Environmental DNA as an emerging tool in botanical research.

Over the past quarter century, environmental DNA (eDNA) has been ascendant as a tool to detect, measure, and monitor biodiversity (species and communities), as a means of elucidating biological interaction networks, and as a window into understanding past patterns of biodiversity. However, only recently has the potential of eDNA been realized in the botanical world. Here we synthesize the state of eDNA applications in botanical systems with emphases on aquatic, ancient, contemporary sediment, and airborne systems, and focusing on both single-species approaches and multispecies community metabarcoding. Further, we describe how abiotic and biotic factors, taxonomic resolution, primer choice, spatiotemporal scales, and relative abundance influence the utilization and interpretation of airborne eDNA results. Lastly, we explore several areas and opportunities for further development of eDNA tools for plants, advancing our knowledge and understanding of the efficacy, utility, and cost-effectiveness, and ultimately facilitating increased adoption of eDNA analyses in botanical systems.

A reappraisal of the phylogeny and historical biogeography of Sparganium (Typhaceae) using complete chloroplast genomes.

BACKGROUND: Sparganium (Typhaceae) is a widespread temperate genus of ecologically important aquatic plants. Previous reconstructions of the phylogenetic relationships among Sparganium species are incompletely resolved partly because they were based on molecular markers comprising < 7,000 bp. Here, we sequenced and assembled the complete chloroplast genomes from 19 Sparganium samples representing 15 putative species and three putative subspecies in order to explore chloroplast genome evolution in this genus, clarify taxonomic lineages, estimate the divergence times of Sparganium species, and reconstruct aspects of the biogeographic history of the genus. RESULTS: The 19 chloroplast genomes shared a conserved genome structure, gene content, and gene order. Our phylogenomic analysis presented a well-resolved phylogeny with robust support for most clades. Non-monophyly was revealed in three species: S. erectum, S. eurycarpum, and S. stoloniferum. Divergence time estimates suggest that the two subgenera of Sparganium split from each other ca. 30.67 Ma in the middle Oligocene. The subgenus Xanthosparganium diversified in the late Oligocene and Miocene, while the subgenus Sparganium diversified in the late Pliocene and Pleistocene. Ancestral area reconstruction suggested that the two subgenera may have originated in East Eurasia and North America. CONCLUSION: The non-monophyletic nature of three putative species underscores the necessity of taxonomic revision for Sparganium: S. stoloniferum subsp. choui may be more appropriately identified as S. choui, and subspecies of S. erectum may be in fact distinct species. The estimated diversification times of the two subgenera correspond to their species and nucleotide diversities. The likely ancestral area for most of subgenus Xanthosparganium was East Eurasia and North America from where it dispersed into West Eurasia and Australia. Most of subgenus Sparganium likely originated in North America and then dispersed into Eurasia. Our study demonstrates some of the ways in which complete chloroplast genome sequences can provide new insights into the evolution, phylogeny, and biogeography of the genus Sparganium.

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.

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.

The importance of molecular markers and primer design when characterizing biodiversity from environmental DNA.

Environmental DNA (eDNA) comprises DNA fragments that have been shed into the environment by organisms, and which can be extracted from environmental samples such as water or soil. Characterization of eDNA can allow researchers to infer the presence or absence of species from a particular site without the need to locate and identify individuals, and therefore may provide an extremely valuable tool for quantifying biodiversity. However, as is often the case with relatively new protocols, methodological challenges remain. A number of earlier reviews have discussed these challenges, but none have provided extensive treatment of the critical decisions surrounding molecular markers and primer development for use in eDNA assays. This review discusses a number of options and approaches that can be used when determining which primers and gene regions are most appropriate for either targeted species detection or metabarcoding macro-organisms from eDNA. The latter represents a new field that is growing rapidly, and which has the potential to revolutionize future assessments of community and ecosystem diversity.

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.

Development of species-specific primers with potential for amplifying eDNA from imperilled freshwater unionid mussels.

Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.

Contrasting patterns of pollen and seed flow influence the spatial genetic structure of sweet vernal grass (Anthoxanthum odoratum) populations.

