Chromosome-level genome and high nitrogen stress response of the widespread and ecologically important wetland plant Typha angustifolia.

Typha angustifolia L., known as narrowleaf cattail, is widely distributed in Eurasia but has been introduced to North America. Typha angustifolia is a semi-aquatic, wetland obligate plant that is widely distributed in Eurasia and North America. It is ecologically important for nutrient cycling in wetlands where it occurs and is used in phytoremediation and traditional medicine. In order to construct a high-quality genome for Typha angustifolia and investigate genes in response to high nitrogen stress, we carried out complete genome sequencing and high-nitrogen-stress experiments. We generated a chromosomal-level genome of T. angustifolia, which had 15 pseudochromosomes, a size of 207 Mb, and a contig N50 length of 13.57 Mb. Genome duplication analyses detected no recent whole-genome duplication (WGD) event for T. angustifolia. An analysis of gene family expansion and contraction showed that T. angustifolia gained 1,310 genes and lost 1,426 genes. High-nitrogen-stress experiments showed that a high nitrogen level had a significant inhibitory effect on root growth and differential gene expression analyses using 24 samples found 128 differentially expressed genes (DEGs) between the nitrogen-treated and control groups. DEGs in the roots and leaves were enriched in alanines, aspartate, and glutamate metabolism, nitrogen metabolism, photosynthesis, phenylpropanoid biosynthesis, plant-pathogen interaction, and mitogen-activated protein kinase pathways, among others. This study provides genomic data for a medicinal and ecologically important herb and lays a theoretical foundation for plant-assisted water pollution remediation.

Phylogenomic Analyses of Alismatales Shed Light into Adaptations to Aquatic Environments.

Land plants first evolved from freshwater algae, and flowering plants returned to water as early as the Cretaceous and multiple times subsequently. Alismatales is the largest clade of aquatic angiosperms including all marine angiosperms, as well as terrestrial plants. We used Alismatales to explore plant adaptations to aquatic environments by analyzing a data set that included 95 samples (89 Alismatales species) covering four genomes and 91 transcriptomes (59 generated in this study). To provide a basis for investigating adaptations, we assessed phylogenetic conflict and whole-genome duplication (WGD) events in Alismatales. We recovered a relationship for the three main clades in Alismatales as (Tofieldiaceae, Araceae) + core Alismatids. We also found phylogenetic conflict among the three main clades that was best explained by incomplete lineage sorting and introgression. Overall, we identified 18 putative WGD events across Alismatales. One of them occurred at the most recent common ancestor of core Alismatids, and three occurred at seagrass lineages. We also found that lineage and life-form were both important for different evolutionary patterns for the genes related to freshwater and marine adaptation. For example, several light- or ethylene-related genes were lost in the seagrass Zosteraceae, but are present in other seagrasses and freshwater species. Stomata-related genes were lost in both submersed freshwater species and seagrasses. Nicotianamine synthase genes, which are important in iron intake, expanded in both submersed freshwater species and seagrasses. Our results advance the understanding of the adaptation to aquatic environments and WGDs using phylogenomics.

Landscape Genomics Provides Evidence of Ecotypic Adaptation and a Barrier to Gene Flow at Treeline for the Arctic Foundation Species Eriophorum vaginatum.

Global climate change has resulted in geographic range shifts of flora and fauna at a global scale. Extreme environments, like the Arctic, are seeing some of the most pronounced changes. This region covers 14% of the Earth's land area, and while many arctic species are widespread, understanding ecotypic variation at the genomic level will be important for elucidating how range shifts will affect ecological processes. Tussock cottongrass (Eriophorum vaginatum L.) is a foundation species of the moist acidic tundra, whose potential decline due to competition from shrubs may affect ecosystem stability in the Arctic. We used double-digest Restriction Site-Associated DNA sequencing to identify genomic variation in 273 individuals of E. vaginatum from 17 sites along a latitudinal gradient in north central Alaska. These sites have been part of 30 + years of ecological research and are inclusive of a region that was part of the Beringian refugium. The data analyses included genomic population structure, demographic models, and genotype by environment association. Genome-wide SNP investigation revealed environmentally associated variation and population structure across the sampled range of E. vaginatum, including a genetic break between populations north and south of treeline. This structure is likely the result of subrefugial isolation, contemporary isolation by resistance, and adaptation. Forty-five candidate loci were identified with genotype-environment association (GEA) analyses, with most identified genes related to abiotic stress. Our results support a hypothesis of limited gene flow based on spatial and environmental factors for E. vaginatum, which in combination with life history traits could limit range expansion of southern ecotypes northward as the tundra warms. This has implications for lower competitive attributes of northern plants of this foundation species likely resulting in changes in ecosystem productivity.

