Interspecific variation in Rubisco CO2/O2 specificity along the leaf economic spectrum across 23 woody angiosperm plants in the Pacific islands.

The coordinated interspecific variation in leaf traits and leaf lifespan is known as the leaf economic spectrum (LES). The limitation of CO2 diffusion to chloroplasts within the lamina is significant in C3 photosynthesis, resulting in a shortage of CO2 for Rubisco. Although Rubisco CO2/O2 specificity (SC/O) should be adaptively adjusted in response to the interspecific variation in CO2 concentrations [CO2] associated with Rubisco, SC/O variations across species along the LES remain unknown. We investigated the coordination among leaf traits, including SC/O, CO2 conductance, leaf protein content, and leaf mass area, across 23 woody C3 species coexisting on an oceanic island through phylogenetic correlation analyses. A high SC/O indicates a high CO2 specificity of Rubisco. SC/O was negatively correlated with [CO2] at Rubisco and total CO2 conductance within lamina, while it was positively correlated with leaf protein across species, regardless of phylogenetic constraint. A simulation analysis shows that the optimal SC/O for maximizing photosynthesis depends on both [CO2] at Rubisco sites and leaf protein per unit leaf area. SC/O is a key parameter along the LES axis and is crucial for maximizing photosynthesis across species and the adaptation of woody plants.

Plant biodiversity assessment through pollen DNA metabarcoding in Natura 2000 habitats (Italian Alps).

Monitoring biodiversity is of increasing importance in natural ecosystems. Metabarcoding can be used as a powerful molecular tool to complement traditional biodiversity monitoring, as total environmental DNA can be analyzed from complex samples containing DNA of different origin. The aim of this research was to demonstrate the potential of pollen DNA metabarcoding using the chloroplast trnL partial gene sequencing to characterize plant biodiversity. Collecting airborne biological particles with gravimetric Tauber traps in four Natura 2000 habitats within the Natural Park of Paneveggio Pale di San Martino (Italian Alps), at three-time intervals in 1 year, metabarcoding identified 68 taxa belonging to 32 local plant families. Metabarcoding could identify with finer taxonomic resolution almost all non-rare families found by conventional light microscopy concurrently applied. However, compared to microscopy quantitative results, Poaceae, Betulaceae, and Oleaceae were found to contribute to a lesser extent to the plant biodiversity and Pinaceae were more represented. Temporal changes detected by metabarcoding matched the features of each pollen season, as defined by aerobiological studies running in parallel, and spatial heterogeneity was revealed between sites. Our results showcase that pollen metabarcoding is a promising approach in detecting plant species composition which could provide support to continuous monitoring required in Natura 2000 habitats for biodiversity conservation.

Model-Based Detection of Whole-Genome Duplications in a Phylogeny.

Ancient whole-genome duplications (WGDs) leave signatures in comparative genomic data sets that can be harnessed to detect these events of presumed evolutionary importance. Current statistical approaches for the detection of ancient WGDs in a phylogenetic context have two main drawbacks. The first is that unwarranted restrictive assumptions on the "background" gene duplication and loss rates make inferences unreliable in the face of model violations. The second is that most methods can only be used to examine a limited set of a priori selected WGD hypotheses and cannot be used to discover WGDs in a phylogeny. In this study, we develop an approach for WGD inference using gene count data that seeks to overcome both issues. We employ a phylogenetic birth-death model that includes WGD in a flexible hierarchical Bayesian approach and use reversible-jump Markov chain Monte Carlo to perform Bayesian inference of branch-specific duplication, loss, and WGD retention rates across the space of WGD configurations. We evaluate the proposed method using simulations, apply it to data sets from flowering plants, and discuss the statistical intricacies of model-based WGD inference.

The assessment of cadmium, chromium, copper, and nickel tolerance and bioaccumulation by shrub plant Tetraena qataranse.

