RAPD analysis of genome polymorphism in the family Lemnaceae

The multilocus RAPD analysis of intergeneric, inter-and intraspecific nuclear genome polymorphism was used for the first time to assess intergeneric, interspecific, and intraspecific polymorphism in Lemnaceae growing on the territory of Russia. The origin of the chosen accessions overlapped with the natural range of duckweeds in Russia. Seventy-five Lemnaceae accessions representing eight species (L. minor, L. gibba, L. turionifera, L. japonica, L. trisulca, L. aequinoctialis, S. polyrhiza, and L. punctata) from three genera (Lemna, Spirodela, and Landoltia), were analyzed. The highest variability levels were revealed in L. minor accessions (0.03–0.20). Species L. trisulca and S. polyrhiza were characterized by values of genetic distance 0.01–0.18 and 0.03–0.16, respectively. The lowest polymorphism levels were detected for L. turionifera (0.01–0.11). The dendrogram based on RAPD data showed that L. aequinoctialis was the most genetically distant species of the genus Lemna. Accessions of species L. turionifera and L. japonica, as well as L. minor and L. gibba, did not form separate species-specific subclusters; rather, they fell into clusters with L. japonica/L. turionifera and L. minor/L. gibba. Accessions of the genera Spirodela and Landoltia formed two separate clusters combined into one group.     

Schottarum (Schismatoglottideae: Araceae) substantiated based on combined nuclear and plastid DNA sequences

Recent studies on Schismatoglottideae have resulted in the recognition of four new monophyletic genera, the resurrection of two additional genera following clarification of their monophyly, and the publication of many taxonomically novel species. However, generic boundaries among some parts of Schismatoglottideae remain unclear owing to several reasons: (1) more taxa are being revealed through our on-going fieldwork, now expanded to previously unsampled localities on Borneo; (2) established occurrence of a high level of homoplasies among the morphological characteristics hitherto used to delimitate genera; and (3) gene regions used in previous studies contradicted some of current taxonomic placements. Among the unsolved groups from previous studies a clade comprising Schismatoglottis sarikeensis and S. josefii needs further investigation. Therefore, phylogenetic analyses were carried out to investigate the position of these two species using the nuclear region, internal transcribed spacer and combined plastid regions: trnL intron and trnL-F intergenic spacer, coding matK+partial 3′ trnK, intergenic spacer trnH-psbA. A total of 23 accessions representing 16 taxa of Schismatoglottideae and Philonotieae were included in the study. Phylogenetic analyses of a total 4,658 bp combined dataset using parsimony, maximum likelihood, and Bayesian methods revealed that S. sarikeensis and S. josefii do not belong to Schismatoglottis, and therefore are transferred to Schottarum (≡Hottarum sarikeense ≡ Schismatoglottis sarikeense). Flowering mechanism, pollination strategy, and fruitset of S. sarikeense are also presented in the paper.     

The discovery and phylogenetic implications of a novel 41 bp plastid DNA deletion in wild potatoes

Insertions and deletions (indels) are common in intergenic spacer regions of plastid DNA and can provide important phylogenetic characters for closely related species. For example, a 241-bp plastid DNA deletion in the trnV-UAC/ndhC intergenic spacer region has been shown to have major phylogenetic importance in determining the origin of the cultivated potato. As part of a phylogenetic study of the wild potato Solanum series Piurana group we screened 199 accessions of 38 wild potato species in nine of the 19 tuber-bearing (Solanum section Petota) series that have not been examined before for indels in the trnV-UAC/ndhC intergenic spacer region. A novel 41 bp deletion (but no 241 bp deletion) was discovered for 30 accessions of three species: S. chiquidenum (5 of 10 accessions), S. chomatophilum (19 of 28), and S. jalcae (6 of 6). Accessions with and without this deletion are found throughout much of the north-south range of all three species in northern and central Peru, but not east of the Marañón River. Multivariate morphological analyses of these 44 accessions showed no morphological associations to the deletion. The results suggest extensive interspecific gene flow among these three species, or a common evolutionary history among species that have never been suggested to be interrelated.     

