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Pollen dispersal regulates the formation of the invasive, wind-pollinated hybrid cattail Typha × glauca Godron, the F1 offspring of the broadleaf ( Typha latifolia L.) and narrowleaf ( Typha angustifolia L.) cattail. An earlier study suggested that pollen dispersal by T. latifolia might be spatially restricted, with most dispersal occurring over distances less than 2 m. Restricted pollen dispersal would imply that hybrid formation primarily occurs within mixed stands of cattails. Hybrid formation might also be affected by preferential receipt of conspecific pollen, but this has not been investigated for cattails. We compared patterns of pollen dispersal for T. latifolia and T. angustifolia using a wind tunnel. We then tested whether patterns of pollen receipt were biased toward the capture of conspecific versus heterospecific pollen using monospecific cattail stands with a single local pollen source. Results from the wind tunnel partially supported the previous finding of spatially restricted pollen dispersal for T. latifolia, the paternal parent of F1 hybrids. Pollen receipt by T. angustifolia was biased toward the capture of conspecific pollen. Localized pollen dispersal by T. latifolia and preferential conspecific pollen capture by T. angustifolia should reduce rates of hybrid formation below that expected under random mating.

A critical knowledge gap in freshwater plants research is the lack of genetic resources necessary to answer fundamental questions about their demographic histories, adaptation, and taxonomy. One example of this isTypha, a macrophyte genus essential to wetlands that is also becoming an increasingly problematic biological invader in numerous regions worldwide; while important insights have been discovered for this genus, currently available genetic markers are insufficient to resolve its phylogenetic relationships, population structure, and hybridization dynamics. We performed a cost and time–accessible library preparation for high–throughput sequencing to develop a suite of genomic resources forTypha. Genome– wide nuclear SNPs of 140Typhasamples from North America, temperate Eurasia and Africa revealed three independent genetic clusters corresponding toT. angustifolia, T. domingensisandT. latifolia. Data from ∼40% of the nuclear genome was obtained, permitting the characterization of 119,324 nuclear diagnostic markers (SNPs that differentiate the three species). A reference–guided workflow to reconstruct whole–chloroplast–genome sequences was implemented, recovering ∼60% of the genome per sample. Three genetic lineages were identified from the cpDNA phylogenetic analysis, agreeing with those determined from nrDNA. With a cost below 15 USD per sample and a processing time of two hours for the library preparation, this is a rapid and cost–effective protocol for population genomic studies. The genomic locations of diagnostic markers and the chloroplast sequences produced in this study will be permanent resources that can be incorporated into future studies ofTypha, a globally important and evolutionarily dynamic genus.

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.

Documenting species occurrences and habitat occupancy of unionid mussels can often be challenging. Environmental DNA (eDNA) has been shown to be a reliable tool for detecting unionids with comparable or greater sensitivity than conventional sampling and has the added advantages of not disturbing individuals or occupied habitats. However, single‐species eDNA assays are limited to targeting individual species of interest and are functionally blind to the presence of other species. Community eDNA assays have the potential to characterize local species assemblages simultaneously but are currently less extensively developed and implemented than single‐species eDNA testing. We tested the effectiveness of community eDNA markers to identify unionid species assemblages, using two overlapping conserved primers that target the maternal mitochondrial 16S rDNA region. Both primer sets were optimized using three mock communities and successfully amplified 62.5%–81.6% of species with largely consistent results between the primer sets. Following optimization, eDNA from water samples from 24 reference sites with known mussel communities was amplified and sequenced to quantify species richness and diversity within and among sites. Metabarcoding results from the monitoring sites largely mirrored those from the mock communities, with >80% of species detections identified by both assays. The results were broadly consistent with species data from quadrat‐based manual field surveys, although both community eDNA and conventional sampling detected some species that the other method did not. These results demonstrate that community eDNA assays using conserved primers and next‐generation sequencing have the potential to simultaneously target eDNA from multiple unionid species and provide a powerful tool for complementing or augmenting conventional field surveys to characterize and monitor unionid species assemblages.

