Hybridization and diversity of aquatic macrophyte Sparganium L. (Typhaceae) as revealed by high-throughput nrDNA sequencing.

Sparganium is an emergent aquatic macrophyte widely spread in temperate and subtropical zones. Taxa of this genus feature high phenotypic plasticity and can produce interspecific hybrids. By means of high-throughput sequencing of the internal transcribed spacer (ITS1) of 35S rDNA, the status of 15 Eurasian Sparganium species and subspecies was clarified and the role of hybridization events in the recent evolution of the genus was investigated. It has been shown that a number of species such as S. angustifolium, S. fallax and S. subglobosum have homogenized rDNA represented by one major ribotype. The rDNA of other taxa is represented by two or more major ribotypes. Species with high rDNA heterogeneity are apparently of hybrid origin. Based on the differences in rDNA patterns, intraspecific diversity was identified in S. probatovae and S. emersum. Thus, we have concluded that Sparganium has extensive interspecific hybridization at the subgenus level, and there may also be occasional hybridization between species from different subgenera.

Evidence of hybrid breakdown among invasive hybrid cattails (Typha × glauca).

Interspecific hybridization has varied consequences for offspring fitness, with implications for the maintenance of species integrity. Hybrid vigour, when it occurs, can peak in first-generation (F1) hybrids and then decline in advanced-generation (F2+) hybrids. This hybrid breakdown, together with the processes affecting patterns of hybridization and hybrid fitness, determine the evolutionary stability of hybrid zones. An extensive hybrid zone in North America involving the cattails Typha latifolia, T. angustifolia, and their invasive hybrid T. × glauca is characterized by hybrid vigour among F1s, but the fitness of advanced-generation hybrids has not been studied. We compared seed germination and plant growth of T. latifolia (parental L), F1 T. × glauca (F1), hybrid backcrosses to T. angustifolia (bcA) and T. latifolia (bcL), and advanced-generation (F2) hybrids. Consistent with expectations under hybrid breakdown, we found reduced plant growth for F2 hybrids in comparison with F1s (plant height and above-ground biomass) and parental Ls (above-ground biomass). Backcrossed hybrids had intermediate measures of plant growth and bcLs were characterized by reduced seed germination in comparison with parental Ls. Hybrid breakdown could make the formation of F1s in North America finite because (1) hybridization among cattails is asymmetric, with T. angustifolia but not T. latifolia subject to genetic swamping, and (2) T. angustifolia is less common and subject to competitive displacement by F1s. Hybrid breakdown is therefore expected to reduce hybrid frequencies over time, contributing to the long-term maintenance of T. latifolia - the only native cattail in the study region.

Molecular confirmation of the hybrid origin of Sparganium longifolium (Typhaceae).

Sparganium longifolium was reported as a hybrid between S. emersum and S. gramineum based on its intermediate type or the common characteristics of its parent species. Its hybrid origin needs to be confirmed using molecular technology. We investigated the origin of S. longifolium based on 10 populations of S. emersum, S. gramineum and S. longifolium from five lakes in European Russia, using sequences of six nuclear loci and one chloroplast DNA fragment. Haplotype network, principal coordinate analysis and genetic clustering based on data of nuclear loci confirmed that S. longifolium is the hybrid between S. emersum and S. gramineum. We found that the natural hybridization between S. emersum and S. gramineum is bidirectional but asymmetrical, and the latter mainly acts as maternal species. We also found that all samples of S. longifolium were F1 generations, and thus hypothesized that S. emersum and S. gramineum could likely maintain their species boundary through the post-zygote reproductive isolation mechanism of F1 generation sterility.

Genome assembly, annotation, and comparative analysis of the cattail Typha latifolia.