Responses of root phenology in ecotypes of Eriophorum vaginatum to transplantation and warming in the Arctic.

The effect of climate change on phenology and growth is less understood for belowground plant tissues than for aboveground plant tissues, particularly in high-latitude regions. Ecotypes within a species adapted to a locality may display different responses to climate change. We established two common garden plots in the Arctic tundra north of the Brooks Range in northern Alaska. Three ecotypes of Eriophorum vaginatum along a latitudinal gradient were transplanted into common gardens, and half of the transplants were warmed using open-top chambers (OTCs). Minirhizotrons were used to track the root phenology during the growing seasons of 2016 and 2017. Warming with OTCs (approximately +1 °C in air) did not affect the root biomass, root production or root phenology. The southern ecotype (from 67°16'N) of Eriophorum vaginatum transplanted northward experienced delayed startup and root production compared to two northern ecotypes (from 68°38'N and 69°25'N), although significant differences were not observed in the three ecotypes in terms of root production, root biomass and growth duration at the two sites. Our results suggest that as the climate warms, ecotypes of Eriophorum vaginatum may be able to adjust their duration of root growth and root productivity by phenotypic plasticity, although the degree of plasticity controlling the root startup time may vary between southern and northern ecotypes.

Nothing in Evolution Makes Sense Except in the Light of Biology.

A key question in biology is the predictability of the evolutionary process. If we can correctly predict the outcome of evolution, we may be better equipped to anticipate and manage species' adaptation to climate change, habitat loss, invasive species, or emerging infectious diseases, as well as improve our basic understanding of the history of life on Earth. In the present article, we ask the questions when, why, and if the outcome of future evolution is predictable. We first define predictable and then discuss two conflicting views: that evolution is inherently unpredictable and that evolution is predictable given the ability to collect the right data. We identify factors that generate unpredictability, the data that might be required to make predictions at some level of precision or at a specific timescale, and the intellectual and translational value of understanding when prediction is or is not possible.

Comparative transcriptomics of an arctic foundation species, tussock cottongrass (Eriophorum vaginatum), during an extreme heat event.

Tussock cottongrass (Eriophorum vaginatum) is a foundation species for much of the arctic moist acidic tundra, which is currently experiencing extreme effects of climate change. The Arctic is facing higher summer temperatures and extreme weather events are becoming more common. We used Illumina RNA-Seq to analyse cDNA libraries for differential expression of genes from leaves of ecologically well-characterized ecotypes of tussock cottongrass found along a latitudinal gradient in the Alaskan Arctic and transplanted into a common garden. Plant sampling was performed on a typical summer day and during an extreme heat event. We obtained a de novo assembly that contained 423,353 unigenes. There were 363 unigenes up-regulated and 1,117 down-regulated among all ecotypes examined during the extreme heat event. Of these, 26 HSP unigenes had >log2-fold up-regulation. Several TFs associated with heat stress in previous studies were identified that had >log2-fold up- or down-regulation during the extreme heat event (e.g., DREB, NAC). There was consistent variation in DEGs among ecotypes, but not specifically related to whether plants originated from taiga or tundra ecosystems. As the climate changes it is essential to determine ecotypic diversity at the genomic level, especially for widespread species that impact ecosystem function.

Differential responses of ecotypes to climate in a ubiquitous Arctic sedge: implications for future ecosystem C cycling.