In much of the United States and Canada, the common reed consists of both a native subspecies Phragmites australis americanus, and a highly invasive introduced subspecies P. australis australis. DNA testing is generally used to distinguish them definitively and is necessary to detect hybridization. We report a group of single nucleotide polymorphisms and indels in the nuclear NRT2 gene of Phragmites that differentiate North American native and European-introduced populations. All native samples tested were identical in NRT2 sequence over 1564 bases except for two positions. There were nine positions, consisting of seven base substitutions and two indels, at which all introduced samples were fixed for a different allele than the native samples. For the two indels, samples collected from northern Europe were also fixed for the same allele as the introduced samples collected across North America. One of the indels was easily detected by a PCR-RFLP assay and provides a rapid and inexpensive way to screen for hybrids between native and European-introduced populations of Phragmites and thus can facilitate more widespread surveillance for hybrids between native and introduced populations in North America.

The Prairie Pothole Region (PPR) of North America has experienced extreme changes in wetland habitat due to proliferation of invasive plants. Typha × glauca is a highly competitive hybrid between native T. latifolia and non-native T. angustifolia, and it is likely the predominant taxon in PPR wetlands. Genetics-based studies are limited, and distributions are poorly known for the first-generation (F1) hybrid and advanced-generation hybrids from F1 mating. Information pertaining to the distribution of T. × glauca could benefit efforts to understand the mechanisms of its spread and to develop management strategies to limit hybrid expansion and preserve progenitors. We used microsatellite markers of field-collected tissue samples from 131 wetlands spread over approximately 350,000 km2 in the PPR to assess the distribution of hybrid T. × glauca relative to its parental species and to examine the prevalence of F1 hybrids and advanced-generation hybrids. Typha × glauca was found in over 80% of wetlands throughout the PPR, compared to 26 and 18% of wetlands with T. latifolia and T. angustifolia, respectively. Advanced-generation hybrids were more common than F1 hybrids, suggesting that hybridization is not a recent phenomenon. Hybrids were significantly taller than T. latifolia, indicating heterosis. Only 7% of sampled individual genets were pure T. latifolia. These results suggest that T. × glauca is pervasive throughout the PPR and may spread independently of both parents. In addition, limited prevalence of native T. latifolia indicates the need for active management to preserve the species.

Invasive species can significantly impact native wildlife by structurally altering habitats and access to resources. Understanding how native species respond to habitat modification by invasive species can inform effective habitat restoration, avoiding inadvertent harm to species at risk. The invasive graminoids Phragmites australis australis (hereafter Phragmites) and Typha × glauca are increasingly dominating Nearctic wetlands, often outcompeting native vegetation. Previous research suggests that turtles may avoid invasive Phragmites when moving through their home ranges, but the mechanisms driving avoidance are unclear. We tested two hypotheses that could explain avoidance of invaded habitat: (1) that stands of invasive macrophytes (Phragmites and Typha x glauca) impede movement, and (2) that they provide inadequate thermal conditions for turtles. We quantified active-season movements of E. blandingii (n = 14, 1328 relocations) and spotted turtles (Clemmys guttata; n = 12, 2295 relocations) in a coastal wetland in the Laurentian Great Lakes. Neither hypothesis was supported by the data. Phragmites and mixed-species Typha stands occurred within the home ranges of mature, active E. blandingii and C. guttata, and were used similarly to most other available habitats, regardless of macrophyte stem density. Turtles using stands of invasive macrophytes did not experience restricted movements or cooler shell temperatures compared to other wetland habitat types. Control of invasive macrophytes can restore habitat heterogeneity and benefit native wetland species. However, such restoration work should be informed by the presence of at-risk turtles, as heavy machinery used for control or removal may injure turtles that use these stands as habitat.

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., The invasive cattail hybrid Typha × glauca dominates wetlands around the Laurentian Great Lakes. We investigated why it is much less common in the maritime provinces of Canada and found that salinity inhibits seed germination and hence the establishment of hybrids in coastal wetlands.

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.

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.

Cattails (Typha species) comprise a genus of emergent wetland plants with a global distribution. Typha latifolia and T. angustifolia are two of the most widespread species, and in areas of sympatry can interbreed to produce the hybrid Typha x glauca. In some regions the relatively high fitness of T. x 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 1,189 scaffolds, with an N50 of 8.706 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 2,700 RNA sequences were putatively identified. Comparative analysis detected over 9,000 shared orthologues with related taxa and phylogenomic analysis supporting Typha 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.

