Plant-Pollinator Specialization: Origin and Measurement of Curvature.

AbstractA feature of biodiversity is the abundance of curves displayed by organs and organisms. Curvature is a widespread, convergent trait that has important ecological and evolutionary implications. In pollination ecology, the curvature of flowers and pollinator mouthparts (e.g., hummingbird bills) along the dorsiventral plane has been associated with specialization, competition, and species coexistence. Six differing methods have historically been used to measure curvature in pollination systems; we provide a solution to this inconsistency by defining curvature using well-established concepts from differential geometry. Intuitively, curvature is the degree to which a line is not straight, but more formally it is the rate at which the tangent of a curve changes direction with respect to arc length. Here, we establish a protocol wherein a line is fitted against landmarks placed on an image of a curved organ or organism, then curvature is computed at many points along the fitted line and the sum taken. The protocol is demonstrated by studying the development of nectar spur curvature in the flowering plant genus Epimedium (Berberidaceae). By clarifying the definition of curvature, our aim is to make the language of comparative morphology more precise and broadly applicable to capture other curved structures in nature.

Dark extinction: the problem of unknown historical extinctions.

The extinction of species before they are discovered and named (dark extinction, DE) is widely inferred as a significant part of species loss in the 'pre-taxonomic' period (approx. 1500-1800 CE) and, to some extent, in the 'taxonomic period' (approx. 1800-present) as well. The discovery of oceanic islands and other pristine habitats by European navigators and the consequent introduction of destructive mammals, such as rats and goats, started a process of anthropogenic extinction. Much ecosystem change happened before systematic scientific recording, so has led to DE. Statistical methods are available to robustly estimate DE in the 'taxonomic period'. For the 'pre-taxonomic period', simple extrapolation can be used. The application of these techniques to world birds, for example, suggests that approximately 56 DEs occurred in the 'taxonomic period' (1800-present) and approximately 180 in the 'pre-taxonomic period' (1500-1800). Targeting collection activities in extinction hotspots, to make sure organisms are represented in collections before their extinction, is one way of reducing the number of extinct species without a physical record (providing that collection efforts do not themselves contribute to species extinction).

Introgression from Populus balsamifera underlies adaptively significant variation and range boundaries in P. trichocarpa.

Introgression can be an important source of adaptive phenotypes, although conversely it can have deleterious effects. Evidence for adaptive introgression is accumulating but information on the genetic architecture of introgressed traits lags behind. Here we determine trait architecture in Populus trichocarpa under introgression from P. balsamifera using admixture mapping and phenotypic analyses. Our results reveal that admixture is a key driver of clinal adaptation and suggest that the northern range extension of P. trichocarpa depends, at least in part, on introgression from P. balsamifera. However, admixture with P. balsamifera can lead to potentially maladaptive early phenology, and a reduction in growth and disease resistance in P. trichocarpa. Strikingly, an introgressed chromosome 9 haplotype block from P. balsamifera restores the late phenology and high growth parental phenotype in admixed P. trichocarpa. This epistatic restorer block may be strongly advantageous in maximizing carbon assimilation and disease resistance in the southernmost populations where admixture has been detected. We also confirm a previously demonstrated case of adaptive introgression in chromosome 15 and show that introgression generates a transgressive chlorophyll-content phenotype. We provide strong support that introgression provides a reservoir of genetic variation associated with adaptive characters that allows improved survival in new environments.

Scale and direction of adaptive introgression between black cottonwood (Populus trichocarpa) and balsam poplar (P. balsamifera).

Introgression can introduce novel genetic variation at a faster rate than mutation alone and result in adaptive introgression when adaptive alleles are maintained in the recipient genome over time by natural selection. A previous study from our group demonstrated adaptive introgression from Populus balsamifera into P. trichocarpa in a target genomic region. Here we expand our local ancestry analysis to the whole genome of both parents to provide a comprehensive view of introgression patterns and to identify additional candidate regions for adaptive introgression genomewide. Populus trichocarpa is a large, fast-growing tree of mild coastal regions of the Pacific Northwest, whereas P. balsamifera is a smaller stature tree of continental and boreal regions with intense winter cold. The species hybridize where they are parapatric. We detected asymmetric patterns of introgression across the whole genome of these two poplar species adapted to contrasting environments, with stronger introgression from P. balsamifera to P. trichocarpa than vice versa. Admixed P. trichocarpa individuals contained more genomic regions with unusually high levels of introgression (19 regions) and also the largest introgressed genome fragment (1.02 Mb) compared with admixed P. balsamifera (nine regions). Our analysis also revealed numerous candidate regions for adaptive introgression with strong signals of selection, notably related to disease resistance, and enriched for genes that may play crucial roles in survival and adaptation. Furthermore, we detected a potential overrepresentation of subtelomeric regions in P. balsamifera introgressed into P. trichocarpa and possible protection of sex-determining regions from interspecific gene flow.

