Reconstruction of Phylogenetic Relationships of Larch Larix sukaczewii Dyl. Based on Chloroplast DNAtrnK Intron Sequences

To reconstruct the systematic relationships of larch Larix sukaczewiiwe used the chloroplast trnK intron sequences of L. decidua, L. sukaczewii, L. sibirica, L. czekanovskii, and L. gmelinii.Analysis of phylogenetic trees constructed using the maximum parsimony and maximum likelihood methods showed a clear divergence of the trnK intron sequences between L. sukaczewii and L. sibirica. This divergence reaches intraspecific level, which supports a previously published hypothesis on the taxonomic isolation of L. sukaczewii.     

Phylogenetic signal in matK vs. trnK: a case study in early diverging eudicots (angiosperms)

The matK gene has been among the most useful loci for resolving plant phylogenetic relationships at different evolutionary time-scales, but much less is known about the phylogenetic utility of the flanking trnK intron, especially for deep level phylogenetics. We compared the relative performance of matK and trnK intron regions for resolving the relationships of the early diverging eudicots (angiosperms). The two regions display similar nucleotide compositions and distributions of rate variation among sites. The trnK intron sequences also provide similar levels of phylogenetic information per-site as matK. Combining the trnK intron sequences with matK increases overall bootstrap support for the early diverging eudicots compared to analyses of matK alone. MP, ML and Bayesian analyses provide strong support for eudicots, the sister group relationship of Ranunculales to remaining eudicots, and a Buxales+Trochodendraceae+core eudicots clade. matK and the trnK intron support conflicting positions for Buxales and Trochodendrales in relation to the core eudicots.     

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

五种落叶松遗传关系的等位酶分析

由于种间形态上的微弱区别,落叶松属的系统分类一直很混乱,落叶松属的系统发生也知之甚少。本文分析了西伯利亚落叶松(Larix sibirica Ledeb.),卡氏落叶松(L.cajanderi Mayr.),兴安落叶松(L.gmelinii Rupr.),苏氏落叶松(L.sukaczewii Dil.)和杂交种切氏落叶松L.czekanowskii(L.gmelinii×L.sibirica)天然种群的遗传结构。采用水平切片淀粉凝胶电泳技术,等位酶分析手段对5个酶系统(AAT,IDH,DIA,PGM,SKDH)的8个基因位点进行了遗传结构分析。结果表明各种间遗传距离(D)在0.067~0.260之间,明显大于各种群内居群间的遗传距离。等位酶的分析结果揭示了5种落叶松的遗传关系。结合以上每种落叶松的形态学、生物学和生态学特性,等位酶的证据了支持兴安落叶松、西伯利亚落叶松、卡氏落叶松、苏氏落叶松作为独立种的观点。     

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

Revisiting the phylogeny of Ranunculeae: Implications for divergence time estimation and historical biogeography

Previous studies based on different molecular datasets have generated conflicting topologies for Ranunculeae. Here, we revisit the phylogeny of Ranunculeae by analyzing the individual matK/trnK, psbJ-petA, and internal transcribed spacer (ITS) data, the combined matK/trnK, psbJ-petA, and ITS dataset, and the combinedrbcL, trnL-F, matK/trnK, psbJ-petA, and ITS dataset. Based on the tree-based comparisons, blast searches against NCBI of the sequences, and close examination of the alignment, we found that 10 psbJ-petA sequences previously used were questionable (erroneous or problematic) and responsible for previous conflicting topologies. After omitting these questionable sequences, we provide a new phylogeny for Ranunculeae, in which Beckwithia–Cyrtorhyncha, Kumlienia, andPeltocalathos were replaced. These new replacements are supported by corresponding morphological characters. Moreover, three previously proposed intercontinental disjunct distributions within Ranunculus were also refuted. In our framework, our divergence time and biogeographic analyses indicate that divergence time estimates and the ancestral areas reconstructed for 10 of the 15 nodes in the genus-level phylogeny were influenced by elimination of the questionable sequences. The most recent common ancestor of Ranunculeae was inferred to be present in Europe and North America during the late Eocene. Clades I and II began to diversify in Europe and North America, respectively, and subsequently migrated to other continents. This study shows that it is necessary to analyze individual chloroplast DNA region datasets separately to detect questionable sequences early in the study. The combined dataset including the questionable sequences resulted in an erroneous phylogenetic tree, and the use of this tree subsequently affected age estimates and biogeographic analyses.     

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.     

Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria

We present phylogenetic analyses of 37 taxa of Fritillaria (Liliaceae), 15 species of Lilium, and several outgroup taxa from Liliaceae s.s. to investigate the generic delimitation of Fritillaria in relation to Lilium as well as infrageneric relationships within Fritillaria. We used DNA sequences from the maturase-coding plastid matK gene and the trnK intron, the intron of the ribosomal protein-coding rpl16 plastid gene, and the nuclear ribosomal internal transcribed spacers (ITS). Phylogenetic analysis using maximum parsimony defined Fritillaria and Lilium (the latter including Nomocharis) as sister taxa. Fritillaria sections Fritillaria and Liliorhiza are supported in part, and some of the most enigmatic species usually included in Fritillaria (sections Petilium and Theresia and the monotypic genus Korolkowia) are closely related. The results support the new classification of Fritillaria proposed by Rix. We postulate independent origins of the underground bulbils found in Fritillaria davidii and the remainder of subgenus Liliorhiza.     

Cryptic goldfields: a molecular phylogenetic reinvestigation of Lasthenia californica sensu lato and close relatives (Compositae: Heliantheae sensu lato)

Maximum parsimony analysis of DNA sequence data from the internal and external transcribed spacer (ITS and ETS) regions of 18S-26S nuclear ribosomal DNA and the 3' trnK intron of chloroplast DNA from over 60 populations of Lasthenia sect. Amphiachaenia yielded a well-supported tree showing that the most common species of Lasthenia, L. californica sensu lato (s.l.), is not monophyletic. Members of Lasthenia californica s.l. belong to two well-supported but morphologically cryptic clades. One clade includes members of L. macrantha; the other represents a basally divergent lineage in L. sect. Amphiachaenia. Members of each clade can be diagnosed by pappus morphology and by geographic distribution, except for epappose plants that occur in a broad region of sympatry in central California. Overall diversification in the clade corresponding to L. sect. Amphiachaenia has been accompanied by minimal morphological divergence, which has resulted in previously underappreciated cryptic diversity.     

Nucleotide sequence variation of the chloroplast trnK/matK region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice

Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intraspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (pi), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.     

A geographical pattern of Antirrhinum (Scrophulariaceae) speciation since the Pliocene based on plastid and nuclear DNA polymorphisms

Aim To infer phylogenetic relationships among Antirrhinum species and to reconstruct the historical distribution of observed sequence polymorphism through estimates of haplotype clades and lineage divergence. Location Antirrhinum is distributed primarily throughout the western Mediterranean, with 22 of 25 species in the Iberian Peninsula. Methods Plastid (83 trnS‐trnG and 83 trnK‐matK) and nuclear (87 ITS) sequences were obtained from 96 individuals representing 24 of the 25 Antirrhinum species. Sequences were analysed using maximum parsimony, Bayesian inference and statistical parsimony networking. Molecular clock estimates were obtained for plastid trnK‐matK sequences using the penalized likelihood approach. Results Phylogenetic results gave limited support for monophyletic groups within Antirrhinum. Fifty‐one plastid haplotypes were detected and 27 missing haplotypes inferred, which were all connected in a single, star‐like network. A significant number of species shared both the same haplotypes and the same geographical areas, primarily in eastern Iberia. Furthermore, many species harboured populations with unrelated haplotypes from divergent haplotype clades. Plastid haplotype distribution, together with nucleotide additivity in 59 of the 86 nuclear ribosomal ITS sequences, is interpreted as evidence of extensive hybridization. Lineage divergence estimates indicated that differentiation within Antirrhinum post‐dates the Miocene, when the Mediterranean climate was established. Main conclusions Incongruence between plastid sequences, nuclear sequences and taxonomic delimitation is interpreted as strong evidence of limited cladogenetic processes in Antirrhinum. Rather, extensive nucleotide additivities in ITS sequences in conjunction with haplotype and haplotype‐clade distributions related to geographical areas support both recent and ancient hybridization. This geographical pattern of Antirrhinum speciation, particularly in eastern Iberia, is congruent with isolation–contact–isolation processes in the Pleistocene.     