Background: Alliaria petiolata (garlic mustard) is a widespread allelopathic invasive species in North America, but despite extensive research we do not fully understand how it impacts plan...

Typha is an iconic wetland plant found worldwide. Hybridization and anthropogenic disturbances have resulted in large increases in Typha abundance in wetland ecosystems throughout North America at a cost to native floral and faunal biodiversity. As demonstrated by three regional case studies, Typha is capable of rapidly colonizing habitats and forming monodominant vegetation stands due to traits such as robust size, rapid growth rate, and rhizomatic expansion. Increased nutrient inputs into wetlands and altered hydrologic regimes are among the principal anthropogenic drivers of Typha invasion. Typha is associated with a wide range of negative ecological impacts to wetland and agricultural systems, but also is linked with a variety of ecosystem services such as bioremediation and provisioning of biomass, as well as an assortment of traditional cultural uses. Numerous physical, chemical, and hydrologic control methods are used to manage invasive Typha, but results are inconsistent and multiple methods and repeated treatments often are required. While this review focuses on invasive Typha in North America, the literature cited comes from research on Typha and other invasive species from around the world. As such, many of the underlying concepts in this review are relevant to invasive species in other wetland ecosystems worldwide.

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.

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), KEY POINTS We assessed the response of nontarget, endangered macrophytes and associated mycorrhizal fungi to glyphosate exposures that mimicked realistic scenarios encountered during invasive plant control.Glyphosate toxicity to macrophytes varied with the exposure pathway, where spray at low concentrations reduced native macrophyte growth, but no effects were detected from low‐level surface water residues or adjacent wick‐application.Mycorrhizal fungi in macrophyte roots were not affected by glyphosate spray.We recommend that glyphosate‐based invasive plant management implements measures to limit off‐target spray drift, and that risk assessment procedures for nontarget macrophytes consider adding spray exposure tests with emergent species to their repertoire.

Invasive species affect native vegetation through combinations of direct interactions (e.g. competition) and indirect interactions (e.g. altered pollination regimes) mediated through the occupation of shared environments. Hybrid cattails (Typha × glauca) are rapidly displacing their parental species, the native broadleaf cattail (T.latifolia) and the introduced narrow-leaf cattail (T. angustifolia), but the mechanisms involved are poorly understood. We evaluated the potential for direct and indirect interactions among cattails using a series of experiments. First, we evaluated whether seed germination was affected by hybrid leaf litter using a factorial experiment in which we tested for indirect effects of leaf-litter leachate and shading. Second we tested whether these indirect effects of hybrid leaf litter affected seedling performance using a factorial common-garden experiment. Finally, we tested for direct competitive interactions between hybrid and native cattail seedlings. One or more components of hybrid leaf-litter leachate strongly reduced the germination of the parental cattail species, but not that of the hybrid. There was no evidence that direct competitive interactions affected seedling performance. Because regular recruitment by seeds appears to be important in shaping patterns of genetic diversity in natural populations, our findings provide a mechanism for explaining the dominance of hybrid cattails in eastern North America.

Spatial separation between hybridizing taxa can limit hybridization and introgression when mating occurs between near neighbours. Water depth can be an important factor structuring wetland plant communities, which often include cattails (Typha spp., Typhaceae). In eastern North America, native Typha latifolia regularly hybridizes with introduced T. angustifolia to produce the invasive F1 hybrid T. × glauca, which can backcross to parental species. Hybridization in this wind-pollinated species complex may be facilitated by overlapping niches among taxa, but previous studies have yielded contradictory results, possibly because these studies were limited to shallow sites or single locations. We investigated patterns of depth occupancy in ditches (mean depth 3.4 cm) and permanent wetlands (mean depth 25.0 cm) with various mixtures of T. latifolia, T. angustifolia, and T. × glauca across a 1700 km transect from Michigan, USA, to Nova Scotia, Canada. Overall, we found that water depth cannot on its own explain patterns of cattail occupancy. We therefore conclude that niche partitioning by water depth does not promote mating isolation in Typha. Previous findings of habitat segregation by depth may be attributable to a combination of idiosyncratic features of individual sites, multi-dimensional niches, and phenotypic plasticity, which could prevent consistent niche segregation among Typha taxa. A lack of spatial segregation between Typha taxa should promote hybridization and introgression. This, coupled with the competitive superiority of hybrids, appears to be facilitating the displacement of native T. latifolia across a wide geographical range.