Cattails (Typha species) comprise a genus of emergent wetland plants with a global distribution. Typha latifolia and Typha angustifolia are two of the most widespread species, and in areas of sympatry can interbreed to produce the hybrid Typha × glauca. In some regions, the relatively high fitness of Typha × glauca allows it to outcompete and displace both parent species, while simultaneously reducing plant and invertebrate biodiversity, and modifying nutrient and water cycling. We generated a high-quality whole-genome assembly of T. latifolia using PacBio long-read and high coverage Illumina sequences that will facilitate evolutionary and ecological studies in this hybrid zone. Genome size was 287 Mb and consisted of 1158 scaffolds, with an N50 of 8.71 Mb; 43.84% of the genome were identified as repetitive elements. The assembly has a BUSCO score of 96.03%, and 27,432 genes and 2700 RNA sequences were putatively identified. Comparative analysis detected over 9000 shared orthologs with related taxa and phylogenomic analysis supporting T. latifolia as a divergent lineage within Poales. This high-quality scaffold-level reference genome will provide a useful resource for future population genomic analyses and improve our understanding of Typha hybrid dynamics.

Genetic Diversity and Connectivity in Plant Species Differing in Clonality and Dispersal Mechanisms in Wetland Island Habitats.

In plants, long-distance dispersal is both attenuated and directed by specific movement vectors, including animals, wind, and/or water. Hence, movement vectors partly shape metapopulation genetic patterns that are, however, also influenced by other life-history traits such as clonal growth. We studied the relationship between area, isolation, plant-species richness, reproduction, and dispersal mechanisms with genetic diversity and divergence in 4 widespread wetland plant-species in a total of 20 island-like kettle-hole habitats surrounded by an intensive agricultural landscape. Our results showed that genetic parameters reflect the reproduction strategies with the highest genetic diversity being observed in the non-clonal, outcrossing Oenanthe aquatica compared to the clonal Lycopus europaeus, Typha latifolia, and Phragmites australis. Lycopus showed a positive relationship between genetic diversity and kettle-hole area, but a negative relationship with the number of neighboring kettle holes (less isolation). Genetic diversity increased with plant-species richness in the clonal species Phragmites and Lycopus; while it decreased in the non-clonal Oenanthe. Finally, genetic divergence and, therefore, connectivity differed between alternative dispersal strategies, where wind-dispersed Typha and Phragmites had a higher gene flow between the analyzed kettle holes compared with the insect-pollinated, hydrochorous Lycopus and Oenanthe. Our study provides information on genetic patterns related to reproduction and dispersal mechanisms of 4 common wetland species contributing to the understanding of the functioning of plant metacommunities occurring in kettle holes embedded in agricultural landscapes.

Genetic structure in hybrids and progenitors provides insight into processes underlying an invasive cattail (Typha × glauca) hybrid zone.

Traditional models of hybrid zones have assumed relatively low hybrid fitness, and thus focussed more on interspecific gene flow than on hybrid dispersal. Therefore, when hybrids have high fitness and the potential for autonomous dispersal, we have limited understanding of whether hybrid dispersal or repeated local hybrid formation is more important for maintaining hybrid zones. The invasive hybrid cattail Typha × glauca occupies an extensive hybrid zone in northeastern North America where it is sympatric with its progenitors T. latifolia and T. angustifolia. We characterized genetic diversity and genetic structure of the three taxa across a broad spatial scale where the maternal parent is relatively rare, and tested the hypothesis that the hybrid shows stronger evidence of gene flow than its progenitor species, particularly among disturbed sites (ditches) compared with established wetlands. Support for this hypothesis would suggest that dispersal, rather than repeated local formation, is more important for maintaining hybrid zones. Within each taxon, genetic differentiation among ditches was comparable to that among wetlands, although clonal richness was consistently greater in ditches, suggesting more frequent seed establishment. Genetic structure across sites was more pronounced in the hybrid compared with either progenitor species. Overall, our data reflect relatively low gene flow in hybrids, and suggest that hybrids are more likely to be created in situ than to be introduced from other sites. Despite the high fitness of invasive T. × glauca and its potential for autonomy, local processes appear more important than dispersal in maintaining this hybrid zone.