The response of vegetation to climate change has implications for the carbon cycle and global climate. It is frequently assumed that a species responds uniformly across its range to climate change. However, ecotypes - locally adapted populations within a species - display differences in traits that may affect their gross primary productivity (GPP) and response to climate change. To determine if ecotypes are important for understanding the response of ecosystem productivity to climate we measured and modeled growing season GPP in reciprocally transplanted and experimentally warmed ecotypes of the abundant Arctic sedge Eriophorum vaginatum. Transplanted northern ecotypes displayed home-site advantage in GPP that was associated with differences in leaf area index. Southern ecotypes exhibited a greater response in GPP when transplanted. The results demonstrate that ecotypic differentiation can impact the morphology and function of vegetation with implications for carbon cycling. Moreover they suggest that ecotypic control of GPP may limit the response of ecosystem productivity to climate change. This investigation shows that ecotypes play a substantial role in determining GPP and its response to climate. These results have implications for understanding annual to decadal carbon cycling where ecotypes could influence ecosystem function and vegetation feedbacks to climate change.

Effect of growth temperature on photosynthetic capacity and respiration in three ecotypes of Eriophorum vaginatum.

Ecotypic differentiation in the tussock-forming sedge Eriophorum vaginatum has led to the development of populations that are locally adapted to climate in Alaska's moist tussock tundra. As a foundation species, E. vaginatum plays a central role in providing topographic and microclimatic variation essential to these ecosystems, but a changing climate could diminish the importance of this species. As Arctic temperatures have increased, there is evidence of adaptational lag in E. vaginatum, as locally adapted ecotypes now exhibit reduced population growth rates. Whether there is a physiological underpinning to adaptational lag is unknown. Accordingly, this possibility was investigated in reciprocal transplant gardens. Tussocks of E. vaginatum from sites separated by ~1° latitude (Coldfoot: 67°15'N, Toolik Lake: 68°37', Sagwon: 69°25') were transplanted into the Toolik Lake and Sagwon sites and exposed to either an ambient or an experimental warming treatment. Five tussocks pertreatment combination were measured at each garden to determine photosynthetic capacity (i.e., Vcmax and Jmax) and dark respiration rate (Rd) at measurement temperatures of 15, 20, and 25°C. Photosynthetic enhancements or homeostasis were observed for all ecotypes at both gardens under increased growth temperature, indicating no negative effect of elevated temperature on photosynthetic capacity. Further, no evidence of thermal acclimation in Rd was observed for any ecotype, and there was little evidence of ecotypic variation in Rd. As such, no physiological contribution to adaptational lag was observed given the increase in growth temperature (up to ~2°C) provided by this study. Despite neutral to positive effects of increased growth temperature on photosynthesis in E. vaginatum, it appears to confer no lasting advantage to the species.

Population genetic analysis reveals barriers and corridors for gene flow within and among riparian populations of a rare plant.

Landscape features and life-history traits affect gene flow, migration and drift to impact on spatial genetic structure of species. Understanding this is important for managing genetic diversity of threatened species. This study assessed the spatial genetic structure of the rare riparian Grevillea sp. Cooljarloo (Proteaceae), which is restricted to a 20 km2 region impacted by mining in the northern sandplains of the Southwest Australian Floristic Region, an international biodiversity hotspot. Within creek lines and floodplains, the distribution is largely continuous. Models of dispersal within riparian systems were assessed by spatial genetic analyses including population level partitioning of genetic variation and individual Bayesian clustering. High levels of genetic variation and weak isolation by distance within creek line and floodplain populations suggest large effective population sizes and strong connectivity, with little evidence for unidirectional gene flow as might be expected from hydrochory. Regional clustering of creek line populations and strong divergence among creek line populations suggest substantially lower levels of gene flow among creek lines than within creek lines. There was however a surprising amount of genetic admixture in floodplain populations, which could be explained by irregular flooding and/or movements by highly mobile nectar-feeding bird pollinators. Our results highlight that for conservation of rare riparian species, avoiding an impact to hydrodynamic processes, such as water tables and flooding dynamics, may be just as critical as avoiding direct impacts on the number of plants.