Cattails (Typha spp.) are essential components of wetland around the world, although they can also be problematic invaders. In some regions of North America, T. latifolia and T. angustifolia hybridize to produce T. x glauca, which is invading and dominating wetlands around the Laurentian Great Lakes. However, in other regions such as China and the maritime provinces of Canada, hybrids are either non-existent or maintained at very low frequencies, and cattails are not considered invasive. Little is known about cattail hybrids in Europe. We used microsatellite markers to characterize T. latifolia, T. angustifolia, T. laxmannii, and T. domingensis sampled from 63 sites across nine European countries. Hybrids were very uncommon (

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.

Although cattails (Typha spp.) are important components of wetlands around the world, the three most widespread species (T. angustifolia, T. domingensis, T. latifolia) are becoming increasingly dominant. We used global phylogenetic and phylogeographic assessments to test the hypotheses that each species has experienced multiple introductions of divergent lineages into North America and that commercial retailers are aiding long-distance dispersal. Our analyses identified T. angustifolia as a paraphyletic species with a highly divergent lineage. We found evidence for at least one introduced T. angustifolia lineage in wild populations and garden centres of North America. Although potentially complicated by incomplete lineage sorting, our data suggest dispersal of T. domingensis between Europe and Australia, and further investigation should assess a possible introduction of a non-native T. domingensis lineage into North America. T. latifolia has experienced bidirectional dispersal between North America and Europe, and a sample of T. latifolia purchased in a Canadian garden centre was an Asian lineage. Interspecific hybridization and novel intraspecific admixture have been repeatedly implicated in biological invasions, including invasions by the hybrid cattail Typha × glauca, and future work should focus on the potential contributions of non-native lineages to regional patterns of invasion by Typha spp. in North America.

Hybridization can result in the formation of highly competitive lineages that displace their progenitor species and other native organisms. In eastern North America, hybridization between native Typha latifolia and introduced Typha angustifolia yields the vigorous hybrid species T. × glauca. The hybrid has become increasingly prevalent over the past century in eastern North America, where there are indications it displaces native plants. Moreover, it has been suggested that water depth influences the growth performance of hybrid vs. native cattails but there have been no comparisons of plant performance under contrasting water depths. Using a common-garden experiment with two levels of a water-depth treatment we compared the above- and below-ground growth performance of T. latifolia vs. hybrid cattails collected from 12 sites in southern Ontario. We found that hybrids outperformed all other plants in terms of total and above-ground biomass production and, depending on the method of species assignment used, in terms of the number of ramets produced. However, there was no difference in the mass of rhizomes produced between species. Consistent with a previous study of natural populations from the same region, growth performance of the different species was similar across the different water depths used in the experiment. Collectively, our results indicate that enhanced hybrid performance was driven by the production of larger and more numerous ramets and not via increased investment in rhizomes.

Rapid evolution following interspecific hybridization can facilitate biological invasions. Around the Great Lakes in North America, the hybrid cattail Typha × glauca is dominating wetlands and displacing both parental species. We measured water depth and height of T. × glauca and its parental species (Typha angustifolia and Typha latifolia) throughout the growing season at a site near Lake Ontario that harbors both parental species plus hybrids. We found no evidence of niche partitioning by water depth, nor was there evidence that water depth was influencing plant height. At the beginning of the growing season, T. latifolia comprised the tallest plants, but this potential advantage was short-lived, and for most of the growing season, F1 hybrids were taller than all or most other taxa (T. angustifolia, T. latifolia, and advanced-generation/backcrossed hybrids). Heterosis, inferred from height, is therefore evident in F1 hybrids, but not in advanced-generation/backcrossed hybrids. Typha stands often achieve high densities, and the competitive advantage of superior height is likely contributing to the invasive success of T. × glauca F1 hybrids in the Great Lakes region.