Heavy metals constitute some of the most significant environmental contaminants today. The abundance of naturally growing Tetraena qataranse around Ras Laffan oil and gas facilities in the state of Qatar reflects its toxitolerant character. This study examined the desert plant's tolerance to Ba, Cd, Cr, Cu, Ni and Pb relative to soil concentration. Analysis by inductively coupled plasma - optical emission spectroscopy (ICP-OES) showed that the plant biomass accumulates higher Cd, Cr, Cu and Ni concentration than the soil, particularly in the root. The bioconcentration factor (BCF) of all metals in the root and shoot indicates the plant's capacity to accumulate these metals. Cd had a translocation factor (TF) greater than one; however, it is less than one for all other metals, suggesting that the plant remediate Cd by phytoextraction, where it accumulates in the shoot and Cr, Cu and Ni through phytostabilization, concentrating the metals in the root. Metals phytostabilization restrict transport, shield animals from toxic species ingestion, and consequently prevent transmission across the food chain. Fourier Transform Infrared Spectroscopy (FTIR) analysis further corroborates ICP-OES quantitative data. Our results suggest that T. qataranse is tolerant of Cd, Cr, Cu, and Ni. Potentially, these metals can accumulate at higher concentration than shown here; hence, T. qataranse is a suitable candidate for toxic metals phytostabilization.

NAMS: Noncoding Assessment of long RNAs in Magnoliophyta Species.

Regulation of plant transcriptional machinery has been recently demonstrated to be widely regulated by a class of long noncoding RNAs (lncRNAs) with size larger than 200 nt. The lncRNAs have been demonstrated to play key roles in abiotic stress. Taking into account the rapid pace in the development of the sequencing technologies, accelerated identification of lncRNAs with potential involvement in regulating the gene expression has been witnessed. Although progress has been made to identify the lncRNAs, however, accurate classification of the lncRNAs particularly in the case of plants is still challenging. In this protocol chapter, we present NAMS, which provides large-scale noncoding assessment of the lncRNAs specifically designed for Magnoliophyta species. We describe the approach and the usage of NAMS with potential applications for the lncRNA discovery.

Does intraspecific variability matter in ecological risk assessment? Investigation of genotypic variations in three macrophyte species exposed to copper.

To limit anthropogenic impact on ecosystems, regulations have been implemented along with global awareness that human activities are harmful to the environment. Ecological risk assessment (ERA) is the main procedure which allows to assess potential impacts of stressors on the environment as a result of human activities. ERA is typically implemented through different steps of laboratory testing. The approaches taken for ERA evolve along with scientific knowledge, to improve predictions on ecological risks for ecosystems. We here address the importance of intraspecific variability as a potential source of error in the laboratory evaluation of pollutants. To answer this question, three aquatic macrophyte species with different life-history traits but with their leaves directly in contact with the water were chosen; Lemna minor and Myriophyllum spicatum, two OECD model species, and Ceratophyllum demersum. For each species, three or four genotypes were exposed to 7-8 copper concentrations (up to 1.9 mg/L, 2 mg/L or 36 mg/L for C. demersum, L. minor and M. spicatum, respectively). To assess species sensitivity, growth-related endpoints such as Relative Growth Rate (RGR), based either on biomass production or on length/frond production, and chlorophyll fluorescence Fv/Fm, were measured. For each endpoint, the effective concentration 50% (EC50) was calculated. Almost all endpoints were affected by Cu exposure, except Fv/Fm of M. spicatum, and resulted in significant differences among genotypes for Cu sensitivity. Genotypes of L. minor exhibited up to 35% of variation in EC50 values based on Fv/Fm, showing differential sensivity among genotypes. Significant differences in EC50 values were found for RGR based on length for M. spicatum, with up to 72% of variation. Finally, C. demersum demonstrated significant sensitivity differences among genotypes with up to 78% variation for EC50 based on length. Overall, interspecific variation was higher than intraspecific variation, and explained 77% of the variation found among genotypes for RGR based on biomass, and 99% of the variation found for Fv/Fm. Our results highlight that depending on the endpoint, sensitivity can vary greatly within a species, and that pollutant- and species-specific endpoints should be considered in ERA.

A Phylogenomic Assessment of Ancient Polyploidy and Genome Evolution across the Poales.