Hybrid status of Tibouchina urvilleana Cogn. revealed by ITS sequences of nuclear ribosomal DNA

Tibouchina urvilleana Cogn. is native to southern Brazil and currently cultivated as an important ornamental shrub in frost free areas around the world. Its rapid vegetative growth and sterility suggests that it might be of hybrid origin. In this study, Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA and chloroplast trnL-trnF spacer from T. urvilleana and three other congeneric species were sequenced to test its hybrid status. Cloning sequencing revealed two distinct types of ITS sequences from T. urvilleana, with one type almost identical with Tibouchina aspera. Genetic distance between the two types was much larger than the average interspecific genetic distances calculated from other Tibouchina species. Sequencing of chloroplast trnL-trnF spacer showed that T. urvilleana has identical sequence with T. aspera, but differed from other congeneric species by one nucleotide substitution and two indels. Molecular data demonstrated clearly that T. urvilleana indeed was a hybrid, with T. aspera or closely related species acting as the maternal parent.     

Evaluating species nonmonophyly as a trait affecting genetic diversity: a case study of three endangered species of Antirrhinum L. (Scrophulariaceae)

Molecular markers are routinely used to assess levels of diversity within and among populations, particularly with regard to species of conservation concern. However, when interpreting the level and partitioning of diversity observed, an implicit assumption is often made that the populations of the species in question form a monophyletic group. We tested this assumption in three endemics of Antirrhinum (A. charidemi, A. subbaeticum, and A. valentinum) using 79 nuclear [internal transcribed spacer (ITS)] and 85 plastid (psbA-trnH, trnT-trnL, trnK-matK, trnS-trnG) sequences representing multiple accessions of each of 24 Antirrhinum species (single accession of A. cirrhigerum). These species share six life history traits implicated in levels of genetic diversity, and have been the subject of previous population genetic studies. Populations of all three species formed monophyletic groups on ITS analysis. In contrast, none of the three species formed monophyletic groups on plastid sequence analysis: populations of A. charidemi fall in a monophyletic group including one accession of A. mollissimum, populations of A. subbaeticum form a polyphyletic group with plastid sequences shared with A. pulverulentum, and populations of A. valentinum are unresolved within a clade containing six other species. Lack of monophyly using plastid sequences is interpreted as a combination of shared ancestral polymorphism and hybridization in a reticulate evolutionary history of these species. Monophyly in the ITS tree may reflect a more recent sequence homogenization. We draw attention to the evaluation of species monophyly alongside the contribution of other life history traits in the historical interpretation of the level and partitioning of genetic diversity, and its use in recommendations for species conservation programs.     

Phylogenetic analysis of the genus Sorghum based on combined sequence data from cpDNA regions and ITS generate well-supported trees with two major lineages

Background and Aims

Wild Sorghum species provide novel traits for both biotic and abiotic stress resistance and yield for the improvement of cultivated sorghum. A better understanding of the phylogeny in the genus Sorghum will enhance use of the valuable agronomic traits found in wild sorghum.

Methods

Four regions of chloroplast DNA (cpDNA; psbZ-trnG, trnY-trnD, trnY-psbM and trnT-trnL) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA were used to analyse the phylogeny of sorghum based on maximum-parsimony analyses.

Key Results

Parsimony analyses of the ITS and cpDNA regions as separate or combined sequence datasets formed trees with strong bootstrap support with two lineages: the Eu-sorghum species S. laxiflorum and S. macrospermum in one and Stiposorghum and Para-sorghum in the other. Within Eu-sorghum, S. bicolor-3, -11 and -14 originating from southern Africa form a distinct clade. S. bicolor-2, originally from Yemen, is distantly related to other S. bicolor accessions.

Conclusions

Eu-sorghum species are more closely related to S. macrospermum and S. laxiflorum than to any other Australian wild Sorghum species. S. macrospermum and S. laxiflorum are so closely related that it is inappropriate to classify them in separate sections. S. almum is closely associated with S. bicolor, suggesting that the latter is the maternal parent of the former given that cpDNA is maternally inherited in angiosperms. S. bicolor-3, -11 and -14, from southern Africa, are closely related to each other but distantly related to S. bicolor-2.     

Molecular evidence for hybrid origin of Aster chusanensis,an endemic species of Ulleungdo,Korea

Ulleungdo is a small volcanic island and harbors many endemic plant species. Aster chusanensis Lim et al., a recently discovered endemic species on the island was proposed to be derived from hybridization between A. pseudoglehnii Lim et al. and A. oharai Nakai. To test hybrid origin of A. chusanensis, we sequenced the nrITS and three non-coding regions of a plastid genome (trnL-F, rbcL-accD, and psbA-trnH) from eight accessions of A. chusanensis and 37 accessions of its putative parents. Extensive investigation of the genome of A. chusanensis using 160 nrITS PCR amplicon sequences confirmed co-occurrence of nrITS sequences of the parental species within all accessions sampled. The retention of intact parental nrITS types in the genomes of A. chusanensis suggests that hybrid speciation has occurred recently on the island. The plastid DNA sequence data indicate that all of the hybrid individuals inherit the plastid from A. pseudoglehnii, except for one, which has the plastid of A. oharai, implying bidirectional but highly biased hybridization events during the evolution of A. chusanensis. Results of this study suggest that hybridization is an important process in the diversification of indigenous plants on Ulleungdo.     