Unraveling the biogeographic origins of the Eurasian watermilfoil (Myriophyllum spicatum) invasion in North America.

PREMISE OF THE STUDY: Using phylogeographic analyses to determine the geographic origins of biological invaders is important for identifying environmental adaptations and genetic composition in their native range as well as biocontrol agents among indigenous herbivores. Eurasian watermilfoil (Myriophyllum spicatum) and its hybrid with northern watermilfoil (M. sibiricum) are found throughout the contiguous United States and southern Canada, forming one of the most economically costly aquatic plant invasions in North America, yet the geographic origin of the invasion remains unknown. The objectives of our study included determining the geographic origin of Eurasian watermilfoil in North America as well as the maternal lineage of the hybrids. METHODS: DNA sequence data from a cpDNA intron and the nrDNA ITS region were compiled for accessions from 110 populations of Eurasian watermilfoil and hybrids from North America and the native range (including Europe, Asia, and Africa). Datasets were analyzed using statistical parsimony and Bayesian phylogenetics to assess the geographic origin of the invasion. KEY RESULTS: The two Eurasian watermilfoil cpDNA haplotypes in North America are also found from China and Korea, but not elsewhere in the native range. These haplotypes did not overlap and were limited in native geographic range. The ovule parent for hybrids can come from either parental lineage, and multiple haplotypes from both parental species were found. CONCLUSIONS: The geographic origin of this prolific aquatic plant invasion of North America is in Asia. This provides critical information to better understand the invasion pathway and inform management into the future.

Transcriptome sequencing of three Ranunculus species (Ranunculaceae) reveals candidate genes in adaptation from terrestrial to aquatic habitats.

Adaptation to aquatic habitats is a formidable challenge for terrestrial angiosperms that has long intrigued scientists. As part of a suite of work to explore the molecular mechanism of adaptation to aquatic habitats, we here sequenced the transcriptome of the submerged aquatic plant Ranunculus bungei, and two terrestrial relatives R. cantoniensis and R. brotherusii, followed by comparative evolutionary analyses to determine candidate genes for adaption to aquatic habitats. We obtained 126,037, 140,218 and 114,753 contigs for R. bungei, R. cantoniensis and R. brotherusii respectively. Bidirectional Best Hit method and OrthoMCL method identified 11,362 and 8,174 1:1:1 orthologous genes (one ortholog is represented in each of the three species) respectively. Non-synonymous/synonymous (dN/dS) analyses were performed with a maximum likelihood method and an approximate method for the three species-pairs. In total, 14 genes of R. bungei potentially involved in the adaptive transition from terrestrial to aquatic habitats were identified. Some of the homologs to these genes in model plants are involved in vacuole protein formation, regulating 'water transport process' and 'microtubule cytoskeleton organization'. Our study opens the door to understand the molecular mechanism of plant adaptation from terrestrial to aquatic habitats.

Historical biogeography of Haloragaceae: an out-of-Australia hypothesis with multiple intercontinental dispersals.

Haloragaceae are a cosmopolitan plant family with its centre of diversity in Australia. Here, we investigate the historical biogeography of the family and the role of vicariance or dispersal in shaping its current distribution. DNA sequences from ITS, matK and the trnK 5' and trnK 3' introns were obtained for 102 species representing all 8 genera of Haloragaceae for use in Bayesian molecular dating. Molecular dating was conducted using two macrofossils as calibration points for the analyses. Biogeographic history was investigated using a Bayesian dispersal-vicariance analysis and a dispersal-extinction-cladogenesis model. The results suggest that the earliest diversification of the extant Haloragaceae occurred in Australia during the Eocene (37.3-56.3Ma). Early diversification of the family in the Southern Hemisphere is inferred as resulting from vicariance events among Australia, South America and New Zealand. The results also indicate multiple out of Australia dispersal routes, primarily including (1) from Australia to Asia during the Miocene, with subsequent dispersal to Europe and North America; (2) from Australia to New Zealand, then to South America during the Miocene and Pliocene. Most of the inferred dispersal events occurred throughout the Miocene and later, and are biased towards the aquatic Haloragaceae lineages.