The spatial genetic structure of plant populations is determined by a combination of gene flow, genetic drift, and natural selection. Gene flow in most plants can result from either seed or pollen dispersal, but detailed investigations of pollen and seed flow among populations that have diverged following local adaptation are lacking. In this study, we compared pollen and seed flow among 10 populations of sweet vernal grass (Anthoxanthum odoratum) on the Park Grass Experiment. Overall, estimates of genetic differentiation that were based on chloroplast DNA (cpDNA) and, which therefore resulted primarily from seed flow, were lower (average F(ST) = 0.058) than previously published estimates that were based on nuclear DNA (average F(ST) = 0.095). Unlike nuclear DNA, cpDNA showed no pattern of isolation by adaptation; cpDNA differentiation was, however, inversely correlated with the number of additions (nutrients and lime) that each plot had received. We suggest that natural selection is restricting pollen flow among plots, whereas nutrient additions are increasing seed flow and genetic diversity by facilitating the successful germination and growth of immigrant seeds. This study highlights the importance of considering all potential gene flow mechanisms when investigating determinants of spatial genetic structure, and cautions against the widespread assumption that pollen flow is more important than seed flow for population connectivity in wind-pollinated species.

Clear phylogeographical structures shed light on the origin and dispersal of the aquatic boreal plant Hippuris vulgaris.

Aquatic plants are an important ecological group in the arctic flora; however, their evolutionary histories remain largely unknown. In order to deepen our understanding of the evolution of these plants, we explored the phylogeographical structure of an aquatic boreal plant Hippuris vulgaris in a broad geographical sampling from Eurasia and North America using the chloroplast intergenic spacer psbA-trnH and seven nuclear microsatellite loci. Two closely-related species H. lanceolata and H. tetraphylla were also included because of their taxonomic controversy. Both chloroplast DNA sequences and nuclear microsatellite data revealed three genetic lineages with distinct distribution ranges. Incongruence between nuclear and chloroplast DNA lineages occurred in 14 samples from Russian Far East and Europe caused by inter-lineage hybridization. No private haplotypes or independent genetic clusters were evident in H. lanceolata or H. tetraphylla, suggesting that these two species should be considered conspecific ecotypes of H. vulgaris. Analysis using Approximate Bayesian Computation-Random Forest approach suggests that Hippuris vulgaris originated in China, followed by dispersal into Russia plus Northeast China, then successively westwards into Europe and North America, and finally into the Russian Far East from both North America and Russia plus Northeast China. This study is the first to elucidate the historical dispersal processes of a circumarctic aquatic plant across the entirety of its range.

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.

Long-distance dispersal and high genetic diversity are implicated in the invasive spread of the common reed, Phragmites australis (Poaceae), in northeastern North America.

PREMISE OF THE STUDY: The Eurasian subspecies of the common reed (Phragmites australis subsp. australis, hereafter abbreviated as P. a. australis) was introduced to North America in the late 18(th) century and rapidly expanded its range, posing an ecological threat to wetlands. In this study, we aimed to determine whether admixture among multiple lineages, dispersal mechanisms, and high genetic diversity have contributed to the invasion of P. a. australis in the northeastern part of its range. Understanding mechanisms of the P. a. australis invasion will 1) contribute to a broader understanding of the factors that facilitate plant invasion, and 2) help us to develop effective management strategies for wetlands threatened by P. a. australis invasion. METHODS: We used a population genetics approach incorporating nine microsatellite loci to study genetic diversity and population structure in relation to biogeography of introduced North American Phragmites a. australis stands in the northeastern continental region. KEY RESULTS: Phragmites a. australis is genetically diverse in the region studied here. Significant population structure exists, and population structure is likely influenced by both long-distance dispersal via major waterways, and short-distance dispersal overland. Different lineages sometimes colonize geographically proximate locations leading to opportunities for admixture. Clonal reproduction likely exaggerates geographical structure among some stands, although high genetic and clonal diversity within some stands implies that sexual reproduction occurs frequently in P. a. australis. CONCLUSIONS: A variety of factors, including admixture among multiple lineages, multiple modes of dispersal, and plasticity in reproductive strategy promote the invasion success of Phragmites a. australis. Wetland managers in the St. Lawrence River/Great Lakes region should focus monitoring efforts on the shores of conservation lands to prevent the establishment of propagules from novel lineages.

Applications and implications of neutral versus non-neutral markers in molecular ecology.

The field of molecular ecology has expanded enormously in the past two decades, largely because of the growing ease with which neutral molecular genetic data can be obtained from virtually any taxonomic group. However, there is also a growing awareness that neutral molecular data can provide only partial insight into parameters such as genetic diversity, local adaptation, evolutionary potential, effective population size, and taxonomic designations. Here we review some of the applications of neutral versus adaptive markers in molecular ecology, discuss some of the advantages that can be obtained by supplementing studies of molecular ecology with data from non-neutral molecular markers, and summarize new methods that are enabling researchers to generate data from genes that are under selection.

