Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

Phylogenetic analysis of Leucojum and Galanthus (Amaryllidaceae) based on plastid matK and nuclear ribosomal spacer (ITS) DNA sequences and morphology

Phylogenetic analyses of the monocotyledonous genera Leucojum and Galanthus (Amaryllidaceae, Asparagales), using plastid (trnL-F and matK) and largely non-coding nuclear ribosomal (ITS) DNA sequences show the two to be closely related to Lapiedra, Narcissus, Vagaria, Pancratium and Sternbergia. We compare the results obtained with a combined parsimony analysis of these nucleotide sequences with that of a matrix of morphological characters. The sampling included all species of Leucojum and most species of Galanthus (representing all series and subseries of the genus) and used as outgroup the above mentioned genera of Amaryllidaceae shown to be close relatives. The plastid, nuclear and morphological data were analysed independently and in combination, showing that the boundaries between the two genera are not appropriate. Galanthus is monophyletic but embedded in Leucojum. On the basis of chromosome numbers and floral characters Leucojum has been previously divided into four subgenera, which have been accepted as genera by some authors. In our phylogenetic analyses (separate as well as combined), Leucojum species are separated in two primary clades corresponding to L. subgenera Ruminia + Acis and L. Leucojum + Aerosperma. The taxonomic implications of this pattern are discussed, and an alternative classification is proposed. Finally, biogeographic relationships of species of both Leucojum and Galanthus are discussed, emphasising the possible origin of the narrowly distributed taxa of Leucojum relative to the widespread species.     

Physical mapping of plastid DNA variation among eleven Nicotiana species

Summary Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.Abbreviations ims isonuclear male sterile - ptDNA plastid chloroplast DNA - Rubisco ribulosebisphosphate carboxylase/oxygenase - kbp kilobase pairs - LSU large subunit of Rubisco     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

Variation in culture,isoenzyme patterns and plastid DNA in the genusDaucus

Summary To identify markers for fusion and transformation studies, cell suspension cultures of four members of theDaucus genus were examined to determine differences in culture conditions, isoenzyme patterns, and plastid DNA. The four were:D. carota subsp.sativus cv. Danvers,D. carota subsp.gummifer, D. capillifolius, andD. pusillus. Under appropriate conditions, all four grew well as liquid cell suspension cultures and regenerated from protoplasts into plants. Enzyme activities of homoserine dehydrogenase (HSDH) and alcohol dehydrogenase from cell culture extracts were analyzed on electrophoretic gels. Although only one form of HSDH was present in eachDaucus line, the rate of migration of HSDH from cv. Danvers was different from that of the other cell lines. Multiple isoenzymic forms of ADH were present in eachDaucus cultivar. Camparison of endonuclease restriction fragment patterns from plastid DNAs digested by BamHI revealed only small differences between plastid DNAs of cv. Danvers and subsp.Gummifer, whereas large differences were observed between cv. Danvers andD. pusillus plastid DNA patterns. No differences were found between cv. Danvers andD. capillifolius plastid DNA patterns when examined using eight different restriction enzymes. The data indicate that specific isoenzyme and organelle DNA restriction fragment patterns will be useful markers for precise identification of genomes of differentDaucus species in somatic hybridization experiments. This research was supported by the U.S. Department of Agriculture under Agreement 59-2246-1-1-737-0.     

The status of Platyhypnidium mutatum Ochyra & Vanderpoorten and the systematic value of the Donrichardsiaceae based on molecular data

Abstract

Base sequences of 376 bp of chloroplast DNA and of 521 bp of nuclear ribosomal DNA of the newly described Platyhypnidium mutatum Ochyra & Vanderpoorten and Platyhypnidium riparioides (Hedw.) Dixon are identical with the exception of one substitution in the nuclear ribosomal DNA. From these almost identical base sequences and the identical sporophytes found in both species, it is concluded that Platyhypnidium mutatum is a somatic mutant of the latter species differing by a bistratose lamina and a broader nerve. Previous to these results, Platyhypnidium mutatum would have been placed in the genus Donchrichardsia because of its vegetative morphological characters. It is therefore concluded that the family Donrichardsiaceae is artificial in spite of its strong morphological integrity.     