Phylogenomics from Whole Genome Sequences Using aTRAM.

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.].

Adaptive introgression: a plant perspective.

Introgression is emerging as an important source of novel genetic variation, alongside standing variation and mutation. It is adaptive when such introgressed alleles are maintained by natural selection. Recently, there has been an explosion in the number of studies on adaptive introgression. In this review, we take a plant perspective centred on four lines of evidence: (i) introgression, (ii) selection, (iii) phenotype and (iv) fitness. While advances in genomics have contributed to our understanding of introgression and porous species boundaries (task 1), and the detection of signatures of selection in introgression (task 2), the investigation of adaptive introgression critically requires links to phenotypic variation and fitness (tasks 3 and 4). We also discuss the conservation implications of adaptive introgression in the face of climate change. Adaptive introgression is particularly important in rapidly changing environments, when standing genetic variation and mutation alone may only offer limited potential for adaptation. We conclude that clarifying the magnitude and fitness effects of introgression with improved statistical techniques, coupled with phenotypic evidence, has great potential for conservation and management efforts.

Homogenization of Populations in the Wildflower, Texas Bluebonnet (Lupinus texensis).

Wildflower seeds are routinely spread along highways and thoroughfares throughout North America as part of federal beautification policy, but the genetic effect of the introduction of these cultivated populations on wild populations of the same species is unknown. Interbreeding may occur between these seeded and wild populations, resulting in several possible outcomes. Here we sample 187 individuals in 12 matched pairs of neighboring wild and seeded populations of the Texas bluebonnet (Lupinus texensis), a species popular in commercially available wildflower seed mixes used by both the Texas Department of Transportation and the public. We use genotyping by sequencing to identify 11741 genome-wide single nucleotide polymorphisms, as well as a smaller number of SNPs from the chloroplast genome, to analyze population structure and genetic diversity within and between the populations. We find a striking lack of population structure both between wild and seeded populations and amongst wild populations. STRUCTURE analyses indicate that all populations are apparently panmictic. This pattern may be explained by extensive swamping of wild populations by seeded germplasm and increased dispersal of semi-domesticated seed across this species' core native range by humans. We discuss the possible negative and positive ramifications of homogenization on the evolutionary future of this popular wildflower species.

Dissecting the 'bacon and eggs' phenotype: transcriptomics of post-anthesis colour change in Lotus.

Background and Aims: Post-anthesis colour change (PACC) is widely thought to be an adaptation to signal floral suitability to pollinators. Lotus filicaulis and Lotus sessilifolius are insect-pollinated herbaceous legumes with flowers that open yellow, shift to orange and finally red. This study examines the molecular basis for floral colour change in these Lotus species. Methods: Lotus filicaulis was cultivated in a glasshouse from which pollinating insects (bees) were excluded, and the rate of colour change was recorded in both unpollinated and manually pollinated flowers. Unpollinated flowers from both the yellow stage and the red stage were sampled for sequencing. The transcriptomes of L. filicaulis and L. sessilifolius of both colour stages were analysed for differentially expressed genes and enriched ontologies. Key Results: The rate of progression through PACC doubled when L. filicaulis was hand-pollinated. De novo assembly of RNA-Seq reads from non-model Lotus species outperformed heterologous alignment of reads to the L. japonicus genome. Differential expression analysis suggested that the carotenoid biosynthetic pathway is upregulated at anthesis while the flavonoid biosynthetic pathway is upregulated with the onset of PACC in L. filicaulis and L. sessilifolius . Conclusion: Pollination significantly accelerates PACC in L. filicaulis , consistent with the hypothesis that PACC increases pollination efficiency by directing pollinators to unpollinated flowers. RNA-Seq results show the synchronized upregulation of the entire cyanidin biosynthesis pathway in the red stage of PACC in L. filicaulis and L. sessilifolius . The genes implicated offer the basis for further investigations into how gene families, transcription factors and related pathways are likely to be involved in PACC.