The genetic diversity of peripheral populations is potentially important to the future adaptive capacity of species, although may be difficult to predict. A large number of species-at-risk in Canada are at the northern edge of their distribution, and many of these live in fragmented habitat. We used nuclear and chloroplast markers to assess patterns of genetic diversity and differentiation within and among populations of Canadian Magnolia acuminata (Cucumber tree), an endangered species in Canada that extends as far north as the fragmented Carolinian forest in southern Ontario. We also compared the genetic composition of Canadian M. acuminata to populations sampled throughout its central distribution in the USA. We found a high proportion of shared microsatellite alleles, plus a single cpDNA haplotype, distributed throughout the entire M. acuminata range. We also found that despite occupying fragmented habitat at their range periphery, Canadian populations showed little reduction in genetic diversity relative to the USA populations, and we attribute this to effective historical dispersal in a long-lived, polyploid species. However, a combination of private alleles, genetic substructuring, and lower levels of genetic diversity in seedlings compared to mature trees, suggests that current levels of gene flow are relatively low among Canadian populations. Therefore, despite high levels of genetic diversity in Canadian M. acuminata, managers should be aware that without intervention, populations will likely become increasingly isolated and experience a reduction in genetic diversity which in turn may threaten their long-term survival in Canada.

Environmental DNA (eDNA) has recently been employed as a surveillance tool for invasive species, although its use has so far been limited to animals. We investigated the possibility of using eDNA to monitor aquatic invasive plants by assessing the potential utility of three regions of the chloroplast genome- matK, rbcL, and trnH–psbA- in ten aquatic invasive plant species. Of the three regions, matK was most likely to harbor putatively species-specific nucleotides in candidate primer-binding sites, and the potentially diagnostic primers that we designed for this region were least likely to amplify DNA from non-target species. A lack of species-specific nucleotides precluded the design of appropriate rbcL primers in most of our species, and amplification of non-target DNA often precluded the use of trnH–psbA markers. We, therefore, suggest that of the three regions, matK is the most useful starting point for designing eDNA markers for aquatic plants. We also demonstrated that these markers can identify species from eDNA that has been extracted from water samples. Our results should be useful to the ongoing development of eDNA as a tool for the early detection and ongoing surveillance of aquatic invasive plants.

In northeastern North America, an important wetland invader is the cattail Typha × glauca, a hybrid of native Typha latifolia and introduced Typha angustifolia. Although intensively studied in localized wetlands around the Great Lakes, the distributions of the hybrid and its parental species across broad spatial scales are poorly known. We obtained genotypes from plants collected from 61 sites spanning two geographical regions. The first region, near the Great Lakes and St. Lawrence Seaway (GLSL), has experienced substantial Typha increases over the last century, whereas more modest increases have occurred in the second region across Nova Scotia, New Brunswick, and Maine (NSNB). We found that hybrids predominate in the GLSL region, thriving in both disturbed and undisturbed habitats, and are expanding at the expense of both parental species. In contrast, the native T. latifolia is by far the most common of the three taxa across all habitat types in the NSNB region. We found no evidence that the formation of backcrossed and advanced-generation hybrids is limited by the reproductive barriers that are evident in F1 hybrids. However, although backcrossed individuals arise in both regions, they are much less common than F1 hybrids, which may explain why the parental species boundary remains. We conclude that F1 hybrids are playing a key role in the invasion of wetlands in the GLSL region, whereas their low frequency in the NSNB region may explain why Typha appears to be much less invasive further east. An improved understanding of these contrasting patterns of distribution is necessary before we can accurately predict future wetland invasions.

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.

Platanthera leucophaea, the Eastern Prairie Fringed-orchid, is a globally imperilled species native to the USA and Canada, with its Canadian distribution limited to the Province of Ontario. In Canada, where P. leucophaea is listed as endangered, approximately 40 % of populations have been extirpated in recent years, and many remaining populations have experienced substantial declines. In this study, we investigated whether reduced population sizes have led to low genetic diversity and inbreeding. We also investigated the extent to which hybridization with Platanthera psycodes may be threatening the genetic integrity of P. leucophaea populations. We found that overall, genetic diversity is low, and inbreeding is high. This is despite evidence of regular gene flow between proximate populations, although more distant populations show high levels of genetic differentiation. At sites where P. leucophaea is sympatric with P. psycodes, interspecific hybridization occurs in a bidirectional manner, i.e. with both parental species acting as either pollen donor or pollen recipient. Inbreeding and low genetic diversity in all populations, and hybridization in some populations, may pose future threats to P. leucophaea, and should be considered in the future by biodiversity managers.

