Evolution of trnF(GAA) pseudogenes in cruciferous plants

In several studies we used the 5′-trnL(UAA)–trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions, gene diversity calculations and phylogenetic analyses among the genera Arabidopsis and Boechera. Despite the fact that extensive gene duplications are rare within the chloroplast genome of higher plants, within several genera of the Brassicaceae the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with 1–12 tandemly repeated copies in close 5′-proximity of the functional gene. A recent re-examination and additional analysis of trnL(UAA)–trnF(GAA) regions from numerous cruciferous taxa not only reveal extensive trnF gene duplications, but also favour the hypothesis that in cruciferous taxa at least four independent phylogenetic lineages are characterized by these pseudogenes. Among these lineages there is one major clade of taxa carrying pseudogenes indicating an ancient split in crucifer evolution. In two case studies, Boechera and Arabidopsis, intra- and inter-molecular recombinations have been shown to be the reason for the reciprocal exchange of several similar motifs. However, functional constraints might favour two to three or five to six copies as shown for Arabidopsis and Boechera. Herein, we compare the occurrence and distribution of pseudogene copy number in the framework of a comprehensive survey of cpDNA haplotype variation in Boechera, the former genus Cardaminopsis and Arabidopsis thaliana and comment on the value of such kind of mutations in phylogenetic and evolutionary reconstructions.     

Recombination diversifies chloroplast trnF pseudogenes in Arabidopsis lyrata

Extensive intraspecific variation in the chloroplast trnL(UAA)-trnF(GAA) spacer of model plant Arabidopsis lyrata is caused by multiple copies of a tandemly repeated trnF pseudogene undergoing parallel independent changes in copy number. Linkage disequilibrium and secondary structure analyses indicate that the diversification of pseudogene copies is driven by complex processes of structurally mediated illegitimate recombination. Disperse repeats sharing similar secondary structures interact, facilitating reciprocal exchange of structural motifs between copies via intramolecular and intermolecular recombinations, forming chimeric sequences and iterative expansion and contraction in pseudogene copy numbers. Widely held assumptions that chloroplast sequence evolution is simple and structural changes are informative are violated. Our findings have important implications for the use of this highly variable region in Brassicaceae studies. The reticulate evolution and nonindependent nucleotide substitution render the pseudogene inappropriate for standard phylogenetic reconstruction, but over short evolutionary timescales they may be useful for assessing gene flow, hybridization and introgression.     

Molecular evolution of a tandemly repeated trnF(GAA) gene in the chloroplast genomes of Microseris (Asteraceae) and the use of structural mutations in phylogenetic analyses

We sequenced the first ca. 900 bp of the 5'-trnL(UAA)-trnV(UAC)/ndhJ region of the chloroplast DNA of different Microseris accessions in order to resolve homoplasious length variation detected in the trnL(UAA)-trnF(GAA) region. We found two to four tandemly repeated trnF genes in the species of Microseris (Asteraceae, Lactuceae) and two in their sister genus Uropappus. Sequences indicated nonhomologous transitions between two, three, and four trnF genes in different Microseris taxa. Independent origins of similar trnF copy numbers were inferred from a chloroplast phylogeny of Microseris. The taxa involved grow on separate continents, supporting parallel origins of similar length variants. The changes in trnF copy numbers were best explained by interchromosomal recombination with unequal crossing over. The 5' copies of the repeats showed the highest sequence conservation, suggesting that these copies are likely to be functional trnF genes, whereas the other ones probably represent pseudogenes. Our results show that length polymorphisms accumulate once a duplicated sequence has become incorporated. Due to parallel gains of similar trnF copy numbers, homoplasious length variation was introduced into the data matrix. The data demonstrate that length polymorphisms cannot be used as indicators for phylogenetic distance unless they can be analyzed at the sequence level.     

Amplification of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a significant source of characters for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte and monilophyte species to determine the potential utility of PCR amplification primers for 18 noncoding chloroplast DNA regions that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of five cpDNA regions for resolving relationships in Botrychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnL(UAA)-trnF(GAA) intergenic spacer in this group. To these published data we added sequences of the trnS(GCU)-trnG(UUC) intergenic spacer + the trnG(UUC) intron region, the trnS(GGA)-rpS4 intergenic spacer+rpS4 gene, and the rpL16 intron. Both the trnS(GCU)-trnG(UUC) and rpL16 regions are highly variable in angiosperms and the trnS(GGA)-rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the five sampled regions the trnS(GCU)-trnG(UUC) spacer+trnG(UUC) intron region provided the strongest support for the inferred phylogeny.     