Restriction fragment polymorphisms in the rDNA region among seven species ofAlnus andBetula papyrifera

A study of restriction fragment polymorphisms of ribosomal DNA among seven actinorhizal species (Alnus spp.) and a non-actinorhizal species (Betula papyrifera Marsh.) of the Betulaceae was conducted, using a simple method for the extraction of high molecular weight restrictable nuclear DNA from leaf tissues of perennial angiosperms and nine restriction endonucleases. rDNA restriction fragments were variable within and among the species studied, and the variation noted was used to calculate the similarities and infer phenetic relationships among these members of the Betulaceae. The results confirmed the taxonomy of alder based on morphological characters, showing a clear clustering of the species ofAlnus sampled in each of the two different subgeneraAlnus andAlnobetula. Within each subgenus, the closely related taxa often classified as subspecies by their similar morphology and their ability to interhybridize, were similarly shown by restriction fragment polymorphisms to be more closely related to each other than to any other taxon. The analysis also suggested that some alder species may not be more divergent fromBetula papyrifera than from other alder species.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Morphological and molecular characterization of a natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae)

A putative natural hybrid betweenOrchis laxiflora andO. morio (Orchidaceae) from southern Italy, formerly known asO. alata, was characterized both on morphological and molecular grounds in order to confirm its hybrid status and to trace its maternal lineage. The morphological characters of the putative hybrid showed intermediacy between those of the parent species, and restriction fragment length polymorphism (RFLP) analysis of a region of the nuclear ribosomal DNA confirmed its hybrid origin. Chloroplast DNA RFLP analysis indicated thatO. morio provided the maternal genome.     

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron

Standard plant DNA barcodes based on 2–3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3–4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.     

Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy‐to‐access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.     

A molecular investigation of polymorphism in the North Atlantic red alga Chondrus crispus (Gigartinales)*

Seven samples of Chondrus crispus Stackhouse, representing widely contrasting forms from both sides of the North Atlantic, were compared by restriction digestion of their plastid DNA. The similar banding patterns confirmed that the seven forms were conspecific and distinct from Chondrus ocellatus Holmes f. ocellatus from Japan, used as an outgroup. Nucleotide sequences of the internal transcribed spacers (ITS1 and ITS21 and the intervening 5.8S rRNA gene of the nuclear rRNA operon were investigated as a potential indicator of genetic divergence among morphological variants of C. crispus. The combined ITS regions were relatively short in Chondrus (between 719 and 731 base pairs [bp] in C. crispus and 724 bp in C. ocellatus f. ocellatus), and the sequence of the 5.8s rDNA fragment (152 bp) was identical in both species. In the aligned ITS regions, there were 0–18 base pair differences (0–2.18% divergence) in pairwise comparisons of the seven forms of C. crispus but no consistent pattern of variation according to gross morphology or geographic origin. However, the ITS sequence differed at 41–54 sites (6.22-7.56%) between C. crispus and C. ocellatus f. ocellatus, again illustrating the genetic distinctiveness of the latter species.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Analysis of taxonomic and geographic patterns of Turkish Veronica orientalis using nuclear and plastid DNA and morphological data

Veronica orientalis and related species form one of the taxonomically most challenging subgroups within the genus Veronica. Hybridization and polyploidization on the one hand and convergent character evolution on the other have made delimitation of species difficult. Amplified fragment length polymorphisms and plastid DNA markers were used in conjunction with 54 morphological characters to study relationships among 35 accessions of V. orientalis Mill. as well as other closely related species of Veronica occurring in Turkey and adjacent areas of Georgia and Armenia. In addition, ploidy levels were estimated for 15 accessions using flow cytometry. Diploid to hexaploid individuals were detected in V. orientalis. Analysis of DNA markers demonstrated the non-monophyly of V. orientalis, especially with regard to V. fuhsii and V. multifida from Eastern Turkey, but nuclear and plastid DNA markers are largely incongruent. Neither demonstrates a clear biogeographic pattern. The morphological analysis reveals the distinction of V. orientalis subsp. carduchorum, which is weakly retrieved in some molecular analyses and some clustering according to geography. V. multifida is not monophyletic likely due to parallel evolution of pinnatifid leaves east and west of the Anatolian diagonal.     