Phylogenetic analysis of the genus <Emphasis Type="Italic">Petunia</Emphasis> (Solanaceae) based on the sequence of the <Emphasis Type="Italic">Hf1</Emphasis> gene

Polymerase chain reaction fragment length polymorphisms and nucleotide sequences for a cytochrome P450 gene encoding flavonoid-3',5'-hydroxylase, Hf1, were studied in 19 natural taxa of Petunia. Natural Petunia taxa were classified into six groups based on major insertion or deletion events that occurred only in intron II of the locus. The maximum parsimony method was used to calculate strict consensus trees based on nucleotide sequences in selected regions of the Hf1 locus. Petunia taxa were divided into two major clades in the phylogenetic trees. Petunia axillaris (including three subspecies), P. exserta, and P. occidentalis formed a clade with 100% bootstrap support. This clade is associated with a consistently inflexed pedicel, self-compatibility in most taxa, and geographical distribution in southern and western portions of the genus range. The other clade, which comprised the remainder of the genus is, however, less supported (up to 71% bootstrap); it is characterized by a deflexed pedicel in the fruiting state (except P. inflata), self-incompatibility, and a northeastern distribution. A nuclear gene, Hf1, seems to be a useful molecular marker for elucidating the phylogeny of the genus Petunia when compared with the nucleotide sequence of trnK intron of chloroplast DNA.     

Phylogeny and systematics of the genus Lophozia s. str. (Dumort.) Dumort. (Hepaticae) and related taxa from nuclear ITS1-2 and chloroplast trnL-F sequences

Nuclear ITS1-2 and chloroplast trnL-F were sequenced for 21 taxa of Lophozia s. str., two species of Protolophozia, five species of Schistochilopsis, three species of Barbilophozia and Obtusifolium obtusum. The topologies of phylogenetic trees for 49 taxa constructed from combined sequences of these regions by maximum parsimony, maximum likelihood and Bayesian methods are similar. The species of Lophozia s. str., excluding Lophozia sudetica, combine into two main clades and these contradict subdivisions of Lophozia s. str. based on morphology. The species status of Lophozia lantratoviae is confirmed, whereas Lophozia austro-sibirica is almost identical to Lophozia ventricosa var. guttulata. The genus Schistochilopsis is paraphyletic and occupies basal position to Lophozia s. str., while O. obtusum is clearly separated from Schistochilopsis. A low level of divergence was found between L. sudetica and Protolophozia debiliformes, which are closer to Barbilophozia than to Lophozia s. str. Molecular divergence between geographically remote populations of L. sudetica, Lophozia silvicoloides and Protolophozia debiliformis are low as opposed to those of Lophozia polaris, Lophozia pellucida or Lophozia excisa. Consideration of the trnL intron P8 region indels alone can adequately assign some clades revealed by tree building. A consensus secondary structure of the trnL intron P8 region could not be inferred for taxa studied mainly due to high sequence length diversity originated from deletions.     

Group II introns as phylogenetic tools: structure, function, and evolutionary constraints

Group II introns comprise the majority of noncoding DNA in many plant chloroplast genomes and include the commonly sequenced regions trnK/matK, the rps16 intron, and the rpl16 intron. As demand increases for nucleotide characters at lower taxonomic levels, chloroplast introns may come to provide the bulk of plastome sequence data for assessment of evolutionary relationships in infrageneric, intergeneric, and interfamilial studies. Group II introns have many attractive properties for the molecular systematist: they are confined to organellar genomes in eukaryotes and the majority are single-copy; they share a well-defined and empirically tested secondary and tertiary structure; and many are easily amplified due to highly conserved sequence in flanking exons. However, structure-linked mutation patterns in group II intron sequences are more complex than generally supposed and have important implications for aligning nucleotides, assessing mutational biases in the data, and selecting appropriate models of character evolution for phylogenetic analysis. This paper presents a summary of group II intron function and structure, reviews the link between that structure and specific mutational constraints in group II intron sequences, and discusses strategies for accommodating the resulting complex mutational patterns in subsequent phylogenetic analyses.     

Long-distance colonization of the Western Mediterranean by Cistus ladanifer (Cistaceae) despite the absence of special dispersal mechanisms