Sorting through the chaff, nDNA gene trees for phylogenetic inference and hybrid identification of annual sunflowers (Helianthus sect. Helianthus).

The annual sunflowers (Helianthus sect. Helianthus) present a formidable challenge for phylogenetic inference because of ancient hybrid speciation, recent introgression, and suspected issues with deep coalescence. Here we analyze sequence data from 11 nuclear DNA (nDNA) genes for multiple genotypes of species within the section to (1) reconstruct the phylogeny of this group, (2) explore the utility of nDNA gene trees for detecting hybrid speciation and introgression; and (3) test an empirical method of hybrid identification based on the phylogenetic congruence of nDNA gene trees from tightly linked genes. We uncovered considerable topological heterogeneity among gene trees with or without three previously identified hybrid species included in the analyses, as well as a general lack of reciprocal monophyly of species. Nonetheless, partitioned Bayesian analyses provided strong support for the reciprocal monophyly of all species except H. annuus (0.89 PP), the most widespread and abundant annual sunflower. Previous hypotheses of relationships among taxa were generally strongly supported (1.0 PP), except among taxa typically associated with H. annuus, apparently due to the paraphyly of the latter in all gene trees. While the individual nDNA gene trees provided a useful means for detecting recent hybridization, identification of ancient hybridization was problematic for all ancient hybrid species, even when linkage was considered. We discuss biological factors that affect the efficacy of phylogenetic methods for hybrid identification.

Patterns of genetic diversity and candidate genes for ecological divergence in a homoploid hybrid sunflower, Helianthus anomalus.

Natural hybridization accompanied by a shift in niche preference by hybrid genotypes can lead to hybrid speciation. Natural selection may cause the fixation of advantageous alleles in the ecologically diverged hybrids, and the loci experiencing selection should exhibit a reduction in allelic diversity relative to neutral loci. Here, we analyzed patterns of genetic diversity at 59 microsatellite loci associated with expressed sequence tags (ESTs) in a homoploid hybrid sunflower species, Helianthus anomalus. We used two indices, ln RV and ln RH, to compare variation and heterozygosity (respectively) at each locus between the hybrid species and its two parental species, H. annuus and H. petiolaris. Mean values of ln RV and ln RH were significantly lower than zero, which implies that H. anomalus experienced a population bottleneck during its recent evolutionary history. After correcting for the apparent bottleneck, we found six loci with a significant reduction in variation or with heterozygosity in the hybrid species, compared to one or both of the parental species. These loci should be viewed as a ranked list of candidate loci, pending further sequencing and functional analyses. Sequence data were generated for two of the candidate loci, but population genetics tests failed to detect deviations from neutral evolution at either locus. Nonetheless, a greater than eight-fold excess of nonsynonymous substitutions was found near a putative N-myristoylation motif at the second locus (HT998), and likelihood-based models indicated that the protein has been under selection in H. anomalus in the past and, perhaps, in one or both parental species. Finally, our data suggest that selective sweeps may have united populations of H. anomalus isolated by a mountain range, indicating that even low gene-flow species may be held together by the spread of advantageous alleles.

Phylogenetic systematics and character evolution in the angiosperm family Haloragaceae.

The poorly known Haloragaceae R. Br. (Saxifragales) are highly diverse in habit (small trees to submerged aquatics) and labile in floral merosity (2-4), both uncommon among the core eudicots. This family has a cosmopolitan distribution, but taxonomic diversity is concentrated in Australia. An explicit phylogenetic approach has not previously been utilized to examine relationships or character evolution in this family. We used molecular evidence from nrDNA ITS and cpDNA trnK and matK regions under both Bayesian and parsimony analyses to address phylogenetic relationships. Combined molecular analyses defined a monophyletic Haloragaceae with the woody genera (Haloragodendron, Glischrocaryon) sister to the rest. Relationships among many genera were well resolved, with genera as currently delimited generally well supported, although there were notable exceptions; a new genus (Trihaloragis) is recognized, and the aquatic genus Meionectes is again distinct from Haloragis. Three new species combinations are also recognized. There are multiple (two or three) origins of the submerged aquatic habit in the family and potentially an intermediate reversal to the terrestrial habit, neither previously demonstrated in a core eudicot family using an explicit phylogenetic hypothesis. Ancestral character analyses suggest two origins of trimerous flowers and multiple reductions to dimerous flowers throughout Haloragaceae.