绣球属(Hydrangea L.)植物分子系统学及系统发育分析

该研究基于对绣球属(Hydrangea L.)的大尺度取样,选取国内外61种绣球属和近缘属植物,分别基于核基因片段(ITS)和叶绿体基因片段(rbcL,trnL-F,atpB)重建了绣球属及其近缘种属的系统发育关系。结果表明:(1)核基因与叶绿体基因树之间在树形上没有明显的冲突,进而基于核基因和叶绿体基因联合数据重建了绣球属及其近缘种属的系统发育关系。(2)基于联合数据构建的系统树确认了2个大分支,并得到了果实顶端截平与否这一形态学证据的强力支持;每个大分枝又分为4个类群,共确定了8个类群。部分类群也得到了广义宏观形态性状的支持,如第1类群得到了叶形、花粉以及种子形态的支持。因此,该系统发育关系的重建对于全面理解绣球属及其近缘种属的演化关系具有重要的启发。     

A molecular method for identification of the morphologically plastic invasive algal genera Eucheuma and Kappaphycus (Rhodophyta, Gigartinales) in Hawaii

A paucity of diagnostic morphological characters for identification and high morphological plasticity within the genera Eucheuma and Kappaphycus has led to confusion about the distributions and spread of three introduced eucheumoid species in Hawaii. Entities previously identified as E. denticulatum, K. alvarezii, and K. striatum have had profound negative effects on Oahu’s coral reef ecosystems. The use of molecular tools to aid identification of algal species has been promising in other morphologically challenging taxa. We used three molecular markers (partial nuclear 28S rRNA, partial plastid 23S rRNA, and mitochondrial 5′ COI) and followed a DNA barcoding-like approach to identify Eucheuma and Kappaphycus samples from Hawaii. Neighbor-joining analyses were congruent in their separation of Eucheuma and Kappaphycus, and the resulting clusters were consistent with those revealed for global comparisons with the mitochondrial cox2-3 spacer and GenBank data. Based on these results, new insights were revealed into the distribution of these groups in Hawaii.     

Phylogeny of Veronica in the Southern and Northern Hemispheres based on plastid,nuclear ribosomal and nuclear low-copy DNA

The cosmopolitan and ecologically diverse genus Veronica with approximately 450 species is the largest genus of the newly circumscribed Plantaginaceae. Previous analyses of Veronica DNA sequences were in stark contrast to traditional systematics. However, analyses did not allow many inferences regarding the relationship between major groups identified, hindering further analysis of diversification and evolutionary trends in the genus. To resolve the backbone relationships of Veronica, we added sequences from additional plastid DNA regions to existing data and analyzed matching data sets for 78 taxa and more than 5000 aligned characters from nuclear ribosomal DNA and plastid DNA regions. The results provide the best resolved and supported estimate of relationships among major groups in the Northern (Veronica s. str.) and Southern Hemisphere (hebes). We present new informal names for the five main species groups within the Southern Hemisphere sect. Hebe. Furthermore, in two instances we provide morphological and karyological characters supporting these relationships. Finally, we present the first evidence from nuclear low-copy CYCLOIDEA2-region to compare results from the plastid genome with the nuclear genome.     

Temperature,Herbivory and Epibiont Acquisition as Factors Controlling the Distribution and Ecological Role of an Invasive Seaweed

The invasive canopy alga, Codium fragile ssp. tomentosoides, first observed at the Isles of Shoals in 1983, has become the dominant canopy species to 8 m throughout the islands. Codium populations are replacing themselves at most sites in what appears to be a new, climax, canopy species. However, Codium densities have declined in protected Gosport Harbor areas where it first became established. Codium has only slowly expanded its presence in adjacent nearshore subtidal habitats. Recent studies suggest a combination of factors that may be influencing the relative success of populations between habitats. The herbivorous sea slug, Placida dendritica, may be reducing populations in protected areas in spite of predators such as the green crab, Carcinus maenas, while surge may inhibit herbivore buildup in exposed habitats. Temperature instability due to localized, wind-driven upwelling may be slowing the buildup of subtidal Codium populations in nearshore sites. The combination of Codium dominance and the acquisition of increasing epibiont diversity are producing a new, potentially more complex community state than the previous kelp-dominated climax typical of the Gulf of Maine.     

