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.

Chloroplast markers for the Malvaceae and the plastome of Henderson's checkermallow (Sidalcea hendersonii S.Wats.), a rare plant from the Pacific Northwest.

OBJECTIVE: Sidalcea is a genus of flowering plants restricted to the west coast of North America, commonly known as checkermallows. Remarkably, of the ~ 30 recognized species, 16 are of conservation concern (vulnerable, imperilled or critically imperilled). To facilitate biological studies in this genus, and in the wider Malvaceae, we have sequenced the whole plastid genome of Sidalcea hendersonii. This will allow us both to check those regions already developed as general Malvaceae markers in a previous study, and to search for new regions. RESULTS: By comparing the Sidalcea genome to that of Althaea, we have identified a hypervariable circa 1 kb region in the short single copy region. This region shows promise for examining phylogeographic pattern, hybridization and haplotype diversity. Remarkably, considering the conservation of plastome architecture between Sidalcea and Althaea, the former has a 237 bp deletion in the otherwise highly conserved inverted repeat region. Newly designed primers provide a PCR assay to determine presence of this indel across the Malvaceae. Screening of previously designed chloroplast microsatellite markers indicates two markers with variation within S. hendersonii that would be useful in future population conservation genetics.

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.

Floral phenology of an Andean bellflower and pollination by buff-tailed sicklebill hummingbird.

The Andean bellflowers comprise an explosive radiation correlated with shifts to specialized pollination. One diverse clade has evolved with extremely curved floral tubes and is predicted to be pollinated exclusively by one of two parapatric species of sicklebill hummingbirds (Eutoxeres). In this study, we focused on the floral biology of Centropogon granulosus, a bellflower thought to be specialized for pollination by Eutoxeres condamini, in a montane cloud forest site in southeastern Peru. Using camera traps and a pollination exclusion experiment, we documented E. condamini as the sole pollinator of C. granulosus. Visitation by E. condamini was necessary for fruit development. Flowering rates were unequivocally linear and conformed to the "steady-state" phenological type. Over the course of >1800 h of monitoring, we recorded 12 E. condamini visits totaling 42 s, indicating traplining behavior. As predicted by its curved flowers, C. granulosus is exclusively pollinated by buff-tailed sicklebill within our study area. We present evidence for the congruence of phenology and visitation as a driver of specialization in this highly diverse clade of Andean bellflowers.

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).

A targeted sequence capture array for phylogenetics and population genomics in the Salicaceae.

PREMISE: The family Salicaceae has proved taxonomically challenging, especially in the genus Salix, which is speciose and features frequent hybridization and polyploidy. Past efforts to reconstruct the phylogeny with molecular barcodes have failed to resolve the species relationships of many sections of the genus. METHODS: We used the wealth of sequence data in the family to design sequence capture probes to target regions of 300-1200 bp of exonic regions of 972 genes. RESULTS: We recovered sequence data for nearly all of the targeted genes in three species of Populus and three species of Salix. We present a species tree, discuss concordance among gene trees, and present population genomic summary statistics for these loci. CONCLUSIONS: Our sequence capture array has extremely high capture efficiency within the genera Populus and Salix, resulting in abundant phylogenetic information. Additionally, these loci show promise for population genomic studies.

On the origin of orphan hybrids between Aquilegia formosa and Aquilegia flavescens.

We report the investigation of an Aquilegia flavescens × A. formosa population in British Columbia that is disjunct from its parents-the latter species is present locally but ecologically separated, while the former is entirely absent. To confirm hybridity, we used multivariate analysis of floral characters of field-sampled populations to ordinate phenotypes of putative hybrids in relation to those of the parental species. Microsatellite genotypes at 11 loci from 72 parental-type and putative hybrid individuals were analysed to assess evidence for admixture. Maternally inherited plastid sequences were analysed to infer the direction of hybridization and test hypotheses on the origin of the orphan hybrid population. Plants from the orphan hybrid population are on average intermediate between typical A. formosa and A. flavescens for most phenotypes examined and show evidence of genetic admixture. This population lies beyond the range of A. flavescens, but within the range of A. formosa. No pure A. flavescens individuals were observed in the vicinity, nor is this species known to occur within 200 km of the site. The hybrids share a plastid haplotype with local A. formosa populations. Alternative explanations for this pattern are evaluated. While we cannot rule out long-distance pollen dispersal followed by proliferation of hybrid genotypes, we consider the spread of an A. formosa plastid during genetic swamping of a historical A. flavescens population to be more parsimonious.

Author Correction: Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

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.

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.

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.

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.

Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus †.

The evolution of sexual dimorphism and expansion of sex chromosomes are both driven through sexual conflict, arising from differing fitness optima between males and females. Here, we pair work in poplar (Populus) describing one of the smallest sex-determining regions known thus far in complex eukaryotes (~100 kbp) with comprehensive tests for sexual dimorphism using >1300 individuals from two Populus species and assessing 96 non-reproductive functional traits. Against expectation, we found sexual homomorphism (no non-reproductive trait differences between the sexes), suggesting that gender is functionally neutral with respect to non-reproductive features that affect plant survival and fitness. Combined with a small sex-determining region, we infer that sexual conflict may be effectively stymied or non-existent within these taxa. Both sexual homomorphism and the small sex-determining region occur against a background of strong environmental selection and local adaptation in Populus. This presents a powerful hypothesis for the evolution of dioecious species. Here, we suggest that environmental selection may be sufficient to suppress and stymy sexual conflict if it acts orthogonal to sexual selection, thereby placing limitations on the evolution of sexual dimorphism and genomic expansion of sex chromosomes.

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.].

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.

Evolution of Catkins: Inflorescence Morphology of Selected Salicaceae in an Evolutionary and Developmental Context.

Poplars (Populus sp.) and willows (Salix sp.) are well known woody plants common throughout the northern hemisphere, both with fully sequenced genomes. They bear compact unisexual inflorescences known as "catkins." Closely related genera of the "salicoid clade" within the family Salicaceae include the Asian genera Bennettiodendron, Idesia, Itoa, Poliothyrsis, and Carrierea and the Central American genera Olmediella and Macrohasseltia. Like willow and poplar, most of these genera are dioecious, but unlike willow and poplar they generally have loosely branched panicles rather than catkins, and less highly reduced flowers. However, the early developing inflorescences of Carrierea and Idesia show similarities to catkins which suggest possible pathways by which the salicoid catkin may have evolved.

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.