions were from single‐copy, protein coding genes located on six of the seven chromosome pairs in the diploid H. vulgare genome. The seven plastid regions comprise protein coding genes as well as intergenic regions. Studies of character congruence between data partitions showed no correlation between chromosomal location and congruence among the nuclear sequences and a level of congruence among the plastid sequences comparable with the level among the nuclear sequences. Combined analysis of all data resolved the phylogeny completely with most clades being robust and well supported. However, due to incongruence among data partitions some relationships are still and likely to remain ambiguously inferred. Rather than adding still more genes to the phylogenetic analyses, patterns of incongruence may be better explored by adding data from multiple specimens per taxon. For some species relationships the plastid data appear positively misleading, emphasizing the need for caution if plastid data are the only or dominant type of data used for phylogenetic reconstruction and subsequent re‐classification.
© The Willi Hennig Society 2011.     

Molecular phylogeny of Cotoneaster (Rosaceae) inferred from nuclear ITS and multiple chloroplast sequences

Cotoneaster Medik. (Rosaceae, Maloideae) is distributed in Europe, North Africa, and temperate areas of Asia except Japan. Members of the genus exhibit considerable morphological variation. The infrageneric classification is also obscured by polyploidy, hybridization, and apomixis. In this study, phylogenetic analyses were conducted to test infrageneric classifications of this genus using DNA sequence data from the nuclear ITS (nrITS) region and three chloroplast intergenic spacer regions. Maximum parsimony and Bayesian inference analyses of both datasets agreed with the two sections/subgenera of Koehne’s classification system, and suggested that four subsections (Microphylli, Chaenopetalum, Adpressi, and Cotoneaster) and the series of Koehne’s classification system were non-monophyletic. The incongruence length difference test indicated that the nrITS and cpDNA datasets were significantly incongruent (P = 0.001), and the placement of 14 species was discordant in phylogenetic trees derived from the two datasets. Within Cotoneaster, hybridization was indicated to be an important factor contributing to the incongruence between the nrITS and cpDNA data. By mapping nine morphological characters onto the combined nrITS–cpDNA phylogenetic tree, we inferred that a deciduous habit, glabrous fruit, white anthers, erect and light pink petals, and white filaments are plesiomorphic character states in Cotoneaster.     

The first complete plastid genome of Burmannia disticha L. from the mycoheterotrophic monocot family Burmanniaceae

Burmanniaceae is one major group within the monocot order Dioscoreales that has not had its plastome sequenced. Members of Burmanniaceae are mostly achlorophyllous, although the genus Burmannia also includes autotrophs. Here, we report sequencing and analysis of the first Burmanniaceae plastid genome from Burmannia disticha L.. This plastome is 157,480 bp and was assembled as a circular sequence with the typical quadripartite structure of plant plastid genomes. This plastome has a regular number of potentially functional genes with a total of 111, including 78 protein coding genes, 4 ribosomal RNA (rRNA) genes, and 29 tRNA genes. The ratio of the total length of genic:intergenic DNA is 1.58:1, and the mean length of intergenic regions is 398 bp, the longest being 1918 bp. The overall GC content of the B. disticha plastome is 34.90%, and the IR regions in B. disticha are more GC rich (39.50%) than the LSC (32.30%) and SSC (28.80%) regions. Phylogenetic analysis of protein-coding sequences from plastomes of related species in the order Dioscoreales support a clade comprising Burmanniaceae and Dioscoreaceae. This phylogenetic placement is congruent with previous findings based on nuclear and mitochondrial evidence.     