The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcL sequences

Direct sequencing of polymerase chain reaction products is now an expanding area of plant systematics and evolution. Within angiosperms the rbcL gene has been widely sequenced and used for inferring plant phylogenies at higher taxonomic levels. Unfortunately rbcL does not usually contain enough information to resolve relationships between closely related genera, such as Hordeum, Triticum, and Aegilops. One solution to this problem could be to analyze noncoding regions of chloroplast DNA, which are supposed to evolve more rapidly than coding regions. Here we present pairwise comparisons among dicots and monocots for rbcL and two noncoding sequences of cpDNA (the trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene). It appears that these regions evolve faster (more than three times faster, on average) than rbcL, as previously reported, and that the trnL intron evolves at a rate that is the same as that of the intergenic spacer. By the analysis of these regions, the genera Hordeum, Triticum, and Aegilops clearly could be distinguished. A phylogeny using trnL (UAA) intron sequences is also inferred for some species of the genus Gentiana L., clearly illustrating the phylogenetic utility of these zones at the generic level. The advantages and the disadvantages of the use of these regions to resolve plant phylogenies are discussed, as well as the desirability of a preliminary study before every large-scale analysis.      

Supernetwork identifies multiple events of plastid trnF(GAA) pseudogene evolution in the Brassicaceae

The occurrence of nonfunctional trnF pseudogenes has been rarely described in flowering plants. However, we describe the first large-scale supernetwork for the Brassiccaeae built from gene trees for 5 loci (adh, chs, matK, trnL-F, and ITS) and report multiple independent origins for trnF pseudogenes in crucifers. The duplicated regions of the original trnF gene are comprised of its anticodon domain and several other highly structured motifs not related to the original gene. Length variation of the trnL-F intergenic spacer region in different taxa ranges from 219 to 900 bp as a result of differences in pseudocopy number (1-14). It is speculated that functional constraints favor 2-3 or 5-6 copies, as found in Arabidopsis and Boechera. The phylogenetic distribution of microstructural changes for the trnL-F region supports ancient patterns of divergence in crucifer evolution for some but not all gene loci.     

Interspecific and progeny relationships in the genus Stylosanthes inferred from chloroplast DNA sequence variation.

The chloroplast trnL (UAA) intron and trnL (UAA)-trnF (GAA) intergenic spacer region have been sequenced from 37 samples, 36 of which representing 19 Stylosanthes species and one from the related genus Zornia. The DNA sequences were used to study phylogenetic relationships in the tropical forage legume genus Stylosanthes, by means of parsimony analysis using the heuristic search method of the computer program PAUP. The resulting cladograms divide Stylosanthes into four separate clades. Within the clades, species are poorly resolved owing to low sequence divergence. Small intra-specific chloroplast DNA variation is observed in S. humilis, S. scabra and the species complex S. guianensis. Variation between S. humilis populations is considered to be geographically structured. The overall results agree well with previously established inter-specific relationships and provide evidence for the genetic origin of the alloploid species S. hamata, S. scabra, S. ingrata, S. sympodialis, S. subsericea, S. capitata and S. fruticosa. This understanding of evolutionary relationships in Stylosanthes, in combination with biogeographical concepts provides a way of discerning isolated habitats in Central and South America, which may therefore contribute to strategies of plant collecting.     

Discordance of chloroplast and nuclear ribosomal DNA data in Osmorhiza (Apiaceae)

Phylogenetic studies were conducted to evaluate interspecific relationships in Osmorhiza (Apiaceae: Apioideae) using sequences of the ITS regions of nuclear ribosomal DNA, the chloroplast ndhF gene, and two noncoding regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer). All data sets suggest the monophyly of the New World taxa and showed that Osmorhiza aristata from Asia is relatively divergent from other members of the genus, even though it is morphologically similar to the eastern North American O. claytonii and O. longistylis. The ITS and chloroplast DNA trees differ in the relationships among the New World taxa, especially the phylogenetic position of O. occidentalis, O. glabrata, and O. depauperata. The lack of congruence between the two data sets may be a result of hybridization or introgression. Although there is high discordance between nrITS and two chloroplast DNA data sets, the latter two show similar topologies.     

