Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Range expansion during the Pleistocene drove morphological radiation of the fir genus (Abies,Pinaceae) in the Qinghai‐Tibet Plateau and Himalayas

Range expansion caused by climate oscillations in the past probably promoted morphological radiation in a few plant groups. In this study, we aim to test this hypothesis through phylogeographical analysis of the cold‐tolerant fir genus (Abies) in the Qinghai‐Tibet Plateau (QTP) and Himalayas, where it comprises 12 described species. We examined sequence variation in two maternally inherited mitochondrial (mt) DNA fragments (nad5‐4 and nad7‐1) and two paternally inherited plastid DNA fragments (trnS‐G and trnL‐F) for 733 individuals from 75 populations of the species in a monophyletic group. Only six mtDNA haplotypes were recovered, but five were shared between multiple species and one occurred at a high frequency, providing strong evidence of range expansion. Forty‐three plastid DNA haplotypes were detected, 19 of which were shared between species and three occurred at high frequency. Network, mismatch and Bayesian skyline plot analyses of all plastid DNA haplotypes from this clade clearly suggested range expansion. This expansion was dated as having occurred during the longest and most extensive glaciation in the Pleistocene. Our results therefore supported the range expansion hypothesis for this clade of Abies during the Pleistocene; expansion probably drove the morphological radiation of the clade in the QTP and Himalayas, although it remains unclear whether the different morphotypes should be acknowledged as independent, reproductively isolated species. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 444–453.     

First evaluation of mitochondrial DNA as a marker for phylogeographic studies of Calcarea: a case study from Leucetta chagosensis

In most animals mitochondrial DNA (mtDNA) evolves much faster than nuclear DNA. Therefore, and because of its shorter coalescent time, mitochondrial (mt) markers provide better resolution to trace more recent evolutionary events compared to nuclear DNA. But in contrast to most other Metazoa, previous studies suggested that in sponges mitochondrial sequence evolution is much slower, making mtDNA less suitable for studies at the intraspecific level. However, these observations were made in the class Demospongiae and so far no data exist for calcareous sponges (Class Calcarea). We here provide the first study that evaluates intraspecific mt sequence variation in Calcarea. We focus on arguably the best-studied species Leucetta chagosensis, for which three nuclear DNA marker datasets existed previously. We here sequenced the partial mitochondrial cytochrome oxidase subunit III gene (cox3). Our analyses reveal an unexpected variability of up to 8.5% in this mitochondrial marker. In contrast to other sponges where this marker evolves considerable slower than the nuclear internal transcribed spacer region (ITS), we found that cox3 in L. chagosensis evolves about five times as fast as ITS. The variability is similar to that of nuclear intron data of the species. The phylogeny inferred with cox3 is congruent with other markers, but separates earlier reported genetic groups much more distinctively than nuclear DNA. This provides further evidence for cryptic speciation in L. chagosensis. All these features make calcarean mtDNA exceptional among sponges and show its suitability for phylogeographic studies and potential as a species-specific (DNA barcoding) marker to distinguish morphologically identical cryptic species.     

Homology-dependent DNA Transfer from Plants to a Soil Bacterium Under Laboratory Conditions: Implications in Evolution and Horizontal Gene Transfer

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.     

Isolation, purification and identification of acetylcholine in Pharbitis nil seedlings

Acetylcholine (ACh), a well known animal neurotransmitter was isolated from tissues of Pharbitis nil using five different methods. Its presence in plant extract was confirmed by infrared spectroscopy and mass spectrometry. For quantitative estimation of ACh in P. nil seedlings pyrolysis-gas chromatography was applied. The presence of ACh was found in all organs of the examined plant: seeds, shoot apex, cotyledons, leaves, shoots and roots. However, the highest level of the investigated substance was noted in the youngest growing parts. In 5-day-old etiolated seedlings they were cotyledons, whereas in 14 day-old green plants - shoot apex and young leaves.     

Evaluating the utility of cox1 for cetacean species identification

The mitochondrial cytochrome c oxidase I (cox1) gene has been promoted as a universal reference gene, or barcode, to identify organisms to the species level. We evaluated whether cox1 would be appropriate to diagnose cetacean species. The 5′ end of cox1 (686 base pairs, bp) was sequenced for 46 of 86 recognized species of cetaceans. In addition, we included 105 sequences from GenBank, increasing our taxonomic coverage to 61 species. Particular focus was placed on sampling two subfamilies that contain closely related taxa: the Delphininae and the Globicephalinae. Species‐specific sequences were observed for all but three taxa (Delphinus delphis, D. capensis, and Stenella coeruleoalba). Although correct assignment was seen for most species, significant overlap between intra‐ and interspecific variation makes cox1 an imperfect barcode for cetaceans. The efficacy of cox1 was compared to the 5′ end of the cytochrome b (cytb) gene, a mitochondrial region routinely used for cetacean species identification. Although cytb performed better than cox1 for some species, this marker could not differentiate other closely related taxa (Eubalaena spp.). Species identification for taxa not reliably identified using cox1 or cytb might be best addressed through use of multiple mitochondrial DNA fragments or other newly developed markers.     