Transcriptome -wide modulation combined with morpho-physiological analyses of Typha orientalis roots in response to lead challenge.

Lead (Pb) is a common pollutant in many environments, including in the soil, water, and/or air. Typha orientalis Presl, a large emergent aquatic plant, has been reported to function as a Pb-tolerant and Pb-accumulating plant; however, very little molecular information regarding the tolerance of T. orientalis towards Pb is known. In this study, Pb accumulation and key factors involved in the Pb stress response at different Pb concentrations were investigated. Pb was primarily accumulated in the roots and was mainly located in the cell wall and membrane systems. Differentially expressed genes (DEGs) were identified in T. orientalis roots after Pb exposure via RNA-seq analyses. In the 0.10 mM and 0.25 mM Pb2+-treated groups, a total of 3275 DEGs were detected relative to the control. Many of these genes were associated with oxidation-reduction processes, metal transport, protein kinase/phosphorylation, and DNA binding transcription factors, which were shown to be Pb-responsive DEGs. Mapping Kyoto Encyclopedia of Genes and Genomes (KEGG) database, "phenylpropanoid biosynthesis" was analyzed as the major pathway of the important modules of overlapping DEGs of 0.10 mM and 0.25 mM Pb2+ treatments. Furthermore, a lead response gene named ToLR1 with unknown function was of particular interest. The full-length of ToLR1 sequence was cloned using rapid amplification of cDNA ends (RACE) and overexpressed in Arabidopsis thaliana, which resulted in enhanced resistance to Pb stress. This is the first report providing genomic information detailing Pb responsive genes in T. orientalis. Moreover, this study provides novel insights into the molecular mechanisms underlying the response of T. orientalis and other accumulators towards Pb stress. The key genes identified in this study may serve as potential targets for genetic engineering targeting phytoremediation.

Enhancement of oil field-produced wastewater remediation by bacterially-augmented floating treatment wetlands.

Plants and bacteria individually as well as in synergism with each other hold a great potential to degrade a wide range of environmental pollutants. Floating treatment wetlands (FTWs) is an efficient and low-cost technology that uses the synergistic interaction between plant roots and microbes for in situ remediation of wastewater. The present study aims to assess the feasibility of FTW-based remediation of oil field-produced wastewater using an interaction between two plant species, Typha domingensis and Leptochloa fusca, in partnership with a consortium of crude oil-degrading bacterial species, Bacillus subtilis LORI66, Klebsiella sp. LCRI87, Acinetobacter Junii TYRH47, and Acinetobacter sp. BRSI56. All the treatments reduced contaminant levels, but T. domingensis, in combination with bacterial inoculation, exhibited the highest reduction in hydrocarbon (95%), COD (90%), and BOD content (93%) as compared to L. fusca. This combination maximally promoted increases in fresh biomass (31%), dry biomass (52%), and length (25%) of plants as well. This effect was further signified by the persistence of bacteria (40%) and considerable abundance (27%) and expression (28.5%) of the alkB gene in the rhizoplane of T. domingensis in comparison to that of L. fusca. The study, therefore, suggests that T. domingensis, in combination with bacterial consortium, has significant potential for treatment of oil field-produced water and can be exploited on large scale in FTWs.

Revised phylogeny and historical biogeography of the cosmopolitan aquatic plant genus Typha (Typhaceae).

Typha is a cosmopolitan aquatic plant genus that includes species with widespread distributions. It is a relatively ancient genus with an abundant fossil record dating back to the Paleogene. However, the details of its biogeographic history have remained unclear until now. In this study, we present a revised molecular phylogeny using sequences of seven chloroplast DNA markers from nine species sampled from various regions in order to infer the biogeographic history of the genus. Two clades were recovered with robust support. Typha minima and T. elephantina comprised one clade, and the other clade included the remaining seven species, which represented a polytomy of four robustly supported subclades. Two widespread species, T. angustifolia and T. domingensis, were revealed to be paraphyletic, indicating the need for taxonomic revision. Divergence time estimation suggested that Typha had a mid-Eocene crown origin, and its diversification occurred in the Middle and Late Miocene. Ancestral area reconstruction showed that Typha possibly originated from eastern Eurasia. Both dispersal via the Beringian Land Bridge and recent transoceanic dispersal may have influenced the intercontinental distribution of Typha species.