Review of Typha spp. (cattails) as toxicity test species for the risk assessment of environmental contaminants on emergent macrophytes.

Macrophytes play an important role in aquatic ecosystems, and thus are often used in ecological risk assessments of potentially deleterious anthropogenic substances. Risk assessments for macrophyte populations or communities are commonly based on inferences drawn from standardized toxicity tests conducted on floating non-rooted Lemna species, or submerged-rooted Myriophyllum species. These tests follow strict guidelines to produce reliable and robust results with legal credibility for environmental regulations. However, results and inferences from these tests may not be transferrable to emergent macrophytes due to their different morphology and physiology. Emergent macrophytes of the genus Typha L. are increasingly used for assessing phytotoxic effects of environmental stressors, although standardized testing protocols have not yet been developed for this genus. In this review we present a synthesis of previous toxicity studies with Typha, based on which we evaluate the potential to develop standard toxicity tests for Typha spp. with seven selection criteria: ecological relevance to the ecosystem; suitability for different exposure pathways; availability of plant material; ease of cultivation; uniform growth; appropriate and easily measurable toxicity endpoints; and sensitivity toward contaminants. Typha meets criteria 1-3 fully, criteria 4 and 5 partly based on current limited data, and we identify knowledge gaps that limit evaluation of the remaining two criteria. We provide suggestions for addressing these gaps, and we summarize the experimental design of ecotoxicology studies that have used Typha. We conclude that Typha spp. can serve as future standard test species for ecological risk assessments of contaminants to emergent macrophytes.

Glyphosate Toxicity to Native Nontarget Macrophytes Following Three Different Routes of Incidental Exposure.

A major goal of invasive plant management is the restoration of native biodiversity, but effective methods for invasive plant control can be harmful to native plants. Informed application of control methods is required to reach restoration goals. The herbicide glyphosate, commonly applied in invasive plant management, can be toxic to native macrophytes. Our study assessed the response of 2 macrophytes that are endangered in our study area (Ammannia robusta and Sida hermaphrodita) to glyphosate concentrations that mimic incidental exposure from nearby invasive plant control: spray drift of 4 × 10-7 % to 5% glyphosate; pulse and continuous immersion in water containing 2 to 41 µg/L glyphosate; and rhizosphere contact with 5%-glyphosate-wicked invasive plants. We assessed macrophyte sensitivity at 14-d postexposure, and quantified abundance of arbuscular mycorrhizal fungi. Glyphosate spray concentrations as low as 0.1% reduced macrophyte growth. Ammannia was more sensitive overall to glyphosate spray than Sida, although sensitivity varied among measured endpoints. Conversely, macrophytes were not affected by immersion in low concentrations of glyphosate or rhizosphere contact with a glyphosate-wicked plant. Likewise, arbuscular mycorrhizal fungi abundance in roots was similar among glyphosate-sprayed and control plants. Based on our results, we recommend that invasive plant managers reduce risks to native nontarget plants through implementing measures that limit off-target spray drift, and consider the feasibility of more targeted applications, such as with wick equipment. Integr Environ Assess Manag 2021;17:597-613. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Salinity, not genetic incompatibilities, limits the establishment of the invasive hybrid cattail Typha × glauca in coastal wetlands.

Hybrids of a single pair of parent species can be much more common in some geographical regions than in others. The reasons for this are not well understood, but could help explain processes such as species diversification or the range expansion of invasive hybrids. The widespread cattails Typha latifolia and T. angustifolia seldom hybridize in some parts of their range, but in other areas produce the dominant hybrid T. × glauca. We used a combination of field and greenhouse experiments to investigate why T. × glauca has invaded wetlands in the Laurentian Great Lakes region of southern Ontario, Canada, but is much less common in the coastal wetlands of Nova Scotia (NS) in eastern Canada. One potentially important environmental difference between these two regions is salinity. We therefore tested three hypotheses: (1) T. latifolia and T. angustifolia in NS are genetically incompatible; (2) the germination or growth of T. × glauca is reduced by salinity; and (3) T. latifolia, a main competitor of T. × glauca, is locally adapted to saline conditions in NS. Our experiments showed that NS T. latifolia and T. angustifolia are genetically compatible, and that saline conditions do not impede growth of hybrid plants. However, we also found that under conditions of high salinity, germination rates of hybrid seeds were substantially lower than those of NS T. latifolia. In addition, germination rates of NS T. latifolia were higher than those of Ontario T. latifolia, suggesting local adaptation to salinity in coastal wetlands. This study adds to the growing body of literature which identifies the important roles that local habitat and adaptation can play in the distributions and characteristics of hybrid zones.