Does the Mode of Plastid Inheritance Influence Plastid Genome Architecture?

Plastid genomes show an impressive array of sizes and compactnesses, but the forces responsible for this variation are unknown. It has been argued that species with small effective genetic population sizes are less efficient at purging excess DNA from their genomes than those with large effective population sizes. If true, one may expect the primary mode of plastid inheritance to influence plastid DNA (ptDNA) architecture. All else being equal, biparentally inherited ptDNAs should have a two-fold greater effective population size than those that are uniparentally inherited, and thus should also be more compact. Here, we explore the relationship between plastid inheritance pattern and ptDNA architecture, and consider the role of phylogeny in shaping our observations. Contrary to our expectations, we found no significant difference in plastid genome size or compactness between ptDNAs that are biparentally inherited relative to those that are uniparentally inherited. However, we also found that there was significant phylogenetic signal for the trait of mode of plastid inheritance. We also found that paternally inherited ptDNAs are significantly smaller (n = 19, p = 0.000001) than those that are maternally, uniparentally (when isogamous), or biparentally inherited. Potential explanations for this observation are discussed.     

Cytoplasmic inheritance in green algae: patterns,mechanisms and relation to sex type

Cytological and genetic investigations of two major groups of green algae, chlorophyte and streptophyte green algae, show a predominance of uniparental inheritance of the plastid and mitochondrial genomes in most species. However, in some crosses of isogamous species of Ulva compressa, these genomes are transmitted from mt⁺, mt⁻, and both parents. In species with uniparental organelle inheritance, various mechanisms can eliminate organelles and their DNA during male gametogenesis or after fertilization. Concerning plastid inheritance, two major mechanisms are widespread in green algae: (1) digestion of plastid DNA during male gametogenesis, during fertilization, or after fertilization; and (2) disintegration or fusion of the plastid in the zygote. The first mechanism also eliminates the mitochondrial DNA in anisogamous and oogamous species. These mechanisms would ensure the predominantly uniparental inheritance of organelle genomes in green algae. To trace the evolutionary history of cytoplasmic inheritance in green algae, the relations between uniparental inheritance and sex type were considered in isogamous, anisogamous, and oogamous species using sex-specific features that might be nearly universal among Chlorophyta.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome

Summary In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain ana ^r -14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome - c oxidase - cob gene encoding apocytochrome b - cox1I1, cox1I2a, cox1I2b, cox1I3 introns in cox1 - cox1Ix ^+/– indicates the presence or absence of the intron either in the native gene or after intron DNA excision - cox1Ix is a deletion in the intron leading to respiratory deficiency     

IS POLYPLOIDY NECESSARY FOR TISSUE DIFFERENTIATION IN HIGHER PLANTS?

Measurements of relative DNA per nucleus of cells from various tissues show that cell differentiation can occur in the absence of polyploidy in higher plants. In Pisum polyploidy was present in roots, sepals, pods, pistils, and stamens but not in petals or leaves. In Triticum cells of leaves exhibited some polyploidy, but no polyploid cells were present in mature roots. No polyploid cells were found in any tissue of Helianthus examined (roots, cotyledons, stems, leaves, sepals, petals, pistils, and stamens). Therefore, as a general rule, polyploidy should not be considered essential in tissue or organ differentiation of higher plants. In Helianthus polyploidy is unnecessary for the completion of the life cycle.     

Heteroblasty in Arabidopsis thaliana (L.) Heynh

 Heteroblasty in Arabidopsis thaliana was analyzed in a variety of plants with mutations in leaf morphology using a tissue-specific β-glucuronidase gene marker. Some mutants exhibited their mutant phenotypes specifically in foliage leaves. The phenotypes associated with the foliage-leaf-specific mutations were also found to be induced ectopically in cotyledons in the presence of the lec1 mutation. Moreover, the features of an emf1lec1 double mutant showed that cotyledons can be partially converted into carpelloids. When heteroblastic traits were examined in foliage leaves in the presence of certain mutations or natural deviations by histochemical analysis of the expression of the tissue-specific marker gene, it was found that ectopic expression of the developmental program for the first foliage leaves in lec1 cotyledons seemed to affect the heteroblastic features of the first set of foliage leaves, while foliage leaves beyond the third position appeared normal. Similarly, in wild-type plants, discrepancies in heteroblastic features, relative to standard features, of foliage leaves at early positions seemed to be eliminated in foliage leaves at later positions. These results suggest that heteroblasty in foliage leaves might be affected in part by the heteroblastic stage of the preceding foliage leaves but is finally controlled autonomously at each leaf position. Received: 9 July 1999 / Accepted: 17 August 1999     