De novo transcriptomic analysis to identify differentially expressed genes during the process of aerenchyma formation in Typha angustifolia leaves.

Lysigenous aerenchyma is formed through programmed cell death (PCD) in Typha angustifolia leaves. However, the genome and transcriptome data for this species are unknown. To further elucidate the molecular basis of PCD during aerenchyma formation in T. angustifolia leaves, transcriptomic analysis of T. angustifolia leaves was performed using Illumina sequencing technology, revealing 73,821 unigenes that were produced by assembly of the reads in T1, T2 and T3 samples. The important pathways, such as programmed cell death (PCD), aerenchyma formation, and ethylene responsiveness were regulated by these unigenes. 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO) were highly up-regulated as key enzymes for ethylene synthesis, along with respiratory burst oxidase homolog (RBOH), metallothionein, calmodulin-like protein (CML), and polygalacturonase (PG), may collectively explain the PCD involved in T. angustifolia aerenchyma formation. We hypothesize that fermentation, metabolism and glycolysis generate ATP for PCD. We searched the 73,821 unigenes against protein databases, and 24,712 were annotated. Based on sequence homology, 16,012 of the 73,821 annotated unigenes were assigned to one or more Gene Ontology (GO) terms. Meanwhile, a total of 9537 unigenes were assigned to 126 pathways in the KEGG database. In summary, this investigation provides important guidelines for exploring the molecular mechanisms of aerenchyma formation in aquatic plants.

Asymmetric Hybridization in Cattails (Typha spp.) and Its Implications for the Evolutionary Maintenance of Native Typha latifolia.

Cattails (Typha spp.) have become an increasingly dominant component of wetlands in eastern North America and this dominance is largely attributable to the high frequency of Typha × glauca, the hybrid of native Typha latifolia and putatively introduced Typha angustifolia. Hybridization in this group is asymmetric, with T. angustifolia nearly always the maternal parent of F1 hybrids. However, the magnitude of hybrid infertility and whether mating asymmetries extend to the formation of advanced-generation hybrids have not been examined. We used hand-crosses to measure seed set and germination success. We found that mating asymmetries extend to the formation of back-crosses, with ~0 seeds set when T. latifolia was pollinated by hybrid cattails. Seed set was unaffected by pollen source for T. × glauca or T. angustifolia. However, seed production by T. angustifolia was consistently high while that of T. × glauca was variable and when pollinated by other T. × glauca more than 75% lower than for any other intraspecific cross indicating reduced hybrid fertility. We used these results to parameterize a model of hybrid zone evolution in which mating patterns and fertility were governed by interactions between alleles at nuclear and cytoplasmic loci. The model revealed that asymmetric mating and reduced hybrid fertility should favor the maintenance of T. latifolia over T. angustifolia compared to null expectations. However, the model also indicated restrictive conditions for the long-term maintenance of T. latifolia within populations, indicating that asymmetric mating might only stall rather than prevent the displacement of native cattails by hybrids.

Transcriptome-Wide Identification of miRNAs and Their Targets from Typha angustifolia by RNA-Seq and Their Response to Cadmium Stress.

MicroRNAs (miRNAs) play important roles in plant responses to environmental stress. In this work, we used high-throughput sequencing to analyze transcriptome and small RNAs (sRNAs) in Typha angustifolia under cadmium (Cd) stress. 57,608,230 raw reads were obtained from deep sequencing of a pooled cDNA library. Sequence assembly and analysis yielded 102,473 unigenes. We subsequently sequenced two sRNA libraries from T. angustifolia with or without Cd exposure respectively. Based on transcriptome data of T. angustifolia, we catalogued and analyzed the sRNAs, resulting in the identification of 114 conserved miRNAs and 41 novel candidate miRNAs in both small RNA libraries. In silico analysis revealed 764 targets for 89 conserved miRNAs and 21 novel miRNAs. Statistical analysis on sequencing reads abundance and experimental validation revealed that 4 conserved and 6 novel miRNAs showed specific expression. Combined with function of target genes, these results suggested that miRNAs might play a role in plant Cd stress response. This study provided the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in T. angustifolia, which provide a framework for further analysis of miRNAs and their role in regulating plant responses to Cd stress.