Reconciling gene and genome duplication events: using multiple nuclear gene families to infer the phylogeny of the aquatic plant family Pontederiaceae

Most plant phylogenetic inference has used DNA sequence data from the plastid genome. This genome represents a single genealogical sample with no recombination among genes, potentially limiting the resolution of evolutionary relationships in some contexts. In contrast, nuclear DNA is inherently more difficult to employ for phylogeny reconstruction because major mutational events in the genome, including polyploidization, gene duplication, and gene extinction can result in homologous gene copies that are difficult to identify as orthologs or paralogs. Gene tree parsimony (GTP) can be used to infer the rooted species tree by fitting gene genealogies to species trees while simultaneously minimizing the estimated number of duplications needed to reconcile conflicts among them. Here, we use GTP for five nuclear gene families and a previously published plastid data set to reconstruct the phylogenetic backbone of the aquatic plant family Pontederiaceae. Plastid-based phylogenetic studies strongly supported extensive paraphyly of Eichhornia (one of the four major genera) but also depicted considerable ambiguity concerning the true root placement for the family. Our results indicate that species trees inferred from the nuclear genes (alone and in combination with the plastid data) are highly congruent with gene trees inferred from plastid data alone. Consideration of optimal and suboptimal gene tree reconciliations place the root of the family at (or near) a branch leading to the rare and locally restricted E. meyeri. We also explore methods to incorporate uncertainty in individual gene trees during reconciliation by considering their individual bootstrap profiles and relate inferred excesses of gene duplication events on individual branches to whole-genome duplication events inferred for the same branches. Our study improves understanding of the phylogenetic history of Pontederiaceae and also demonstrates the utility of GTP for phylogenetic analysis.     

Phylogeny and Classification of Prunus sensu lato （Rosaceae）

The classification of the economically important genus Prunus L. sensu lato （s.L） is controversial due to the high levels of convergent or the parallel evolution of morphological characters. In the present study, phylogenetic analyses of fifteen main segregates of Prunus s.I. represented by eighty-four species were conducted with maximum parsimony and Bayesian approaches using twelve chloroplast regions （atpB- rbcL, matK, ndhF, psbA-trnH, rbcL, rpL 16, rpoC1, rps16, trnS-G, trnL, trnL-F and ycfl） and three nuclear genes （ITS, s6pdh and Sbel） to explore their infrageneric used to develop a new, phylogeny-based classification relationships. The results of these analyses were of Prunus s.I. Our phylogenetic reconstructions resolved three main clades of Prunus s.I. with strong supports. We adopted a broad-sensed genus, Prunus, and recognised three subgenera corresponding to the three main clades： subgenus Padus, subgenus Cerasus and subgenus Prunus. Seven sections of subgenus Prunus were recognised. The dwarf cherries, which were previously assigned to subgenus Cerasus, were included in this subgenus Prunus. One new section name, Prunus L. subgenus Prunus section Persicae （T. T. yu ＆ L. T. Lu） S. L. Zhou and one new species name, Prunus tianshanica （Pojarkov） S. Shi, were proposed.     

Evidence for replication slippage in the evolution of Oenothera chloroplast DNA.

Evolutionary relationships of four plastid genomes (plastomes) from different Oenothera species have been assessed by sequence comparisons of two intergenic regions that separate the ribosomal protein genes rpl16, rpl14, and rps8. Sequence changes include base substitutions, the occurrence of a 29-base tandem duplication, and variation in the length of two poly-A stretches. Additions/deletions in chloroplast DNA may not be useful for evolutionary comparisons more distant than these, particularly if the sequences undergo divergence after the initial event, but the length mutations reported here allow a finer resolution of the phylogeny of the closely related Oenothera plastomes than would have been possible if only base substitutions had been considered. Comparisons with the orthogous sequence from tobacco chloroplast DNA indicate the direction of change at most of the sites. The results suggest that plastomes I and II are closely related to each other, as are plastomes III and IV. Replication slippage is proposed as a mechanism to explain the length mutations.     