Genomic and functional approaches reveal a case of adaptive introgression from Populus balsamifera (balsam poplar) in P. trichocarpa (black cottonwood).

Natural hybrid zones in forest trees provide systems to study the transfer of adaptive genetic variation by introgression. Previous landscape genomic studies in Populus trichocarpa, a keystone tree species, indicated genomic footprints of admixture with its sister species Populus balsamifera and identified candidate genes for local adaptation. Here, we explored the patterns of introgression and signals of local adaptation in P. trichocarpa and P. balsamifera, employing genome resequencing data from three chromosomes in pure species and admixed individuals from wild populations. Local ancestry analysis in admixed P. trichocarpa revealed a telomeric region in chromosome 15 with P. balsamifera ancestry, containing several candidate genes for local adaptation. Genomic analyses revealed signals of selection in certain genes in this region (e.g. PRR5, COMT1), and functional analyses based on gene expression variation and correlations with adaptive phenotypes suggest distinct functions of the introgressed alleles. In contrast, a block of genes in chromosome 12 paralogous to the introgressed region showed no signs of introgression or signatures of selection. We hypothesize that the introgressed region in chromosome 15 has introduced modular or cassette-like variation into P. trichocarpa. These linked adaptive mutations are associated with a block of genes in chromosome 15 that appear to have undergone neo- or subfunctionalization relative to paralogs in a duplicated region on chromosome 12 that show no signatures of adaptive variation. The association between P. balsamifera introgressed alleles with the expression of adaptive traits in P. trichocarpa supports the hypothesis that this is a case of adaptive introgression in an ecologically important foundation species.

The transcriptional landscape of insect galls: psyllid (Hemiptera) gall formation in Hawaiian Metrosideros polymorpha (Myrtaceae).

BACKGROUND: Recent studies show that galling Hymenoptera and Diptera are able to synthesize the plant hormone indole-3-acetic acid (auxin) from tryptophan and that plant response to insect-produced auxin is implicated in gall formation. We examined the leaf transcriptome of galled and ungalled leaves of individuals of the Hawaiian endemic plant Metrosideros polymorpha (Myrtaceae) subject to infestation by psyllid (Hemiptera) gall-makers in the genus Trioza (Triozidae). RESULTS: Transcript libraries were sequenced using Illumina technology and the reads assembled de novo into contigs. Functional identification of contigs followed a two-step procedure, first identifying contigs by comparison to the completely sequenced genome of the related Eucalyptus, followed by identifying the equivalent Arabidopsis gene using a pre-computed mapping between Eucalyptus and Arabidopsis genes. This allowed us to use the rich functional annotation of the Arabidopsis genome to assess the transcriptional landscape of galling in Metrosideros. Comparing galled and ungalled leaves, we find a highly significant enrichment of expressed genes with a gene ontology (GO) annotation to auxin response in the former. One gene consistently expressed in all galled trees examined but not detected in any libraries from ungalled leaves was the Metrosideros version of SMALL AUXIN UPREGULATED (SAUR) 67 which appears to be a marker for leaf-galling in Metrosideros. CONCLUSIONS: We conclude that an auxin response is involved in galling by Metrosideros psyllids. The possibility should therefore be considered that psyllids (like other insects examined) are able to synthesize auxin.

Extensive functional pleiotropy of REVOLUTA substantiated through forward genetics.

In plants, genes may sustain extensive pleiotropic functional properties by individually affecting multiple, distinct traits. We discuss results from three genome-wide association studies of approximately 400 natural poplar (Populus trichocarpa) accessions phenotyped for 60 ecological/biomass, wood quality, and rust fungus resistance traits. Single-nucleotide polymorphisms (SNPs) in the poplar ortholog of the class III homeodomain-leucine zipper transcription factor gene REVOLUTA (PtREV) were significantly associated with three specific traits. Based on SNP associations with fungal resistance, leaf drop, and cellulose content, the PtREV gene contains three potential regulatory sites within noncoding regions at the gene's 3' end, where alternative splicing and messenger RNA processing actively occur. The polymorphisms in this region associated with leaf abscission and cellulose content are suggested to represent more recent variants, whereas the SNP associated with leaf rust resistance may be more ancient, consistent with REV's primary role in auxin signaling and its functional evolution in supporting fundamental processes of vascular plant development.