Evidence of hybridity in invasive watermilfoil (Myriophyllum) populations.

Invasions of nonindigenous species have caused ecological devastation to natural communities worldwide, yet the biological bases for invasiveness remain poorly understood. Our studies of invasive watermilfoil (Myriophyllum) populations revealed widespread polymorphisms in biparentally inherited nuclear ribosomal DNA sequences, which were not detected in populations of native North American species. Subclones of the polymorphic regions revealed the occurrence of distinct sequences matching those acquired from both nonindigenous and native North American species. Molecular data demonstrate clearly that invasive watermilfoil populations in North America have resulted from hybridization between nonindigenous and native species. These observations suggest that invasiveness in these aggressive aquatic weeds may be linked to heterosis maintained by vegetative propagation.

Relationships within Cornales and circumscription of Cornaceae-matK and rbcL sequence data and effects of outgroups and long branches.

Phylogenetic relationships in Cornales were assessed using sequences rbcL and matK. Various combinations of outgroups were assessed for their suitability and the effects of long branches and outgroups on tree topology were examined using RASA 2.4 prior to conducting phylogenetic analyses. RASA identified several potentially problematic taxa having long branches in individual data sets that may have obscured phylogenetic signal, but when data sets were combined RASA no longer detected long branch problems. t(RASA) provides a more conservative measurement for phylogenetic signal than the PTP and skewness tests. The separate matK and rbcL sequence data sets were measured as not containing phylogenetic signal by RASA, but PTP and skewness tests suggested the reverse [corrected]. Nonetheless, the matK and rbcL sequence data sets suggested relationships within Cornales largely congruent with those suggested by the combined matK-rbcL sequence data set that contains significant phylogenetic signal as measured by t(RASA), PTP, and skewness tests. Our analyses also showed that a taxon having a long branch on the tree may not be identified as a "long-branched" taxon by RASA. The long branches identified by RASA had little effect on the arrangement of other taxa in the tree, but the placements of the long-branched taxa themselves were often problematic. Removing the long-branched taxa from analyses generally increased bootstrap support, often substantially. Use of non-optimal outgroups (as identified by RASA) decreased phylogenetic resolution in parsimony analyses and suggested different relationships in maximum likelihood analyses, although usually weakly supported clades (less than 50% support) were impacted. Our results do not recommend using t(RASA) as a sole criterion to discard data or taxa in phylogenetic analyses, but t(RASA) and the taxon variance ratio obtained from RASA may be useful as a guide for improved phylogenetic analyses. Results of parsimony and ML analyses of the sequence data using optimal outgroups suggested by RASA revealed four major clades within Cornales: (1) Curtisia-Grubbia, (2) Cornus-Alangium, (3) Nyssa-Camptotheca-Davidia-Mastixia-Diplopanax, and (4) Hydrangeaceae-Loasaceae, with clades (2) and (3) forming a monophyletic group sister to clade (4) and clade (1) sister to the remainder of Cornales. However, there was not strong bootstrap support for relationships among the major clades. The placement of Hydrostachys could not be reliably determined, although most analyses place the genus within Hydrangeaceae; ML analyses, for example, placed the genus as the sister of Hydrangeeae. Our results supported a Cornales including the systematically problematic Hydrostachys, a Cornaceae consisting of Cornus and Alangium, a Nyssaceae consisting of Nyssa and Camptotheca, a monogeneric Davidiaceae, a Mastixiaceae consisting of Mastixia and Diplopanax, and an expanded Grubbiaceae consisting of Grubbia and Curtisia, and two larger families, Hydrangeaceae and Loasaceae.