Comparisons of flowering plant genomes reveal multiple rounds of ancient polyploidy characterized by large intragenomic syntenic blocks. Three such whole-genome duplication (WGD) events, designated as rho (ρ), sigma (σ), and tau (τ), have been identified in the genomes of cereal grasses. Precise dating of these WGD events is necessary to investigate how they have influenced diversification rates, evolutionary innovations, and genomic characteristics such as the GC profile of protein-coding sequences. The timing of these events has remained uncertain due to the paucity of monocot genome sequence data outside the grass family (Poaceae). Phylogenomic analysis of protein-coding genes from sequenced genomes and transcriptome assemblies from 35 species, including representatives of all families within the Poales, has resolved the timing of rho and sigma relative to speciation events and placed tau prior to divergence of Asparagales and the commelinids but after divergence with eudicots. Examination of gene family phylogenies indicates that rho occurred just prior to the diversification of Poaceae and sigma occurred before early diversification of Poales lineages but after the Poales-commelinid split. Additional lineage-specific WGD events were identified on the basis of the transcriptome data. Gene families exhibiting high GC content are underrepresented among those with duplicate genes that persisted following these genome duplications. However, genome duplications had little overall influence on lineage-specific changes in the GC content of coding genes. Improved resolution of the timing of WGD events in monocot history provides evidence for the influence of polyploidization on functional evolution and species diversification.

Comparative Genomics of NAC Transcriptional Factors in Angiosperms: Implications for the Adaptation and Diversification of Flowering Plants.

NAC proteins constitute one of the largest groups of plant-specific transcription factors and are known to play essential roles in various developmental processes. They are also important in plant responses to stresses such as drought, soil salinity, cold, and heat, which adversely affect growth. The current knowledge regarding the distribution of NAC proteins in plant lineages comes from relatively small samplings from the available data. In the present study, we broadened the number of plant species containing the NAC family origin and evolution to shed new light on the evolutionary history of this family in angiosperms. A comparative genome analysis was performed on 24 land plant species, and NAC ortholog groups were identified by means of bidirectional BLAST hits. Large NAC gene families are found in those species that have experienced more whole-genome duplication events, pointing to an expansion of the NAC family with divergent functions in flowering plants. A total of 3,187 NAC transcription factors that clustered into six major groups were used in the phylogenetic analysis. Many orthologous groups were found in the monocot and eudicot lineages, but only five orthologous groups were found between P. patens and each representative taxa of flowering plants. These groups were called basal orthologous groups and likely expanded into more recent taxa to cope with their environmental needs. This analysis on the angiosperm NAC family represents an effort to grasp the evolutionary and functional diversity within this gene family while providing a basis for further functional research on vascular plant gene families.

Plant-Pathogen Interaction-Related MicroRNAs and Their Targets Provide Indicators of Phytoplasma Infection in Paulownia tomentosa × Paulownia fortunei.

Paulownia witches' broom (PaWB) caused by a phytoplasma, has caused extensive losses in the yields of paulownia timber and resulted in significant economic losses. However, the molecular mechanisms in Paulownia that underlie the phytoplasma stress are poorly characterized. In this study, we use an Illumina platform to sequence four small RNA libraries and four degradome sequencing libraries derived from healthy, PaWB-infected, and PaWB-infected 15 mg·L-1 and 30 mg·L-1 methyl methane sulfonate (MMS)-treated plants. In total, 125 conserved and 118 novel microRNAs (miRNAs) were identified and 33 miRNAs responsive to PaWB disease were discovered. Furthermore, 166 target genes for 18 PaWB disease-related miRNAs were obtained, and found to be involved in plant-pathogen interaction and plant hormone signal transduction metabolic pathways. Eleven miRNAs and target genes responsive to PaWB disease were examined by a quantitative real-time PCR approach. Our findings will contribute to studies on miRNAs and their targets in Paulownia, and provide new insights to further understand plant-phytoplasma interactions.

Horizontal gene transfer from a flowering plant to the insular pine Pinus canariensis (Chr. Sm. Ex DC in Buch).

Horizontal gene transfer (HGT) is viewed as very common in the plant mitochondrial (mt) genome, but, to date, only one case of HGT has been found in gymnosperms. Here we report a new case of HGT, in which a mt nad5-1 fragment was transferred from an angiosperm to Pinus canariensis. Quantitative assay and sequence analyses showed that the foreign nad5-1 is located in the mt genome of P. canariensis and is nonfunctional. An extensive survey in the genus Pinus revealed that the angiosperm-derived nad5-1 is restricted to P. canariensis and present across the species' range. Molecular dating based on chloroplast DNA suggested that the HGT event occurred in the late Miocene after P. canariensis split from its closest relatives, and that the foreign copy became fixed in P. canariensis owing to drift during its colonization of the Canary Islands. The mechanism of this HGT is unclear but it was probably achieved through either direct cell-cell contact or external vectors. Our discovery provides evidence for an important role of HGT in plant mt genome evolution.