Cattails ( Typha spp.) in northeastern North American wetlands are increasingly dominant, partly because of hybridization between Typha latifolia and Typha angustifolia which results in the widespread and successful hybrid T. × glauca . Both parental species are protogynous, and previous studies have shown that T. angustifolia flowers earlier than T. latifolia. Nevertheless, experimental studies have shown that hybridization is overwhelmingly asymmetric, involving T. angustifolia female flowers and T. latifolia pollen which should have minimal overlap in the times at which they are produced. In this study we surveyed two Typha stands in eastern North America, and at both sites found substantial overlap in the times at which the parental species and the hybrids shed pollen and presumably are receptive to fertilization; this means that flowering time is not a prezygotic barrier to either the formation of hybrids or their subsequent backcrossing to parental species. Consistent with experimental studies, all hybrids had T. angustifolia as their maternal parent. Our results suggest that reproductive barriers between Typha spp. are evolving.

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.

The full effects of biological invasions may be underestimated in many areas because of cryptogenic species, which are those that can be identified as neither native nor introduced. In North America, the cattails Typha latifolia, T. angustifolia, and their hybrid T. × glauca are increasingly aggressive invaders of wetlands. There is a widespread belief that T. latifolia is native to North America and T. angustifolia was introduced from Europe, although there has so far been little empirical support for the latter claim. We used microsatellite data and chloroplast DNA sequences to compare T. latifolia and T. angustifolia genotypes from eastern North America and Europe. In both species, our data revealed a high level of genetic similarity between North American and European populations that is indicative of relatively recent intercontinental dispersal. More specifically, the most likely scenario suggested by Approximate Bayesian Computation was an introduction of T. angustifolia from Europe to North America. We discuss the potential importance of our findings in the context of hybridization, novel genomes, and increasingly invasive behaviour in North American Typha spp.

In 2011, Vachon and Freeland presented the results of a study that illustrated the importance of considering mutation patterns, including the potential for homoplasy, when deciding whether to include or exclude repetitive sequences from phylogeographic inferences. Saltonstall and Lambertini (2012) criticized this study by suggesting that some of the analyses and interpretations were flawed. In this reply, we explain why we disagree with most of their criticisms and identify some inconsistencies in their analyses. Most importantly, we reiterate the need to examine underlying assumptions, particularly with respect to mutation patterns, when using molecular genetic data to untangle evolutionary relationships.

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 F1s, 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.

A recent increase in the abundance of cattails (Typha spp.) in North American wetlands has been anecdotally linked with hybridization between Typha latifolia and Typha angustifolia. In this study, we used molecular genetic markers (microsatellites) to investigate whether the hybrid lineage (Typha × glauca) is restricted to The Great Lakes region, or exists across a much broader spatial scale. We also investigated the possibility of backcrossing and genetic introgression in natural populations. Parental species could be distinguished from one another based on the distribution of alleles at six microsatellite loci. Species identification based on genetic data corresponded well with species identifications based on leaf width, a key morphological trait that can distinguish the two parental species. We found that hybrids occur in Ontario, Quebec, New Brunswick, and Nova Scotia, but we did not detect hybrids in Maine. F1s are more abundant than backcrossed or intercrossed hybrids, although we also found evidence of backcrossing, particularly in Ontario. This indicates that hybrids are fertile, and are therefore potential conduits of gene flow between the parental species. Further work is needed to determine whether T. × glauca is particularly successful in the Great Lakes region relative to other areas in which the two parental species co-exist, and to assess whether introgression may lead to increased invasiveness in the species complex.

 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. Signifi cant population structure exists, and population structure is likely infl uenced 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.

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.