Whole plastome sequencing reveals deep plastid divergence and cytonuclear discordance between closely related balsam poplars, Populus balsamifera and P. trichocarpa (Salicaceae).

As molecular phylogenetic analyses incorporate ever-greater numbers of loci, cases of cytonuclear discordance - the phenomenon in which nuclear gene trees deviate significantly from organellar gene trees - are being reported more frequently. Plant examples of topological discordance, caused by recent hybridization between extant species, are well known. However, examples of branch-length discordance are less reported in plants relative to animals. We use a combination of de novo assembly and reference-based mapping using short-read shotgun sequences to construct a robust phylogeny of the plastome for multiple individuals of all the common Populus species in North America. We demonstrate a case of strikingly high plastome divergence, in contrast to little nuclear genome divergence, in two closely related balsam poplars, Populus balsamifera and Populus trichocarpa (Populus balsamifera ssp. trichocarpa). Previous studies with nuclear loci indicate that the two species (or subspecies) diverged since the late Pleistocene, whereas their plastomes indicate deep divergence, dating to at least the Pliocene (6-7 Myr ago). Our finding is in marked contrast to the estimated Pleistocene divergence of the nuclear genomes, previously calculated at 75 000 yr ago, suggesting plastid capture from a 'ghost lineage' of a now-extinct North American poplar.

Geographical and environmental gradients shape phenotypic trait variation and genetic structure in Populus trichocarpa.

• Populus trichocarpa is widespread across western North America spanning extensive variation in photoperiod, growing season and climate. We investigated trait variation in P. trichocarpa using over 2000 trees from a common garden at Vancouver, Canada, representing replicate plantings of 461 genotypes originating from 136 provenance localities. • We measured 40 traits encompassing phenological events, biomass accumulation, growth rates, and leaf, isotope and gas exchange-based ecophysiology traits. With replicated plantings and 29,354 single nucleotide polymorphisms (SNPs) from 3518 genes, we estimated both broad-sense trait heritability (H(2)) and overall population genetic structure from principal component analysis. • Populus trichocarpa had high phenotypic variation and moderate/high H(2) for many traits. H(2) ranged from 0.3 to 0.9 in phenology, 0.3 to 0.8 in biomass and 0.1 to 0.8 in ecophysiology traits. Most traits correlated strongly with latitude, maximum daylength and temperature of tree origin, but not necessarily with elevation, precipitation or heat : moisture indices. Trait H(2) values reflected trait correlation strength with geoclimate variables. The population genetic structure had one significant principal component (PC1) which correlated with daylength and showed enrichment for genes relating to circadian rhythm and photoperiod. • Robust relationships between traits, population structure and geoclimate in P. trichocarpa reflect patterns which suggest that range-wide geographical and environment gradients have shaped its genotypic and phenotypic variability.

Genome-wide association implicates numerous genes underlying ecological trait variation in natural populations of Populus trichocarpa.

In order to uncover the genetic basis of phenotypic trait variation, we used 448 unrelated wild accessions of black cottonwood (Populus trichocarpa) from much of its range in western North America. Extensive data from large-scale trait phenotyping (with spatial and temporal replications within a common garden) and genotyping (with a 34 K Populus single nucleotide polymorphism (SNP) array) of all accessions were used for gene discovery in a genome-wide association study (GWAS). We performed GWAS with 40 biomass, ecophysiology and phenology traits and 29,355 filtered SNPs representing 3518 genes. The association analyses were carried out using a Unified Mixed Model accounting for population structure effects among accessions. We uncovered 410 significant SNPs using a Bonferroni-corrected threshold (P<1.7×10(-6)). Markers were found across 19 chromosomes, explained 1-13% of trait variation, and implicated 275 unique genes in trait associations. Phenology had the largest number of associated genes (240 genes), followed by biomass (53 genes) and ecophysiology traits (25 genes). The GWAS results propose numerous loci for further investigation. Many traits had significant associations with multiple genes, underscoring their genetic complexity. Genes were also identified with multiple trait associations within and/or across trait categories. In some cases, traits were genetically correlated while in others they were not.