[Identification of cattail pollen (puhuang), pine pollen (songhuafen) and its adulterants by ITS2 sequence].

DNA barcoding method was conducted for the authentication of pollen materials due to difficulty of discriminating pollen materials bearing morphological similarity. In this study, a specific focus was to identify cattail pollen (Puhuang) and pine pollen (Songhuafen) samples from their adulterants which are frequently mixed-together. Regions of the internal transcribed spacer (ITS2) from 60 samples were sequenced, and new primers for cattail pollen were designed according to the sequence information. The results from the NJ trees showed that the species of pine pollen, Puhuang and their adulterants can be classified as obvious monophyly. Therefore, we propose to adapt DNA barcoding methodology to accurately distinguish cattail pollen, pine pollen and their adulterant materials. It is a great help for drug regulatory agency to supervise the quality of medicinal materials.

Genetic potential for N2O emissions from the sediment of a free water surface constructed wetland.

Removal of nitrogen is a key aspect in the functioning of constructed wetlands. However, incomplete denitrification may result in the net emission of the greenhouse gas nitrous oxide (N(2)O) resulting in an undesired effect of a system supposed to provide an ecosystem service. In this work we evaluated the genetic potential for N(2)O emissions in relation to the presence or absence of Phragmites and Typha in a free water surface constructed wetland (FWS-CW), since vegetation, through the increase in organic matter due to litter degradation, may significantly affect the denitrification capacity in planted areas. Quantitative real-time PCR analyses of genes in the denitrification pathway indicating capacity to produce or reduce N(2)O were conducted at periods of different water discharge. Genetic potential for N(2)O emissions was estimated from the relative abundances of all denitrification genes and nitrous oxide reductase encoding genes (nosZ). nosZ abundance was invariably lower than the other denitrifying genes (down to 100 fold), and differences increased significantly during periods of high nitrate loads in the CW suggesting a higher genetic potential for N(2)O emissions. This situation coincided with lower nitrogen removal efficiencies in the treatment cell. The presence and the type of vegetation, mainly due to changes in the sediment carbon and nitrogen content, correlated negatively to the ratio between nitrate and nitrite reducers and positively to the ratio between nitrite and nitrous oxide reducers. These results suggest that the potential for nitrous oxide emissions is higher in vegetated sediments.

Correlation between pollen aperture pattern and callose deposition in late tetrad stage in three species producing atypical pollen grains.

PREMISE OF THE STUDY: Pollen grains of flowering plants display a fascinating diversity of forms, in spite of their minute size. The observed diversity is determined by the developmental mechanisms implicated in the establishment of pollen morphological features. Pollen grains are generally surrounded by an extremely resistant wall interrupted in places by apertures that play a key role in reproduction, being the places at which pollen tube growth is initiated. Aperture shape, number, and position are determined during microsporogenesis (male meiosis), the earliest step in pollen ontogeny. We investigate in detail the unfolding of microsporogenesis in three species that present uncommon aperture pattern (i.e., disulculate in Calycanthus floridus [Calycanthaceae, magnoliids], tetraporate in Hohenbergia stellata [Bromeliaceae, monocots], and monoporate in Typha latifolia [Typhaceae, monocots]). METHODS: We performed a comparative analysis of microsporogenesis and aperture distribution within tetrads in these species with contrasting aperture arrangements. This was done using aniline blue coloration and UV light microscope observations. KEYS RESULTS: We show that aperture localization and features of callose deposition on intersporal walls produced during cytokinesis coincide in all three species examined. Such a correlation suggests that patterns of callose deposition are strongly involved in determining aperture localization. CONCLUSION: In flowering plants, patterns of male meiosis and especially callose deposition following meiosis may be implicated in the diversity of pollen aperture patterns.