Community genetics: resource addition has opposing effects on genetic and species diversity in a 150-year experiment.

We used the Park Grass Experiment, begun in 1856, to test alternative hypotheses about the relationship between genetic diversity and plant species diversity. The niche variation hypothesis predicts that populations with few interspecific competitors and hence broader niches are expected to contain greater genetic diversity. The coexistence hypothesis predicts that genetic diversity within species favours coexistence among species and therefore species and genetic diversity should be positively correlated. Amplified Fragment Length Polymorphism (AFLP) markers were used to measure the genetic diversity of populations of Anthoxanthum odoratum growing in 10 plots of differing species richness that lie along resource and soil pH gradients. Genetic diversity in A. odoratum was positively correlated with the number of resources added to a plot, but not correlated with species richness. However, separate analyses have shown a negative correlation between resource addition and species richness at Park Grass and elsewhere, so genetic and species diversity appear to respond in opposite directions.

No evidence for niche segregation in a North American Cattail (Typha) species complex.

Interspecific hybridization can lead to a breakdown of species boundaries, and is of particular concern in cases in which one of the parental species is invasive. Cattails (Typha spp.) have increased their abundance in the Great Lakes region of North America over the past 150 years. This increase in the distribution of cattails is associated with hybridization between broad-leaved (Typha latifolia) and narrow-leaved cattails (T. angustifolia). The resulting hybrids occur predominantly as F(1)s, which are known as T. × glauca, although later-generation hybrids have also been documented. It has been proposed that in sympatric populations, the parental species and hybrids are often spatially segregated according to growth in contrasting water depths, and that this should promote the maintenance of parental species. In this study, we tested the hypothesis that the two species and their hybrids segregate along a water-depth gradient at sites where they are sympatric. We identified the two parental species and their hybrids using molecular genetic markers (SSR), and measured shoot elevations (a proxy for water depth) at 18 sites in Southern Ontario, Canada. We found no evidence for niche segregation among species based on elevation. Our data indicate that all three lineages compete for similar habitat where they co-occur suggesting that there is potential for an overall loss of biodiversity in the species complex, particularly if the hybrid lineage is more vigorous compared to the parental species, as has been suggested by other authors.

Phylogeographic inferences from chloroplast DNA: quantifying the effects of mutations in repetitive and non-repetitive sequences.

Phylogeographic inference can be a powerful tool in reconstructing species' evolutionary histories; however, although inferred phylogeographic patterns should depend in part on the underlying types and rates of mutations, the effects of different types of mutations have seldom been quantified. In this study we identified two chloroplast minisatellites in the common reed Phragmites australis, and showed that these are more variable than chloroplast microsatellites. We then recreated parsimony networks of the global phylogeography of P. australis based on data that either included or excluded repetitive sequences (minisatellites and microsatellites), thereby illustrating the influence that these repetitive sequences can have on large-scale phylogeographic inference. The resulting networks differed in the numbers of mutational steps, degrees of uncertainty, and total numbers of haplotypes. In addition, the suggested ancestor-descendant relationships among lineages changed substantially depending on whether repetitive sequences were included. We therefore caution against the inclusion of repetitive sequences in large-scale networks because of their high potential for homoplasy. Nevertheless, we advocate the inclusion of repetitive sequences in other analyses: specifically, we show that the ratio of mutations in repetitive vs. non-repetitive regions can provide insight into the relative ages of lineages.

THE MITOCHONDRIAL AND NUCLEAR GENETIC HOMOGENEITY OF THE PHENOTYPICALLY DIVERSE DARWIN'S GROUND FINCHES.

The most extensively studied group of Darwin's finches is the genus Geospiza, the ground finches, and yet little is known about the evolutionary history and genetic relationships of these birds. Studies using either allozyme or morphological data have been unable to resolve relationships between the six species and numerous populations of ground finches. In this paper we report the results of a study using mitochondrial control region and nuclear internal transcribed spacer (ITS) 1 sequence data. The differentiation of the ground finch species based on morphological data is not reflected in either mitochondrial or nuclear DNA sequence phylogenies. Furthermore, there is little concordance between the mitochondrial haplotypes and ITS alleles found within individuals. We suggest that the absence of species-specific lineages can be attributed to ongoing hybridization involving all six species of Geospiza. There are no long term selective pressures against hybridization within this genus, and therefore a genetically homogenous genus may be maintained indefinitely. Hybridization has apparently played a role in the adaptive radiation of Darwin's finches.