Phylogenetic pinpointing of a paleopolyploidy event within the flax genus (Linum) using transcriptomics.

BACKGROUND AND AIMS: Cultivated flax (Linum usitatissimum) is known to have undergone a whole-genome duplication around 5-9 million years ago. The aim of this study was to investigate whether other whole-genome duplication events have occurred in the evolutionary history of cultivated flax. Knowledge of such whole-genome duplications will be important in understanding the biology and genomics of cultivated flax. METHODS: Transcriptomes of 11 Linum species were sequenced using the Illumina platform. The short reads were assembled de novo and the DupPipe pipeline was used to look for signatures of polyploidy events from the age distribution of paralogues. In addition, phylogenies of all paralogues were assembled within an estimated age window of interest. These phylogenies were assessed for evidence of a paleopolyploidy event within the genus Linum. KEY RESULTS: A previously unknown paleopolyploidy event that occurred 20-40 million years ago was discovered and shown to be specific to a clade within Linum containing cultivated flax (L. usitatissimum) and other mainly blue-flowered species. The finding was supported by two lines of evidence. First, a significant change of slope (peak) was shown in the age distribution of paralogues that was phylogenetically restricted to, and ubiquitous in, this clade. Second, a large number of paralogue phylogenies were retrieved that are consistent with a polyploidy event occurring within that clade. CONCLUSIONS: The results show the utility of multi-species transcriptomics for detecting whole-genome duplication events and demonstrate that that multiple rounds of polyploidy have been important in shaping the evolutionary history of flax. Understanding and characterizing these whole-genome duplication events will be important for future Linum research.

Fungal endophytes can modulate plant invasion.

Symbiotic organisms may contribute to a host plant's success or failure to grow, its ability to maintain viable populations, and potentially, its probability of establishment and spread outside its native range. Intercellular and intracellular microbial symbionts that are asymptomatic in their plant host during some or all of their life cycle - endophytes - can form mutualistic, commensal, or pathogenic relationships, and sometimes novel associations with alien plants. Fungal endophytes are likely the most common endosymbiont infecting plants, with life-history, morphological, physiological, and plant-symbiotic traits that are distinct from other endophytic guilds. Here, we review the community dynamics of fungal endophytes during the process of plant invasion, and how their functional role may shift during the different stages of invasion: transport, introduction (colonisation), establishment, and spread. Each invasion stage presents distinct ecological filters that an alien plant must overcome to advance to the subsequent stage of invasion. Endophytes can alternately aid the host in overcoming stage-specific filters, or contribute to the barriers imposed by filters (e.g. biotic resistance), thereby affecting invasion pathways. A few fungi can be transported as seed endophytes from their native range and be vertically transmitted to future generations in the non-native range, especially in graminoids. In other plant groups, alien plants mostly acquire endophytes via horizontal transmission from the invaded plant community, and the host endophyte community is shaped by host filtering and biogeographic factors (e.g. dispersal limitation, environmental filtering). Endophytes infecting alien plants (both those transported with their host and those accumulated in the non-native range) may influence invasion success by affecting plant growth, reproduction, environmental tolerance, and pathogen and herbivory defences; however, the direction and magnitude of these effects can be contingent upon the host identity, life stage, ecological conditions, and invasion stage. This context dependence may cause endophytic fungi to shift to a non-endophytic (e.g. pathogenic) functional life stage in the same or different hosts, which can modify alien-native plant community dynamics. We conclude by identifying paths in which alien hosts can exploit the context dependency of endophyte function in novel abiotic and biotic conditions and at the different stages of invasion.

The developing xylem transcriptome and genome-wide analysis of alternative splicing in Populus trichocarpa (black cottonwood) populations.