Phylogeny and biogeography of the genus Lycium (Solanaceae): inferences from chloroplast DNA sequences

Lycium comprises approximately 70 species and is disjunctly distributed in temperate to subtropical regions in South America, North America, southern Africa, Eurasia, and Australia. Among them, only Lycium sandwicense A. Gray sporadically occurs widely on oceanic islands in the Pacific Ocean. To investigate phylogenetic and biogeographic relationships of the genus with emphasis on L. sandwicense, the coding region of matK, the two intergenic spacers trnT (UGU)-trnL (UAA) and trnL (UAA)-trnF (GAA), and the trnL (UAA) intron of chloroplast DNA (cpDNA) were sequenced. A strict consensus tree resulting from the phylogenetic analysis indicates the following: (1) New World species comprise a potentially paraphyletic assemblage; (2) southern African, Australian, and Eurasian species together are monophyletic; (3) southern African species are a paraphyletic assemblage; and (4) L. sandwicense is in a clade with certain New World species. The estimated biogeographic events based on the cpDNA analysis indicate that (1) Lycium originated in the New World, (2) all southern African, Australian, and Eurasian species have a common ancestor from the New World, (3) Australian and Eurasian species originated once from a southern African progenitor, and (4) L. sandwicense differentiated from the New World species.     

Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera

The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).     

Molecular phylogeny of Vincetoxicum (Apocynaceae-Asclepiadoideae) based on the nucleotide sequences of cpDNA and nrDNA

Molecular phylogenetic analyses of Vincetoxicum and Tylophora (Apocynaceae-Asclepiadoideae) were conducted based on the nucleotide sequences of cpDNA (two intergenic spacers of trnL (UAA)-trnF (GAA) and psbA-trnH and three introns, i.e., atpF, trnG (UCC) and trnL (UAA)), and nrDNA (ITS and ETS regions). Our phylogenetic analysis revealed two monophyletic groups; one consisted of seven taxa of Tylophora and Vincetoxicum inamoenum, Vincetoxicum magnificum and Vincetoxicum macrophyllum (Clade I) and the other consisted of 17 accessions of Vincetoxicum (Clade II). The monophyly of the genus Vincetoxicum was not supported. Although many nucleotide substitutions were observed in Clade I, the genetic differentiation within Clade II was small. Low genetic diversification but considerable morphological divergence suggests that the species in Clade II had undergone rapid diversification. Although most species in Clade I have tiny flowers, those in Clade II have larger and more nectariferous ones. Thus, we hypothesized that the rapid morphological radiation in Clade II may have been due to the gaining of floral characters such as large flowers and large amounts of nectar corresponding to diverse pollinators.     

Phylogenetic relationships in Monanthes (Crassulaceae) based on morphological,chloroplast and nuclear DNA variation

Phylogenetic relationships of all 10 recognized taxa of the genus Monanthes which is endemic to the Canary Islands and Salvage Islands, were investigated using the four data sets: morphology, sequences of the chloroplast DNA trnL (UAA) — trnF (GAA) intergenic spacer, ITS2 sequences of the nuclear ribosomal region and Random Amplified Polymorphic DNAs (RAPDs). In contrast to the molecular data, the morphological data were internally inconsistent which probably resulted from parallel or convergent evolution of morphological characters. The molecular data sets indicated that the genus is not monophyletic due to inclusion of the annual M. icterica which is the putative sister taxon of Aichryson, that M. polyphylla is the sister taxon of the perennial species of the genus, that M. muralis from Hierro is of allotetraploid origin and that M. lowei, M. minima, M. brachycaulos, M. laxiflora, M. anagensis and M. muralis from La Palma are closely related. Combined ITS2 sequences, trnL — trnF sequences and morphological data indicated that the relationships among three types of perennial growth forms, i.e. tiny rosettes, small, branched shrubs and diffuse branches shrublets, are highly dependent on the outgroup used. After deletion of the most distant outgroup and a taxon of alleged hybrid origin (M. muralis) relationships among the growth forms of Monanthes still could not be consistently resolved.     

Nucleotide diversity and phylogenetic relationships among <Emphasis Type="Italic">Gladiolus</Emphasis> cultivars and related taxa of family Iridaceae

The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA–trnH and trnL–trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA–trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL–trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.     

Phylogenetic relationships among species of Hypochaeris (Asteraceae, Cichorieae) based on ITS, plastid trnL intron, trnL-F spacer, and matK sequences

Nuclear internal transcribed spacer (ITS) regions and chloroplast trnL intron and trnL/trnF spacer and matK sequences were used from 86 accessions to assess relationships among 31 European and South American species of Hypochaeris plus 18 representatives of related genera of tribe Cichorieae. The ITS tree shows high resolution compared to that of the maternally inherited trnL intron, trnL/F spacer, and matK sequences. The ITS and the combined tree reveal clades that agree well with sections of the genus established previously on morphological and cytological grounds, except for H. robertia, which groups with Leontodon helveticus and L. autumnalis. Monophyly of species of Hypochaeris from South America is strongly supported by both ITS and the joint matrix of ITS, trnL, and matK data. European species lie basal to South American taxa, which suggests that species in South America evolved from a single introduction from European progenitors and not from H. robertia as suggested previously. Low levels of sequence divergence among South American taxa suggest a pattern of rapid speciation, in contrast to much greater divergence among European representatives. Different species of Leontodon form two different clades that are also supported by chromosome numbers and morphology. Both nuclear and chloroplast markers suggest that Helminthotheca, Leontodon, and Picris are closely related to each other as well as to Hypochaeris.     

