CAM evolution is associated with gene family expansion in an explosive bromeliad radiation.

The subgenus Tillandsia (Bromeliaceae) belongs to one of the fastest radiating clades in the plant kingdom and is characterized by the repeated evolution of Crassulacean acid metabolism (CAM). Despite its complex genetic basis, this water-conserving trait has evolved independently across many plant families and is regarded as a key innovation trait and driver of ecological diversification in Bromeliaceae. By producing high-quality genome assemblies of a Tillandsia species pair displaying divergent photosynthetic phenotypes, and combining genome-wide investigations of synteny, transposable element (TE) dynamics, sequence evolution, gene family evolution, and temporal differential expression, we were able to pinpoint the genomic drivers of CAM evolution in Tillandsia. Several large-scale rearrangements associated with karyotype changes between the 2 genomes and a highly dynamic TE landscape shaped the genomes of Tillandsia. However, our analyses show that rewiring of photosynthetic metabolism is mainly obtained through regulatory evolution rather than coding sequence evolution, as CAM-related genes are differentially expressed across a 24-h cycle between the 2 species but are not candidates of positive selection. Gene orthology analyses reveal that CAM-related gene families manifesting differential expression underwent accelerated gene family expansion in the constitutive CAM species, further supporting the view of gene family evolution as a driver of CAM evolution.

Drivers of genomic landscapes of differentiation across a Populus divergence gradient.

Speciation, the continuous process by which new species form, is often investigated by looking at the variation of nucleotide diversity and differentiation across the genome (hereafter genomic landscapes). A key challenge lies in how to determine the main evolutionary forces at play shaping these patterns. One promising strategy, albeit little used to date, is to comparatively investigate these genomic landscapes as progression through time by using a series of species pairs along a divergence gradient. Here, we resequenced 201 whole-genomes from eight closely related Populus species, with pairs of species at different stages along the divergence gradient to learn more about speciation processes. Using population structure and ancestry analyses, we document extensive introgression between some species pairs, especially those with parapatric distributions. We further investigate genomic landscapes, focusing on within-species (i.e. nucleotide diversity and recombination rate) and among-species (i.e. relative and absolute divergence) summary statistics of diversity and divergence. We observe relatively conserved patterns of genomic divergence across species pairs. Independent of the stage across the divergence gradient, we find support for signatures of linked selection (i.e. the interaction between natural selection and genetic linkage) in shaping these genomic landscapes, along with gene flow and standing genetic variation. We highlight the importance of investigating genomic patterns on multiple species across a divergence gradient and discuss prospects to better understand the evolutionary forces shaping the genomic landscapes of diversity and differentiation.

Taxon-specific or universal? Using target capture to study the evolutionary history of rapid radiations.

Target capture has emerged as an important tool for phylogenetics and population genetics in nonmodel taxa. Whereas developing taxon-specific capture probes requires sustained efforts, available universal kits may have a lower power to reconstruct relationships at shallow phylogenetic scales and within rapidly radiating clades. We present here a newly developed target capture set for Bromeliaceae, a large and ecologically diverse plant family with highly variable diversification rates. The set targets 1776 coding regions, including genes putatively involved in key innovations, with the aim to empower testing of a wide range of evolutionary hypotheses. We compare the relative power of this taxon-specific set, Bromeliad1776, to the universal Angiosperms353 kit. The taxon-specific set results in higher enrichment success across the entire family; however, the overall performance of both kits to reconstruct phylogenetic trees is relatively comparable, highlighting the vast potential of universal kits for resolving evolutionary relationships. For more detailed phylogenetic or population genetic analyses, for example the exploration of gene tree concordance, nucleotide diversity or population structure, the taxon-specific capture set presents clear benefits. We discuss the potential lessons that this comparative study provides for future phylogenetic and population genetic investigations, in particular for the study of evolutionary radiations.

La captura selectiva de secuencias de ADN ha surgido como una herramienta importante para la filogenética y la genética de poblaciones en taxones no-modelo. Mientras que el desarrollo de sondas de captura específicas para cada taxón requiere un esfuerzo sostenido, las colecciones de sondas universales disponibles pueden tener una potencia disminuida para la reconstrucción de relaciones filogenéticas poco profundas o de radiaciones rápidas. Presentamos aquí un conjunto de sondas para la captura selectiva desarrollado recientemente para Bromeliaceae, una familia de plantas extensa, ecológicamente diversa y con tasas de diversificación muy variables. El conjunto de sondas se centra en 1776 regiones de codificación, incluyendo genes supuestamente implicados en rasgos de innovación clave, con el objetivo de potenciar la comprobación de una amplia gama de hipótesis evolutivas. Comparamos la potencia relativa de este conjunto de sondas diseñado para un taxón específico, Bromeliad1776, con la colección universal Angiosperms353. El conjunto específico da lugar a un mayor éxito de captura en toda la familia. Sin embargo, el rendimiento global de ambos kits para reconstruir árboles filogenéticos es relativamente comparable, lo que pone de manifiesto el gran potencial de los kits universales para resolver las relaciones evolutivas. Para análisis filogenéticos o de genética de poblaciones más detallados, como por ejemplo la exploración de la congruencia de los árboles de genes, la diversidad de nucleótidos o la estructura de la población, el conjunto de captura específico para Bromeliaceae presenta claras ventajas. Discutimos las lecciones potenciales que este estudio comparativo proporciona para futuras investigaciones filogenéticas y de genética de poblaciones, en particular para el estudio de las radiaciones evolutivas.