Molecular phylogeny of the endemic fern genera Cyrtomidictyum and Cyrtogonellum (Dryopteridaceae) from East Asia

Cyrtomidictyum Ching and Cyrtogonellum Ching are two eastern Asian endemic genera whose taxonomic affinities and phylogenetic relationships have long been controversial. The main uncertainty surrounds the separation of the two genera from the species-rich genus Polystichum. Here we present a phylogenetic study focusing on the phylogenetic relationships of these polystichoid ferns. We reconstructed the relationships based on DNA sequence variation in four chloroplast genome regions, rbcL, atpB, and the intergenic spacers (IGS) rps4-trnS and trnL-trnF. Maximum likelihood and Bayesian inference analyses confirm earlier results that were based on less comprehensive taxon sampling and either only a single gene (rbcL) or two IGS (rps4-trnS and trnL-trnF). Cyrtomidictyum is the sister of the clade of polystichoid ferns that includes Cyrtogonellum, Cyrtomium subser. Balansana and three sections of Polystichum. Cyrtogonellum groups with several species of Polystichum, and constitutes the sister taxon to Polystichum sect. Sphaenopolystichum. We support the recognition of Cyrtomidictyum as circumscribed initially, rather than expansion of the genus to include either several Polystichum species or Cyrtogonellum, some Polystichum and Cyrtomium species. The monophyly of Cyrtomidictyum is supported by morphological characters such as once-pinnate leaves, free venation, prolongated leaf apices, and exindusiate sori. Two synapomorphic indels in the chloroplast genome, one 15-bp deletion in rps4-trnS, and one 3-bp insertion in trnL-trnF further differentiate Cyrtomidictyum from other polystichoid ferns. The close affinity of Cyrtogonellum to section Sphaenopolystichum of Polystichum s.s. is highly supported by molecular data. However, no shared morphological characters or molecular indels have been detected, although the distinctness of Cyrtogonellum is shown by a 13-bp insertion in the rps4-trnS alignment.     

Phylogenetic Analyses of Teleki Grapevine Rootstocks Using Three Chloroplast DNA Markers

Teleki rootstocks are used in grapevine-producing countries all over the world. They represent one of the largest groups of available rootstocks but their origin is still in dispute although they have been regarded as Vitis berlandieri × V. riparia hybrids. To investigate their possible origin, we amplified and sequenced three chloroplast regions, two non-coding spacers (trnL-F, trnS-G) and the trnL group I intron in a core collection of Teleki rootstocks representing widespread accessions and related wild North American grape species (V. berlandieri, V. riparia and V. rupestris). Concatenated sequence data coupled with microstructural changes discovered in the chloroplast regions provided data to trace the maternal ancestry of the Teleki lines. All chloroplast regions showed both nucleotide and length variation. Length mutations in the non-coding regions represented mostly simple sequence repeats of poly-A and -T stretches. These indel characters exhibited additional diversity comparable with the nucleotide diversity and increased resolution of the phylogenetic trees. We found that a group of Teleki accessions position together with the wild grape species V. riparia. Another group of Teleki rootstocks formed a sister group to the other North American species V. berlandieri. These clades had moderate support values, and they do not share ancestry with other accessions of Teleki rootstocks resolved with high support value in the V. riparia clade. It seems that Teleki-Kober 5BB and 125 AA accessions might have a V. berlandieri maternal background. We also found great differences within putative clones of Teleki 5C and Teleki-Kober 5BB suggesting that the selection of these accessions was performed on heterogenous or mislabeled plant material collectively maintained under these names.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.     

Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference. The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats, short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability. A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential, the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.     

Study on chloroplast DNA diversity of cultivated and wild pears (<Emphasis Type="Italic">Pyrus</Emphasis> L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (P_i), average number of nucleotide differences (k) and haplotype diversity (H_d) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.     