Genetic fidelity assessment in micropropagated plants using cytogenetical analysis and heterochromatin distribution: a case study with Nepenthes khasiana Hook f.

Rapid clonal propagation of selected genotypes has been one of the most extensively exploited approaches of biotechnology. However, inclusion of somaclonal variations in tissue-culture-derived plants results in the production of undesirable plant off-types which limits its applications in tissue culture industry. Therefore, the most critical concern has been the maintenance of genetic uniformity of micropropagated plants. Assessment of genetic fidelity in tissue-culture-raised plants of three consecutive regenerations of Nepenthes khasiana has been successfully carried out using chromosome counts and heterochromatin distribution pattern wherein changes in the number of chromosomes and the distribution of AT and GC base pairs were recorded. The cells studied in the plantlets of the first regeneration (23.33 %) showed deviant number of chromosome which was increased to 33.33 % and 40 % in the plantlets of the second and the third regenerations, respectively. Also, 4',6-diamidino-2-phenylindole (DAPI)(+) and chromomycin A3 (CMA)(+) binding sites, on an average of 5.74 ± 0.47 and 5.00 ± 0.30, were observed in the plantlets of the first regeneration. Subsequently, DAPI(+) binding sites were increased to 6.61 ± 0.39 and 6.74 ± 0.57 in the plantlets of the second and the third regenerations, respectively, with a corresponding decrease in the CMA(+) binding sites (4.63 ± 0.45 and 4.16 ± 0.47 CMA(+) sites in the plantlets of the second and the third regenerations, respectively). The study reveals an increase in cytological variations in the morphologically similar micropropagated plants of N. khasiana with the subsequent regenerations which further necessitate the determination of genetic integrity of micropropagated plants.

Costs and benefits of reticulate leaf venation.

BACKGROUND: Recent theoretical and empirical work has identified redundancy as one of the benefits of the reticulate form in the evolution of leaf vein networks. However, we know little about the costs of redundancy or how those costs depend on vein network geometry or topology. Here, we examined both costs and benefits to redundancy in 339 individual reticulate leaf networks comprising over 3.5 million vein segments. We compared levels of costs and benefits within reticulate networks to those within analogous networks without loops known as Maximum Spanning Trees (MSTs). RESULTS: We show that network robustness to varying degrees of simulated damage is positively correlated with structural indices of redundancy. We further show that leaf vein networks are topologically, geometrically and functionally more redundant than are MSTs. However, increased redundancy comes with minor costs in terms of increases in material allocation or decreases in conductance. We also show that full networks do not markedly decrease the distance to non-vein tissue in comparison to MSTs. CONCLUSIONS: These results suggest the evolutionary transition to the reticulate type of networks found in modern Angiosperm flora involved a relatively minor increase in material and conductance costs with significant benefits in terms of network redundancy.

Seed coat development in Velloziaceae: primary homology assessment and insights on seed coat evolution.

UNLABELLED: • PREMISE OF THE STUDY: Seed coat characteristics have historically been used to infer taxonomic relationships and are a potential source of characters for phylogenetic reconstruction. In particular, seed coat morphoanatomy has never been studied in detail in Velloziaceae. One character based on seed surface microsculpture has been used in phylogenies, but was excluded from recent studies owing to problems in primary homology. This work aimed to clarify the origin and general composition of seed coat cell layers in Velloziaceae and to propose hypotheses of primary homology among seed characters.• METHODS: Seed coat development of 24 Velloziaceae species, comprising nine genera, and one species of Pandanaceae (outgroup) was studied using standard anatomical methods. Developmental data were interpreted in the light of a recently published phylogeny.• KEY RESULTS: Eight types of seed coat were identified. Whereas the most common type has four distinct cell layers (two-layered tegmen and testa), we encountered much more variation in seed coat composition than previously reported, the analysis of which revealed some potential synapomorphies. For instance, an exotesta with spiral thickenings may be a synapomorphy of Barbacenia.• CONCLUSIONS: Our results showed that the character states previously used in phylogenies are not based on homologous layers and that the same state was misattributed to species exhibiting quite different seed coats. This study is a first step toward a better understanding of seed coat structure evolution in Velloziaceae.