A molecular phylogeny for the large African orchid genus Disa

Phylogenetic relationships were inferred for the African subtribe Disinae (Orchidoideae, Orchidaceae), which include the large genus Disa and the small genus Schizodium. One nuclear (ITS) gene region and two plastid (trnLF and matK) gene regions were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. Our results show that the generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships and should be rejected. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. Morphological divergence, rather than convergence, has hampered previous attempts at a phylogenetic classification of the Disinae. On the basis of our molecular phylogenetic hypothesis, we propose a monotypic subtribe Disinae and a subdivision of the genus Disa into 18 sections.     

Complete Plastid Genome Sequence of the Basal Asterid Ardisia polysticta Miq. and Comparative Analyses of Asterid Plastid Genomes

Ardisia is a basal asterid genus well known for its medicinal values and has the potential for development of novel phytopharmaceuticals. In this genus of nearly 500 species, many ornamental species are commonly grown worldwide and some have become invasive species that caused ecological problems. As there is no completed plastid genome (plastome) sequence in related taxa, we sequenced and characterized the plastome of Ardisia polysticta to find plastid markers of potential utility for phylogenetic analyses at low taxonomic levels. The complete A. polysticta plastome is 156,506 bp in length and has gene content and organization typical of most asterids and other angiosperms. We identified seven intergenic regions as potentially informative markers with resolution for interspecific relationships. Additionally, we characterized the diversity of asterid plastomes with respect to GC content, plastome organization, gene content, and repetitive sequences through comparative analyses. The results demonstrated that the genome organizations near the boundaries between inverted repeats (IRs) and single-copy regions (SCs) are polymorphic. The boundary organization found in Ardisia appears to be the most common type among asterids, while six other types are also found in various asterid lineages. In general, the repetitive sequences in genic regions tend to be more conserved, whereas those in noncoding regions are usually lineage-specific. Finally, we inferred the whole-plastome phylogeny with the available asterid sequences. With the improvement in taxon sampling of asterid orders and families, our result highlights the uncertainty of the position of Gentianales within euasterids I.     

Molecular analysis of the complete set of length mutations found in the plastomes of Triticum-Aegilops species.

Precise location and nature of each of 14 length mutations detected among chloroplast DNAs of Triticum-Aegilops species by RFLP analysis were determined at the nucleotide sequence level. Each mutation was compared with at least three non-mutated wild-type plastomes as standards. These 14 length mutations were classified into 4 duplications and 10 deletions. One duplication occurred in the small single-copy region close to the border of the inverted repeat, and the remaining 13 length mutations took place in the large single-copy region. All length mutations occurred in the intergenic regions, suggesting that these length mutations do not affect plastid gene expression. Saltatory replication was the cause of all duplications, whereas intramolecular recombination mediated by short direct repeats played a substantial role in the deletions. Recurrent occurrences of certain deletion events were found in some AT-rich regions, which constituted hot spots for deletion. Out of four hypervariable regions detected among the grass plastomes, two (downstream of rbcL and a tRNA gene accumulated region) were still active after differentiation of Triticum and Aegilops complex.     

Extensive intraspecific chloroplast DNA (cpDNA) variation in the alpine Draba aizoides L. (Brassicaceae): haplotype relationships and population structure

Chloroplast DNA (cpDNA) sequence variation is currently the most widely used tool for the inference of phylogenetic relationships among plants at all taxonomic levels. Generally, noncoding regions tend to evolve faster than coding sequences and have recently been applied to the study of phylogenetic relationships among closely related taxa. An implicit assumption of many of these studies is that intraspecific cpDNA variation is either absent or low and therefore will not interfere with the reconstruction of interspecific relationships. A survey of cpDNA sequence variation in the common alpine plant species Draba aizoides L. was undertaken to assess levels of intraspecific cpDNA sequence variation. These levels were compared to levels of interspecific sequence divergence between D. aizoides and related alpine Draba species. Intraspecific cpDNA sequence divergence was extensive in D. aizoides, and intraspecific differences were often larger than interspecific differences. cpDNA haplotype relationships were explored using a maximum parsimony approach and minimum-spanning networks. Results from both methods were largely congruent but comparisons provided interesting insights into the presumed evolutionary history of cpDNA haplotypes. A combined effect of cpDNA introgression and complex lineage sorting was inferred to explain the pattern of cpDNA variation found in D. aizoides. Our results suggest that intraspecific cpDNA variation can be extensive and that intraspecific variation needs to be taken into account when inferring phylogenetic relationships among closely related taxa.     

Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species,little divergence

Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species.Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices.Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species.Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with plastid DNA did not help to resolve the phylogenetic tree because the total number of variable sites was much lower than in the entire plastid genome. The geographical clustering of the individuals against a background of overall low sequence divergence could indicate transfer of plastid genomes due to hybridization and introgression following secondary contact.     

用叶绿体ndhF和核核糖体ITS序列推断李属(蔷薇科)的系统发育

Sequences of the chloroplast ndhF gene and the nuclear ribosomal ITS regions are employed to reconstruct the phylogeny of Prunus (Rosaceae), and evaluate the classification schemes of this genus. The two data sets are congruent in that the genera Prunus s.l. and Maddenia form a monophyletic group, with Maddenia nested within Prunus. However, the ndhF data set is incongruent with the ITS data supporting two major groups within Prunus one consisting of subgenera Laurocerasus (including Pygeum) and Padus as well as the genus Maddenia and another of subgenera Amygdalus, Cerasus, and Prunus. The ITS data, on the other hand, support a clade composed of subgenera Amygdalus and Prunus and Prunus sect. Microcerasus in addition to a paraphyletic grade of subgenera Laurocerasus and Padus (and the genus Maddenia) taxa. In general, the subgeneric classifications of Prunus s.l. are not supported. The ITS and ndhF phylogenies differ mainly in interspecific relationships and the relative position of the Padus/Laurocerasus group. Both ITS and ndhF data sets suggest that the formerly recognized genus Pygeum is polyphyletic and that the distinction of the subgenera Padus and Laurocerasus is not supported. The biogeographic interactions of the temperate and tropical members in the Padus/Laurocera- sus/Maddenia alliance including Pygeum are shown to be highly dynamic and complex.     

DNA序列在植物系统学研究中的应用

植物DNA序列由于进化速率上的差异而适用于不同分类阶元的系统发育研究,因此,针对某一特定的系统学问题选择相应合适的分子片段是分子系统学研究中最为关键的一步。在前人研究的基础上,主要讨论了目前分子系统发育和进化研究中一些常用的DNA序列的适用范围,包括nrDNA的18S基因及ITS等非编码区,cpDNA的编码基因（rbcI、matK、ndhF、atpB）及非编码区序列（rpL16、rpoC1、rps16、trnL-F和trnT-L）和应用较少的mtDNA。研究表明,18S、rbcI等编码基因及mtDNA一般适用于较高分类阶元甚至整个种子植物谱系间的系统发育的探讨,而ITS及cpDNA的非编码区序列等因其较快的进化速率多用于较低分类阶元的系统关系研究。     

Comparative plastid genomics of Pinus species: Insights into sequence variations and phylogenetic relationships

Pinus L. is the largest genus of conifers and provides a classical model for studying species divergence and phylogenetic evolution by gymnosperms. However, our poor understanding of sequence divergence in the whole plastid genomes of Pinus species severely hinders studies of their evolution and phylogeny. Thus, we analyzed the sequences of 97 Pinus plastid genomes, including four newly sequenced genomes and 93 previously published plastomes, to explore the evolution and phylogenetic relationships in the genus Pinus. The complete chloroplast genomes of Pinus species ranged in size from 109 640 bp (P. cembra L.) to 121 976 bp (P. glabra Walter), and these genomes comprised circular DNA molecules in a similar manner to those of most gymnosperms. We identified 9108 repeats where most of the repeats comprised the dispersed type with 3983 (44%), followed by tandem repeats with 2999 (33%), and then palindromic repeats with 2126 (23%). Sixteen divergence hotspot regions were identified in Pinus plastid genomes, which could be useful molecular markers for future population genetics studies. Phylogenetic analysis showed that Pinus species could be divided into two diverged clades comprising the subgenera Strobus (single needle section) and Pinus (double needles section). Molecular dating suggested that the genus Pinus originated approximately 130.38 Mya during the late Cretaceous. The two subgenera subsequently split 85.86 Mya, which was largely consistent with the other molecular results based on partial DNA markers. These findings provide important insights into the sequence variations and phylogenetic evolution of Pinus plastid genomes.     