Assessment of genetic diversity of native species in Izu Islands for a discriminate choice of source populations: Implications for revegetation of volcanically devastated sites

In using native species for revegetation, it is necessary choose source populations carefully to reduce the risk of planting suboptimal germplasm. To make preliminary recommendations for native species to use in the revegetation of a volcanically devastated area on Miyake Is., Japan, we investigated the genetic variation of Alnus sieboldiana, Miscanthus sinensis ssp. condensatus, and Polygonum cuspidatum var. terminalis in the Izu Islands and on the Izu Peninsula based on chloroplast DNA (cpDNA) sequence variations and amplified fragment length polymorphisms (AFLPs). The amount and pattern of differentiation differ between organelle and nuclear markers, suggesting the necessity of evaluation based on both types of markers. Within-population diversity did not vary among populations, suggesting that it does not need to be considered in the choice of a source population. The pattern and degree of differentiation varied among species, and geographical proximity did not necessarily accord with genetic similarity, suggesting that the site of an appropriate source population varies among species and should be determined empirically rather than by assuming that close proximity predicts genetic similarity. The Izu Peninsula populations deviated from the island populations in all species. Comparison of cpDNA sequences with those of related species indicates the possibility of hybridization with related species on the Izu Peninsula, suggesting that seeds collected from populations where related species live sympatrically should not be used for revegetation. These findings indicate the need to assess the genetic diversity empirically by using organelle and nuclear markers to avoid unintended consequences of genetic mixing associated with revegetation.     

Molecular phylogeny and species delimitation of Stachyuraceae: Advocating a herbarium specimen‐based phylogenomic approach in resolving species boundaries

Species concept and delimitation are fundamental to taxonomic and evolutionary studies. Both inadequate informative sites in the molecular data and limited taxon sampling have often led to poor phylogenetic resolution and incorrect species delineation. Recently, the whole chloroplast genome sequences from extensive herbarium specimen samples have been shown to be effective to amend the problem. Stachyuraceae are a small family consisting of only one genus Stachyurus of six to 16 species. However, species delimitation in Stachyurus has been highly controversial because of few and generally unstable morphological characters used for classification. In this study, we sampled 69 individuals of seven species (each with at least three individuals) covering the entire taxonomic diversity, geographic range, and morphological variation of Stachyurus from herbarium specimens for genome‐wide plastid gene sequencing to address species delineation in the genus. We obtained high‐quality DNAs from specimens using a recently developed DNA reconstruction technique. We first assembled four whole chloroplast genome sequences. Based on the chloroplast genome and one nuclear ribosomal DNA sequence of Stachyurus, we designed primers for multiplex polymerase chain reaction and high throughput sequencing of 44 plastid loci for species of Stachyurus. Data of these chloroplast DNA and nuclear ribosomal DNA internal transcribed spacer sequences were used for phylogenetic analyses. The phylogenetic results showed that the Japanese species Stachyurus praecox Siebold & Zucc. was sister to the rest in mainland China, which indicated a typical Sino‐Japanese distribution pattern. Based on diagnostic morphological characters, distinct distributional range, and monophyly of each clade, we redefined seven species for Stachyurus following an integrative species concept, and revised the taxonomy of the family based on previous reports and specimens, in particular the type specimens. Furthermore, our divergence time estimation results suggested that Stachyuraceae split from its sister group Crossosomataceae from the New World at ca. 54.29 Mya, but extant species of Stachyuraceae started their diversification only recently at ca. 6.85 Mya. Diversification time of Stachyurus in mainland China was estimated to be ca. 4.45 Mya. This research has provided an example of using the herbarium specimen‐based phylogenomic approach in resolving species boundaries in a taxonomically difficult genus.