Aim There are few biogeographical and evolutionary studies that address plant colonization and lineage origins in the Mediterranean. Cistus serves as an excellent model with which to study diaspore dispersal and distribution patterns of plants exhibiting no special long‐distance dispersal mechanisms. Here we analyse the pattern of genetic variation and divergence times to infer whether the African–European disjunction of C. ladanifer L. is the result of long‐distance dispersal or of vicariance events. Location Principally the Western Mediterranean region, with a focus on the Strait of Gibraltar. Methods We used DNA sequence phylogenetic approaches, based on plastid (rbcL/trnK‐matK) and nuclear (ITS) sequence data sets, and the penalized likelihood method, to date the diversification of the 21 species of Cistus. Phylogenetic relationships and phylogeographical patterns in 47 populations of C. ladanifer were also analysed using two plastid DNA regions (trnS‐trnG, trnK‐matK). These sequence data were analysed using maximum parsimony, Bayesian inference and statistical parsimony. Results Dating estimates indicated divergence dates of the C. ladanifer lineage in the Pleistocene. Eight nucleotide‐substitution haplotypes distributed on the European (four haplotypes) and African (five haplotypes) sides of the Strait of Gibraltar were revealed from C. ladanifer sequences. Both the haplotype network and the phylogenetic analyses depicted two main Cistus lineages distributed in both Europe and North Africa. An Iberian haplotype forms part of the North African lineage, and another haplotype distributed on both continents is related to the European lineage. Haplotype relationships with respect to outgroup sequences supported the hypothesis that the centre of genetic diversity is in northern Africa. Main conclusions Based on lineage divergence‐time estimates and disassociation between geographical and lineage haplotype distributions, we inferred at least two intercontinental colonization events of C. ladanifer post‐dating the opening of the Strait of Gibraltar (c. 5 Ma). This result supports a hypothesis of long‐distance dispersal rather than a hypothesis of vicariance. We argue that, despite limited dispersal abilities, preference for disturbed habitats was integral to historical colonization after the advent of the Mediterranean climate (c. 3.2 Ma), when Cistus species diverged and became established as a dominant element in the Mediterranean scrub.     

Comparative analysis of a translocated copy of the trnK intron in carnivorous family Nepenthaceae

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.     

The effect of mating system differences on nucleotide diversity at the phosphoglucose isomerase locus in the plant genus Leavenworthia.

To test the theoretical prediction that highly inbreeding populations should have low neutral genetic diversity relative to closely related outcrossing populations, we sequenced portions of the cytosolic phosphoglucose isomerase (PgiC) gene in the plant genus Leavenworthia, which includes both self-incompatible and inbreeding taxa. On the basis of sequences of intron 12 of this gene, the expected low diversity was seen in both populations of the selfers Leavenworthia uniflora and L. torulosa and in three highly inbreeding populations of L. crassa, while high diversity was found in self-incompatible L. stylosa, and moderate diversity in L. crassa populations with partial or complete self-incompatibility. In L. stylosa, the nucleotide diversity was strongly structured into three haplotypic classes, differing by several insertion/deletion sequences, with linkage disequilibrium between sequences of the three types in intron 12, but not in the adjacent regions. Differences between the three kinds of haplotypes are larger than between sequences of this gene region from different species. The haplotype divergence suggests the presence of a balanced polymorphism at this locus, possibly predating the split between L. stylosa and its two inbreeding sister taxa, L. uniflora and L. torulosa. It is therefore difficult to distinguish between different potential causes of the much lower sequence diversity at this locus in inbreeding than outcrossing populations. Selective sweeps during the evolution of these populations are possible, or background selection, or merely loss of a balanced polymorphism maintained by overdominance in the populations that evolved high selfing rates.     

Generic phylogeny and historical biogeography of Alismataceae, inferred from multiple DNA sequences

Alismataceae is an aquatic or semi-aquatic herb family with a subcosmopolitan distribution. The family is one of the oldest lineages within monocots and plays an important role in the systematics, biogeography and evolutionary processes of flowering plants. However, the generic relationships of the family are still a subject of debate, and its historical biogeography is less studied. In the present study, we carried out a comprehensive phylogenetic analysis based on multiple DNA sequences (nuclear: ITS; chloroplast: psbA, rbcL, matK, rpoB, rpoC1, trnK 5' intron and trnK 3' intron; mitochondria: cob and atp1). The result supports merging Limnocharitaceae into Alismataceae as one family. Two well-supported clades were obtained based on the combined ITS, psbA, rbcL and matK dataset. Clade B consists of Luronium, Damasonium, Baldellia and Alisma; and clade A consists of the remaining genera of Alismataceae as well as Limnocharitaceae. Biogeographic analysis and bayesian molecular dating suggested that Alismataceae originated in West Palearctic or Afrotropical area during the Late Cretaceous, and subsequently split into two clades. Clade A and clade B diversified in Afrotropical area and West Palearctic area, respectively. The intercontinental distribution of this family mainly resulted from dispersals involving migration across land bridges and long-distance dispersal.