Reedbeds, which are dominated by the common reed Phragmites australis, provide one of the most important aquatic habitats in the United Kingdom, and have been identified as a priority habitat under the UK Biodiversity Action Plan. Ongoing conservation and management of reedbeds is necessary because past eradication and fragmentation have been extensive. However, there has been little consideration of the potential long-term effects that may arise if processes such as founder effects during restoration projects, or genetic bottlenecks following harvesting, are leading to reductions in genetic diversity. In this study, we used microsatellite data to compare the genetic diversity of 19 P. australis stands in the UK, and found that neither size, management, nor habitat disturbance were affecting genetic diversity. Mixed reproduction (clonal and sexual), possibly combined with gene flow across relatively small spatial scales, appears to be maintaining genetic diversity within most stands. However, most stands were highly genetically differentiated from one another, which implies low gene flow. Long-term genetic diversity in managed stands may therefore require periodic bolstering from other populations, combined with the simultaneous cultivation of multiple generations. In addition, low gene flow suggests that managers should actively introduce plants into all areas in which reedbeds are desired. In conclusion, our study shows that reedbed restoration and maintenance do not seem to be adversely affecting the population genetics of P. australis, but the importance of genetic diversity should be factored into long-term projects.

High rates of mutation and homoplasy mean that microsatellites generally are not considered to be useful molecular markers for inferring systematic relationships between species. However, an earlier pilot study suggested that conserved flanking microsatellite sequences, also known as repetitive flanking sequences (ReFS), may form a basis for a dominant marker that can differentiate between species of Lepidoptera. We present data that demonstrate that ReFS are quick and easy to use, and generate highly repeatable banding patterns from a range of Lepidoptera species. Sequence data from a subset of ReFS-amplified bands revealed microsatellite families with flanking sequences that are more conserved within than among species: this is probably attributable to recombination-mediated events, transposition of mobile elements or a combination of the two. Our data support the use of ReFS as dominant interspecific molecular markers, and add to the growing literature on the evolution of microsatellites in Lepidoptera.

Populations at the edges of their geographical ranges are often characterized by reduced genetic diversity and increased genetic differentiation. These patterns may be exacerbated by specialized associations between plants and uncommon habitats. Together, reduced diversity and increased subdivision may increase the risk of population extirpation, an issue that is particularly important for plants classified as rare or threatened. Hill’s thistle (Cirsium hillii (Canby) Fernald), is a perennial herb endemic to the Great Lakes region and is considered threatened in Canada where its northernmost distribution occurs. We used microsatellite markers to genotype plants from 11 populations of Hill’s thistle that span the range of the species distribution within Canada. These data were used to investigate levels of genetic diversity within populations and genetic differentiation between populations, and to evaluate associations between patterns of genetic variation and those expected under ongoing habitat loss. Genetic differentiation among populations was low, although significant pairwise FST values, along with the presence of private alleles in most populations, was consistent with moderate to low gene flow. Nevertheless, in comparison with a congeneric rare species (Cirsium pitcheri) that occurs in the same geographical region in Canada, genetic diversity within most populations was high, and we suggest that this is at least partially attributable to this species’ combination of sexual and clonal reproduction. Despite high levels of genetic diversity, Hill’s thistle should probably remain a designated species at risk because of ongoing habitat loss; however, our data suggest that Hill’s thistle is unlikely to suffer from reduced genetic diversity in the foreseeable future.

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 extent to which divergent selection can drive genome-wide population differentiation remains unclear. Theory predicts that in the face of ongoing gene flow, population differentiation should be apparent only at those markers that are directly or indirectly (i.e. through linkage) under selection. However, if reproductive barriers limit gene flow, genome-wide population differentiation may occur even in geographically proximate populations. Some insight into the link between selection and genetic differentiation in the presence of ongoing gene flow can come from long-term experiments such as The Park Grass Experiment, which has been running for over 150 years, and provides a unique example of a heterogeneous environment with a long and detailed history. Fertilizer treatments applied in the Park Grass Experiment have led to rapid evolutionary change in sweet vernal grass Anthoxanthum odoratum, but until now, nothing was known of how these changes would be reflected in neutral molecular markers. We have genotyped ten A. odoratum populations from the Park Grass Experiment using Amplified Fragment Length Polymorphisms (AFLPs). Our data show that nutrient additions have resulted in genome-wide divergence among plots despite the high potential for ongoing gene flow. This provides a well-documented example of concordance between genomes and environmental conditions that has arisen in continuous populations across a time span of fewer than 75 generations.