Molecular confirmation of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea

The hypothesis of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea, two mangrove species distributed in the Indo-West Pacific region, was proposed in 1970s based on morphological traits; however, no molecular evidence has been reported to support it. In this study, we sequenced two low-copy nuclear genes and one chloroplast intergenic spacer (trnS-trnG) in the two Lumnitzera species and their putative hybrid to test this hypothesis. Our results revealed that there were 9 and 27 nucleotide substitutions at the two nuclear loci, respectively, between one haplotype of L. racemosa and L. littorea, and that the putative hybrid showed additivity in chromatograms at these sites. Sequencing the chloroplast intergenic region trnS-trnG showed that the two Lumnitzera species differed by seven fixed nucleotide substitutions and four fixed insertions/deletions in this region, while the putative hybrid had identical sequences to L. racemosa. Molecular data clearly demonstrated that there indeed existed natural hybridization between L. racemosa and L. littorea and that L. racemosa was the maternal parent in this hybridization event. The uncommon direction of hybridization and F₁ nature of hybrids in this case, and in mangroves in general, is discussed.     

A phylogenetic analysis of Dipsacaceae based on four DNA regions

Authors studied the phylogeny of Dipsacaceae using maximum parsimony and Bayesian analyses on sequence data from chloroplast (trnL intron, trnL–trnF intergenic spacer, psbB–psbH gene complex) and nuclear genomes (ITS1 and ITS2). Both data partitions as well as their combination show that Dipsacaceae is a monophyletic group. Topology in tribe Scabioseae is similar to those of other recent studies, except for the position of Pycnocomon, which is nested in Lomelosia. Pycnocomon, the pollen and epicalyx morphologies of which closely resemble those of Lomelosia, is interpreted as a psammophilous morphotype of Lomelosia, and its nomenclature has been revised accordingly. Exclusion of Pseudoscabiosa, Pterocephalidium, Pterocephalodes (and probably Bassecoia), Succisa, Succisella from Scabioseae is confirmed. Pterocephalodes hookeri is the sister group to the rest of the family. Its remoteness from Pterocephalus has been confirmed on molecular grounds. Lack of evident synapomorphies for various clades is interpreted as a possible consequence of fast adaptative radiation.     

Natural Hybridization and Introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae,Senecioneae) in Four Different Locations

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5''rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.     

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

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

Phylogenetic Analysis of AA-genome Oryza Species (Poaceae) Based on Chloroplast, Mitochondrial, and Nuclear DNA Sequences

Species in the genus Oryza (Poaceae) contain 10 genomic types and are distributed in pan-tropics of the world. To explore phylogenetic relationships of Oryza species having the AA-genome, DNA sequences of the chloroplast trnL intron and trnL-trnF spacer, mitochondrial nad1 intron 2, and nuclear internal transcribed spacer were analyzed, based on materials from 6 cultivated (O. sativa and O. glaberrima) and 13 wild accessions, in addition to a CC-genome species (O. officinalis) that was used as an outgroup. Analyses of the combined sequence data set from different sources provide a much better resolution of the AA-genome species than the individual data set, indicating the limitation of a single gene in phylogenetic reconstruction. The phylogeny based on the combined data set demonstrated an apparent grouping of the AA-genome Oryza species that was well associated with their geographic origin, although the Australian O. meridionalis showed its affinity with the African species. The geographic pattern of the phylogenetic relationship was probably attributed to the frequent genetic exchange and introgression among the AA-genome species from the same continents. In addition, Asian cultivated rice O. sativa showed its close relation to O. rufipogon and O. nivara, whereas African cultivated rice O. glaberrima was closely linked to O. barthii and O. longistaminata, indicating the independent domestication of the two cultivated species in different geographic locations.     

Origin, diversification, and evolution of Samolus valerandi (Samolaceae, Ericales)

The almost cosmopolitan distribution of Samolus valerandi is unique in the genus Samolus L. (Samolaceae), which also includes 12–15 taxa with distributions restricted to smaller areas of the Southern Hemisphere. Recent molecular phylogenetic studies based on chloroplast and nuclear DNA sequences showed that the widespread S. valerandi and the North American S. parviflorus are both part of a strongly supported clade, together with the North American S. vagans, S. spathulatus, and S. latifolius. To better understand the origin, distribution, and diversification of S. valerandi and to clarify the relationships within this clade, we performed molecular phylogenetic analyses based on the plastid trnS-G intergenic spacer and the nuclear ribosomal ITS region. We have also sought further support for relationships by examining flower and leaf characters. On the basis of new results, we propose that S. valerandi, S. parviflorus, and S. vagans are considered as part of a widespread “species complex”, with its centre of diversity in North America. No clear vicariance patterns were found regarding the phylogeography of S. valerandi, which thus seems to have dispersed secondarily to various places around the world, possibly as a result of human activities.