The cost of reinforcement: selection on flower color in allopatric populations of Phlox drummondii.

Reinforcement is the process by which increased reproductive isolation between incipient species evolves due to selection against maladaptive hybrids or costly hybrid mating. Reinforcement is predicted to create a pattern of greater prezygotic reproductive isolation in regions where the two species co-occur, sympatry, than in allopatry. Although most research on reinforcement focuses on understanding the evolutionary forces acting in sympatry, here we consider what prevents the alleles conferring greater reproductive isolation from spreading into allopatry. We investigate flower color divergence in the wildflower Phlox drummondii, which is caused by reinforcement in the regions sympatric with its congener Phlox cuspidata. Specifically, we performed common garden field experiments and pollinator observations to estimate selection acting on flower color variation in allopatry. We combine our estimates of maternal and paternal fitness using simulations and predict how flower color alleles migrating from sympatry will evolve in allopatry. Our results suggest that strong pollinator preference for the ancestral flower color in allopatry can maintain divergence between allopatric and sympatric populations.

The costs and benefits of tolerance to competition in ipomoea purpurea, the common morning glory.

Tolerance to competition has been hypothesized to reduce the negative impact of plant-plant competition on fitness. Although competitive interactions are a strong selective force, an analysis of net selection on tolerance to competition is absent in the literature. Using 55 full/half-sibling families from 18 maternal lines in the crop weed Ipomoea purpurea, we measured fitness and putative tolerance traits when grown with and without competition in an agricultural field. We tested for the presence of genetic variation for tolerance to competition and determined if there were costs and benefits of this trait. We also assessed correlations between tolerance and potential tolerance traits. We uncovered a fitness benefit of tolerance in the presence of competition and a cost in its absence. We failed to detect evidence of additive genetic variation underlying tolerance, but did uncover the presence of a significant maternal-line effect for tolerance, which suggests its evolutionary trajectory is not easily predicted. The cost of tolerance is likely due to later initiation of flowering of tolerant individuals in the absence of competition, whereas relative growth rate was found to positively covary with tolerance in the presence of competition, and can thus be considered a tolerance trait.

Toward a predictive understanding of the fitness costs of heterospecific pollen receipt and its importance in co-flowering communities.

PREMISE OF THE STUDY: While we have a good understanding of how co-flowering plants interact via pollinator foraging, we still know very little about how plants interact via heterospecific pollen (HP) receipt. To fill this gap, we sought to illuminate the extent of HP receipt and quantitatively evaluate the fitness consequences of HP receipt. We consider plant traits that could mediate the fitness costs of HP receipt in an effort to better understand the potential consequences of pollinator sharing in natural communities. • METHODS: We survey the literature for occurrence of HP receipt and assess variation in the fitness effects of a standard HP treatment. We develop a conceptual framework for understanding variation in fitness consequences of HP receipt. • KEY RESULTS: We find evidence for variation in HP receipt and its costs. Our framework predicts that certain traits (self-incompatibility, small, highly aperaturate or allelopathic pollen) will lead to detrimental HP donors, whereas others (self-compatibility, small or wet stigmas, short styles) will lead to vulnerable HP recipients. We also predict that detrimental effects of HP receipt will increase with decreasing phylogenetic distance between donor and recipient. • CONCLUSIONS: Our framework can guide much needed additional work so that we can evaluate whether and which plant traits contribute to the variation in the effects of HP receipt. This will be a step toward predicting the consequences of HP receipt in natural communities, and ultimately transform our understanding of the role of postpollination interactions in floral trait evolution and pollinator sharing.

The evolution of the worldwide leaf economics spectrum.

The worldwide leaf economic spectrum (WLES) is a strikingly consistent pattern of correlations among leaf traits. Although the WLES effectively summarizes variation in plant ecological strategies, little is known about its evolution. We reviewed estimates of natural selection and genetic variation for leaf traits to test whether the evolution of the WLES was limited by selection against unfit trait combinations or by genetic constraints. There was significant selection for leaf traits on both ends of the WLES spectrum, as well as significant genetic variation for these traits. In addition, genetic correlations between WLES traits were variable in strength and direction. These data suggest that genetic constraints have had a smaller role than selection in the evolution of the WLES.