Genome-Scale Profiling Reveals Noncoding Loci Carry Higher Proportions of Concordant Data

Many evolutionary relationships remain controversial despite whole-genome sequencing data. These controversies arise, in part, due to challenges associated with accurately modeling the complex phylogenetic signal coming from genomic regions experiencing distinct evolutionary forces. Here, we examine how different regions of the genome support or contradict well-established relationships among three mammal groups using millions of orthologous parsimony-informative biallelic sites (PIBS) distributed across primate, rodent, and Pecora genomes. We compared PIBS concordance percentages among locus types (e.g. coding sequences (CDS), introns, intergenic regions), and contrasted PIBS utility over evolutionary timescales. Sites derived from noncoding sequences provided more data and proportionally more concordant sites compared with those from CDS in all clades. CDS PIBS were also predominant drivers of tree incongruence in two cases of topological conflict. PIBS derived from most locus types provided surprisingly consistent support for splitting events spread across the timescales we examined, although we find evidence that CDS and intronic PIBS may, respectively and to a limited degree, inform disproportionately about older and younger splits. In this era of accessible wholegenome sequence data, these results:1) suggest benefits to more intentionally focusing on noncoding loci as robust data for tree inference and 2) reinforce the importance of accurate modeling, especially when using CDS data.     

Addressing the "hardest puzzle in American pomology:" Phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions

Prunus subg. Prunus sect. Prunocerasus (Rosaceae) is a North American taxon with 17 commonly recognized taxa. To test the hypothesis of monophyly for the section we sequenced the trnG and rpL16 introns and the trnH-psbA and trnS-trnG intergenic spacers for at least two representatives of each of the five subgenera in Prunus. Additionally we sampled heavily among Prunus subg. Prunus sections Prunus and Armeniaca and Prunus subg. Amygdalus because these groups are putatively most closely related to Prunocerasus. Once monophyly of sect. Prunocerasus was shown we added the sequences of trnL and rpS16 introns and the trnL-trnF spacer in an attempt to increase resolution within the section. The species of sect. Prunocerasus showed an initial split with P. subcordata, the only species from western North America, sister to the rest of the group. The remaining species fell into three primary clades. Within each of the three primary clades there was little phylogenetic resolution. Lastly, we present evidence that P. texana, previously classified in subg. Amygdalus, may be a plum or at least contain a Prunocerasus chloroplast. This is the first phylogenetic hypothesis presented for sect. Prunocerasus, and the clades recovered contrast sharply with previously defined groups based on morphological characters.     

Uninode coding vs gene tree parsimony for phylogenetic reconstruction using duplicate genes

Two different methods of using paralogous genes for phylogenetic inference have been proposed: reconciled trees (or gene tree parsimony) and uninode coding. Gene tree parsimony suffers from 10 serious problems, including differential weighting of nucleotide and gap characters, undersampling which can be misinterpreted as synapomorphy, all of the characters not being allowed to interact, and conflict between gene trees being given equal weight, regardless of branch support. These problems are largely avoided by using uninode coding. The uninode coding method is elaborated to address multiple gene duplications within a single gene tree family and handle problems caused by lack of gene tree resolution. An example of vertebrate phylogeny inferred from nine genes is reanalyzed using uninode coding. We suggest that uninode coding be used instead of gene tree parsimony for phylogenetic inference from paralogous genes.