DIRECT AND INDIRECT ESTIMATES OF SEED VERSUS POLLEN MOVEMENT WITHIN A POPULATION OF PONDEROSA PINE

We examined the spatial distribution of maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA polymorphisms in a permanently marked stand of ponderosa pine (Pinus ponderosa Laws). Movement of maternally inherited mtDNA occurs only via seed dispersal, and mtDNA haplotypes showed significant patch structure. Moreover, individuals within patches identified by mtDNA haplotypes were related approximately as half-sibs based upon analysis of allozyme genotypes. Thus, seed dispersal is limited within the population, and creates matrilineal clusters in space. By contrast, paternally inherited cpDNA is dispersed by movement of both seed and pollen. Chloroplast DNA polymorphisms showed no evidence of patch structure, but rather a weak (and nonsignificant) trend toward hyperdispersion, suggesting nearly unlimited movement of pollen among trees within this stand. Two of the trees had unique allozyme alleles, which were used to directly measure pollen movement away from those trees. Marked pollen was as likely to disperse across the population as it was to fertilize near neighbors.     

Dynamic evolution of eukaryotic mitochondrial and nuclear genomes: a case study in the gourmet pine mushroom Tricholoma matsutake

Fungi, as eukaryotic organisms, contain two genomes, the mitochondrial genome and the nuclear genome, in their cells. How the two genomes evolve and correlate to each other is debated. Herein, taking the gourmet pine mushroom Tricholoma matsutake as an example, we performed comparative mitogenomic analysis using samples collected from diverse locations and compared the evolution of the two genomes. The T. matsutake mitogenome encodes 49 genes and is rich of repetitive and non-coding DNAs. Six genes were invaded by up to 11 group I introns, with one cox1 intron cox1P372 showing presence/absence dynamics among different samples. Bioinformatic analyses suggested limited or no evidence of mitochondrial heteroplasmy. Interestingly, hundreds of mitochondrial DNA fragments were found in the nuclear genome, with several larger than 500 nt confirmed by PCR assays and read count comparisons, indicating clear evidence of transfer of mitochondrial DNA into the nuclear genome. Nuclear DNA of T. matsutake showed a higher mutation rate than mitochondrial DNA. Furthermore, we found evidence of incongruence between phylogenetic trees derived from mitogenome and nuclear DNA sequences. Together, our results reveal the dynamic genome evolution of the gourmet pine mushroom.     

MITOCHONDRIAL DNA IN THE OOGAMOCHLAMYS CLADE (CHLOROPHYCEAE): HIGH GC CONTENT AND UNIQUE GENOME ARCHITECTURE FOR GREEN ALGAE1

Most mitochondrial genomes in the green algal phylum Chlorophyta are AT‐rich, circular‐mapping DNA molecules. However, mitochondrial genomes from the Reinhardtii clade of the Chlorophyceae lineage are linear and sometimes fragmented into subgenomic forms. Moreover, Polytomella capuana, from the Reinhardtii clade, has an elevated GC content (57.2%). In the present study, we examined mitochondrial genome conformation and GC bias in the Oogamochlamys clade of the Chlorophyceae, which phylogenetic data suggest is closely related to the Reinhardtii clade. Total DNA from selected Oogamochlamys taxa, including four Lobochlamys culleus (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian strains, Lobochlamys segnis (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, and Oogamochlamys gigantea (O. Dill) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, was subjected to Southern blot analyses with cob and cox1 probes, and the results suggest that the mitochondrial genome of these taxa is represented by multiple‐sized linear DNA fragments with overlapping homologies. On the basis of these data, we propose that linear mitochondrial DNA with a propensity to become fragmented arose in an ancestor common to the Reinhardtii and Oogamochlamys clades or even earlier in the evolutionary history of the Chlorophyceae. Analyses of partial cob and cox1 sequences from these Oogamochlamys taxa revealed an unusually high GC content (49.9%–65.1%) and provided evidence for the accumulation of cob and cox1 pseudogenes and truncated sequences in the mitochondrial genome of all L. culleus strains examined.     

Plastid-DNA levels in the different tissues of potato

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - LSU large sub-unit of ribulose-1,5-bisphosphate carboxylase - mtDNA mitochondrial DNA - ptDNA chloroplast or plastid DNA