Assessment of genetic diversity in 31 species of mangroves and their associates through RAPD and AFLP markers.

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity in 31 species of mangroves and mangrove associates. Four AFLP primer combinations resulted in the amplification of 840 bands with an average of 210 bands per primer combination and 11 RAPD primers yielded 319 bands with an average of 29 bands per primer. The percentage of polymorphism detected was too high indicating the high degree of genetic variability in mangrove taxa both at inter- and intra-generic levels. In the dendrogram, species belonging to a particular family/ genus, taxa inhabiting similar habitats or having similar adaptations tended to be together. There were exceptions too; as many unrelated species of mangroves form clusters. The intrafamilial classification and inter-relationships of genera in the family Rhizophoraceae could be confirmed by molecular analysis. Both the markers RAPD and AFLP were found equally informative and useful for a better understanding of the genetic variability and genome relationships among mangroves and their associated species.

Towards building the tree of life: a simulation study for all angiosperm genera.

Comprehensive phylogenetic trees are essential tools to better understand evolutionary processes. For many groups of organisms or projects aiming to build the Tree of Life, comprehensive phylogenetic analysis implies sampling hundreds to thousands of taxa. For the tree of all life this task rises to a highly conservative 13 million. Here, we assessed the performances of methods to reconstruct large trees using Monte Carlo simulations with parameters inferred from four large angiosperm DNA matrices, containing between 141 and 567 taxa. For each data set, parameters of the HKY85+G model were estimated and used to simulate 20 new matrices for sequence lengths from 100 to 10,000 base pairs. Maximum parsimony and neighbor joining were used to analyze each simulated matrix. In our simulations, accuracy was measured by counting the number of nodes in the model tree that were correctly inferred. The accuracy of the two methods increased very quickly with the addition of characters before reaching a plateau around 1000 nucleotides for any sizes of trees simulated. An increase in the number of taxa from 141 to 567 did not significantly decrease the accuracy of the methods used, despite the increase in the complexity of tree space. Moreover, the distribution of branch lengths rather than the rate of evolution was found to be the most important factor for accurately inferring these large trees. Finally, a tree containing 13,000 taxa was created to represent a hypothetical tree of all angiosperm genera and the efficiency of phylogenetic reconstructions was tested with simulated matrices containing an increasing number of nucleotides up to a maximum of 30,000. Even with such a large tree, our simulations suggested that simple heuristic searches were able to infer up to 80% of the nodes correctly.

Regeneration mode affects spatial genetic structure of Nothofagus dombeyi forests.

Disturbance may generate population bottlenecks by reducing population size and the number of founders establishing a new colony. We tested the hypothesis that the scale of disturbance affects the levels of genetic diversity and the spatial distribution of genotypes in naturally regenerating stands of Nothofagus dombeyi, an evergreen angiosperm tree, in northwestern Patagonia. At similar spatial scales, we predicted that old-growth stands characterized by fine-scale gap phase dynamics would be genetically diverse due to restricted gene flow among temporal and spatially isolated gaps. In contrast, young massively regenerated postfire cohorts resulting from coarse-scale disturbances would be genetically more homogeneous. At each of three paired old-growth and postfire stands a minimum of 50 trees were mapped and sampled within 1 ha. Fresh tissue was collected for isozyme analysis from a total of 361 trees along with tree cores and diameters. Tree age distributions reflected the dominant modes of regeneration. Six out of nine analysed loci were polymorphic. Mean genetic diversity parameters were greater but not significant in mature stands. Fixation indices suggested significant heterozygous deficit at two-thirds of possible tests indicating a Wahlund effect due to local recruitment of related seeds. F(ST) indicated moderate between-stand divergence. Mature stands concentrated half of positively like joins and yielded significant (P < 0.05) autocorrelation coefficients at small distance classes (< 20 m). Fine-scale patch dynamics within mature stands favours the maintenance of fine-scale genetic structure as a result of shade intolerance and local seed dispersal. Conversely, postfire stands suffer the effects of genetic drift given that a few reproductive trees produce a somewhat impoverished and genetically uniform progeny. Bottleneck effects will depend upon the density of remnant trees which could also be a function of the severity of fire.