BACKGROUND: Alternative splicing (AS) of genes is an efficient means of generating variation in protein structure and function. AS variation has been observed between tissues, cell types, and different treatments in non-woody plants such as Arabidopsis thaliana (Arabidopsis) and rice. However, little is known about AS patterns in wood-forming tissues and how much AS variation exists within plant populations. RESULTS: Here we used high-throughput RNA sequencing to analyze the Populus trichocarpa (P. trichocarpa) xylem transcriptome in 20 individuals from different populations across much of its range in western North America. Deep transcriptome sequencing and mapping of reads to the P. trichocarpa reference genome identified a suite of xylem-expressed genes common to all accessions. Our analysis suggests that at least 36% of the xylem-expressed genes in P. trichocarpa are alternatively spliced. Extensive AS was observed in cell-wall biosynthesis related genes such as glycosyl transferases and C2H2 transcription factors. 27902 AS events were documented and most of these events were not conserved across individuals. Differences in isoform-specific read densities indicated that 7% and 13% of AS events showed significant differences between individuals within geographically separated southern and northern populations, a level that is in general agreement with AS variation in human populations. CONCLUSIONS: This genome-wide analysis of alternative splicing reveals high levels of AS in P. trichocarpa and extensive inter-individual AS variation. We provide the most comprehensive analysis of AS in P. trichocarpa to date, which will serve as a valuable resource for the plant community to study transcriptome complexity and AS regulation during wood formation.

Genome-wide association mapping for wood characteristics in Populus identifies an array of candidate single nucleotide polymorphisms.

Establishing links between phenotypes and molecular variants is of central importance to accelerate genetic improvement of economically important plant species. Our work represents the first genome-wide association study to the inherently complex and currently poorly understood genetic architecture of industrially relevant wood traits. Here, we employed an Illumina Infinium 34K single nucleotide polymorphism (SNP) genotyping array that generated 29,233 high-quality SNPs in c. 3500 broad-based candidate genes within a population of 334 unrelated Populus trichocarpa individuals to establish genome-wide associations. The analysis revealed 141 significant SNPs (α ≤ 0.05) associated with 16 wood chemistry/ultrastructure traits, individually explaining 3-7% of the phenotypic variance. A large set of associations (41% of all hits) occurred in candidate genes preselected for their suggested a priori involvement with secondary growth. For example, an allelic variant in the FRA8 ortholog explained 21% of the total genetic variance in fiber length, when the trait's heritability estimate was considered. The remaining associations identified SNPs in genes not previously implicated in wood or secondary wall formation. Our findings provide unique insights into wood trait architecture and support efforts for population improvement based on desirable allelic variants.

The correct name for an Aquilegia (Ranunculaceae) hybrid of the parentage Aquilegiaflavescens × A.formosa.

Aquilegia×miniana (J.F.Macbr. & Payson) Cronk, hybr. & stat. nov. is the correct name for the hybrid Aquilegiaflavescens S.Watson × A.formosaFisch. & DC.var.formosa. In 1916 Payson and Macbride, while exploring the mountains of Idaho, found populations of Aquilegia that were pink in flower colour and appeared intermediate between the yellow-flowered A.flavescens and red-flowered A.formosa. They named these plants A.flavescensvar.miniana J.F.Macbr. & Payson. There has been uncertainty over whether their type collections (in GH, RM, MO, US, E, CM, CAS, NY) do indeed represent hybrids or pink-flowered morphs of A.flavescens. Using a Wells diagram, the holotype (in the Gray Herbarium of Harvard University) is shown to be intermediate, allowing its identification as a clear hybrid. However, some of the isotype material is indistinguishable from A.flavescens. The holotype matches material from British Columbia that has been determined to be of hybrid origin using molecular and morphological data. A.flavescensvar.miniana J.F.Macbr. & Payson is, therefore, an available name for the hybrid, which is here raised to the status of hybrid binomial.

Report of two distinct ribotypes in ITS sequences of Phalarisarundinacea (Poaceae) in western Canada and Alaska.

Background: Phalarisarundinacea L. (reed canary grass) is a widely occurring grass throughout the Northern Hemisphere. In North America, it is thought to consist of introduced agricultural forms from Europe as well as native populations. New information: During a survey of Phalarisarundinacea in western Canada, we discovered two distinct ribotypes in the sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA: one full length (ITS-long) and one with a seven base pair deletion (ITS-short). In addition, ITS-long plants have fixed heterozygosity indicating possible polyploidy. Phylogenetic analysis reveals that ITS-short is a unique ribotype that characterises an intraspecific clade. We designed an efficient PCR-based assay that allows sizing of a 238/245 base pair fragment in a capillary sequencer. This approach provides a novel marker that could be useful in future surveys of Phalarisarundinacea.