Shifts in root-associated microbial communities of Typha latifolia growing in naphthenic acids and relationship to plant health.

Naphthenic acids (NAs) are a complex mixture of organic acid compounds released during the extraction of crude oil from oil sands operations. The accumulation of toxic NAs in tailings pond water (TPW) is of significant environmental concern, and phytoremediation using constructed wetlands is one remediation option being assessed. Since root-associated microorganisms are an important factor during phytoremediation of organic compounds, this study investigated the impact of NAs on the microbial communities associated with the macrophyte Typha latifolia (cattail). Denaturing gradient gel electrophoresis revealed that the impact of NAs on microbial communities was niche dependent, with endophytic communities being the most stable and bulk water communities being the least stable. The type of NA used was significant to microbial response, with commercial NAs causing greater adverse changes than TPW NAs. In general, plant beneficial bacteria such as diazotrophs were favoured in cattails grown in TPW NAs, while potentially deleterious bacteria such as denitrifying Dechlorospirillum species increased in commercial NA treatments. These findings suggest that NAs may affect plant health by impacting root-associated microbial communities. A better understanding of these impacts may allow researchers to optimize those microbial communities that support plant health, and thus further optimize wetland treatment systems.

Cost-effective, species-specific microsatellite development for the endangered Dwarf Bulrush (Typha minima) using next-generation sequencing technology.

The dwarf bulrush (Typha minima Funck ex Hoppe) is an endangered pioneer plant species of riparian flood plains. In Switzerland, only 3 natural populations remain, but reintroductions are planned. To identify suitable source populations for reintroductions, we developed 17 polymorphic microsatellite markers with perfect repeats using the 454 pyrosequencing technique and tested them on 20 individuals with low-cost M13 labeling. We detected 2 to 7 alleles per locus and found expected and observed heterozygosities of 0.05-0.76 and 0.07-1, respectively. The whole process was finished in less than 6 weeks and cost approximately USD 5000. Due to low costs and reduced expenditure of time, the use of next-generation sequencing techniques for microsatellite development represent a powerful tool for population genetic studies in nonmodel species, as we show in this first application of the approach to a plant species of conservation importance.

Implications of the plastid genome sequence of typha (typhaceae, poales) for understanding genome evolution in poaceae.

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.

Genetics of cattails in radioactively contaminated areas around Chornobyl.

Research on populations from radioactively contaminated areas around Chornobyl has produced ambiguous results for the presence of radiation effects. More studies are needed to provide information on whether radiation exposure at Chornobyl significantly affected genetic diversity in natural populations of various taxa. Eleven and nine variable microsatellite loci were used to test for differences in genetic diversity between reference and Chornobyl populations of two cattail species (Typha angustifolia and Typha latifolia, respectively) from Ukraine. Our purpose was to determine whether radiation had a significant impact on genetic diversities of the Chornobyl Typha populations, or if their genetic composition might be better explained by species demography and/or changes in population dynamics, mainly in sexual and asexual reproduction. Populations closest to the reactor had increased genetic diversities and high number of genets, which likely were due to factors other than radiation including increased gene flow among Chornobyl populations, enhanced sexual reproduction within populations, and/or origin of the genets from seed bank. Both Typha species also demonstrated small but significant effects associated with latitude, geographical regions, and watersheds. Typha's demography in Ukraine possibly varies with these three factors, and the small difference between Chornobyl and reference populations of T. latifolia detected after partitioning the total genetic variance between them is probably due primarily to these factors. However, the positive correlations of several genetic characteristics with radionuclide concentrations suggest that radiation may have also affected genetics of Chornobyl Typha populations but much less than was expected considering massive contamination of the Chornobyl area.