拟南芥属的系统位置: 种皮及分子证据

拟南芥属(Arabidopsis (L.) Heynhold)拥有现代分子生物学研究的模式植物拟南芥(A. thaliana (L.) Heynhold), 但其属的系统位置及与近缘属关系争议较大。根据对拟南芥属及其相关属种的种皮微形态观察, 结合测定分析各属种叶绿体DNA的trnL内含子和trnL-F基因间隔区序列, 结果表明, 拟南芥属的近缘属种包括荠属(Capsella Medic.)、亚麻荠属(Camelina (L.)Crantz)、须弥芥属(Crucihimalaya Al-Shehbaz et al.)、无苞芥属(Olimarabidopsis Al-Shehbaz et al.)、旗杆芥属(Turritis L.)、南芥属(Arabis L.)和糖芥属(Erysimum Kitagawa)。     

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 DNA evidence for the evolution of Microseris (Asteraceae) in Australia and New Zealand after long-distance dispersal from western North America

Restriction site mutations and trnL(UAA)-trnF(GAA) intergenic spacer length variants in the chloroplast genome were used to investigate the phylogenetic relationships among 53 Australian and New Zealand Microseris populations and to assess their position within their primarily North American genus. The study was performed to enhance understanding of evolutionary processes within this unique example of intercontinental dispersal and subsequent adaptive radiation. A southern blot method using four-base restriction enzymes and fragment separation on polyacryamide gels resulted in 55 mutations of which 30 were potentially phylogenetically informative. Most mutations were small indels of <162 bp, 80% of which were <20 bp. The small indels were useful for phylogenetic reconstruction of Australasian Microseris as judged by the high consistency indexes. The results confirmed the monophyly of the Australian and New Zealand Microseris. The occurrence of “hard” basal polytomies in the most parsimonious trees indicated that rapid radiation has occurred early in the history of the taxon. The monophyly of M. lanceolata, which includes the self-incompatible ecotypes of the Australian mainland, was confirmed. Within this species three clades were found that reflect more geographic distribution than morphological entities, suggesting that migration and possibly introgression between different ecotypes, or parallel evolution of similar adaptations, has occurred. One of the three clades was supported by a 162-bp deletion in the trnL-trnF spacer, while a subgroup of this exhibited also a tandemly repeated trnF exon. The data were inconclusive about the monophyly of the second Australasian species, M. scapigera, which comprises the New Zealand, Tasmanian, and autofertile ecotypes of Australia.     

Sedum surculosum andS. jaccardianum (Crassulaceae) share a unique 70 bp deletion in the chloroplast DNA trnL (UAA)-trnF (GAA) intergenic spacer

TrnL (UAA)-trnF (GAA) chloroplast DNA spacer sequences of three species ofMonanthes, Sedum surculosum (=Monanthes atlanticum) andS. jaccardianum were compared.S. surculosum, the systematic position of which has been disputed ever since its discovery, shares a phylogenetically highly significant 70 bp deletion withS. jaccardianum. In addition to this large deletion the two Moroccan species ofS. ser.Monanthoidea differ in three more indels as well as in four nucleotide substitutions from the species ofMonanthes. These data render strong support for the monophyly ofS. ser.Monanthoidea andMonanthes. Spacer length in seven species and one subspecies ofMonanthes is relatively uniform.     

拟南芥属的系统位置：种皮及分子证据

拟南芥属（Arabidopsis（L.）Heynhold）拥有现代分子生物学研究的模式植物拟南芥（A.thaliana（L.）Heynhold）,但其属的系统位置及与近缘属关系争议较大。根据对拟南芥属及其相关属种的种皮微形态观察,结合测定分析各属种叶绿体DNA的trnL内含子和trnL-F基因间隔区序列,结果表明,拟南芥属的近缘属种包括荠属（Capsella Medic.）、亚麻荠属（Camelina（L.）Crantz）、须弥芥属（Crucihimalaya Al-Shehbaz et al.）、无苞芥属（Olimarabidopsis Al-Shehbaz et al.）、旗杆芥属（Turritis L.）、南芥属（Arabis L.）和糖芥属（Erysimum Kitagawa）。     

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.