Spatial and Ecological Drivers of Genetic Structure in Greek Populations of Alkanna tinctoria (Boraginaceae), a Polyploid Medicinal Herb.

Background and Aims: Quantifying genetic variation is fundamental to understand a species' demographic trajectory and its ability to adapt to future changes. In comparison with diploids, however, genetic variation and factors fostering genetic divergence remain poorly studied in polyploids due to analytical challenges. Here, by employing a ploidy-aware framework, we investigated the genetic structure and its determinants in polyploid Alkanna tinctoria (Boraginaceae), an ancient medicinal herb that is the source of bioactive compounds known as alkannin and shikonin (A/S). From a practical perspective, such investigation can inform biodiversity management strategies. Methods: We collected 14 populations of A. tinctoria within its main distribution range in Greece and genotyped them using restriction site-associated DNA sequencing. In addition, we included two populations of A. sieberi. By using a ploidy-aware genotype calling based on likelihoods, we generated a dataset of 16,107 high-quality SNPs. Classical and model-based analysis was done to characterize the genetic structure within and between the sampled populations, complemented by genome size measurements and chromosomal counts. Finally, to reveal the drivers of genetic structure, we searched for associations between allele frequencies and spatial and climatic variables. Key Results: We found support for a marked regional structure in A. tinctoria along a latitudinal gradient in line with phytogeographic divisions. Several analyses identified interspecific admixture affecting both mainland and island populations. Modeling of spatial and climatic variables further demonstrated a larger contribution of neutral processes and a lesser albeit significant role of selection in shaping the observed genetic structure in A. tinctoria. Conclusion: Current findings provide evidence of strong genetic structure in A. tinctoria mainly driven by neutral processes. The revealed natural genomic variation in Greek Alkanna can be used to further predict variation in A/S production, whereas our bioinformatics approach should prove useful for the study of other non-model polyploid species.

Adaptive Introgression Facilitates Adaptation to High Latitudes in European Aspen (Populus tremula L.).

Understanding local adaptation has become a key research area given the ongoing climate challenge and the concomitant requirement to conserve genetic resources. Perennial plants, such as forest trees, are good models to study local adaptation given their wide geographic distribution, largely outcrossing mating systems, and demographic histories. We evaluated signatures of local adaptation in European aspen (Populus tremula) across Europe by means of whole-genome resequencing of a collection of 411 individual trees. We dissected admixture patterns between aspen lineages and observed a strong genomic mosaicism in Scandinavian trees, evidencing different colonization trajectories into the peninsula from Russia, Central and Western Europe. As a consequence of the secondary contacts between populations after the last glacial maximum, we detected an adaptive introgression event in a genome region of ∼500 kb in chromosome 10, harboring a large-effect locus that has previously been shown to contribute to adaptation to the short growing seasons characteristic of Northern Scandinavia. Demographic simulations and ancestry inference suggest an Eastern origin-probably Russian-of the adaptive Nordic allele which nowadays is present in a homozygous state at the north of Scandinavia. The strength of introgression and positive selection signatures in this region is a unique feature in the genome. Furthermore, we detected signals of balancing selection, shared across regional populations, that highlight the importance of standing variation as a primary source of alleles that facilitate local adaptation. Our results, therefore, emphasize the importance of migration-selection balance underlying the genetic architecture of key adaptive quantitative traits.

Evolution of strong reproductive isolation in plants: broad-scale patterns and lessons from a perennial model group.

Many recent studies have addressed the mechanisms operating during the early stages of speciation, but surprisingly few studies have tested theoretical predictions on the evolution of strong reproductive isolation (RI). To help address this gap, we first undertook a quantitative review of the hybrid zone literature for flowering plants in relation to reproductive barriers. Then, using Populus as an exemplary model group, we analysed genome-wide variation for phylogenetic tree topologies in both early- and late-stage speciation taxa to determine how these patterns may be related to the genomic architecture of RI. Our plant literature survey revealed variation in barrier complexity and an association between barrier number and introgressive gene flow. Focusing on Populus, our genome-wide analysis of tree topologies in speciating poplar taxa points to unusually complex genomic architectures of RI, consistent with earlier genome-wide association studies. These architectures appear to facilitate the 'escape' of introgressed genome segments from polygenic barriers even with strong RI, thus affecting their relationships with recombination rates. Placed within the context of the broader literature, our data illustrate how phylogenomic approaches hold great promise for addressing the evolution and temporary breakdown of RI during late stages of speciation. This article is part of the theme issue 'Towards the completion of speciation: the evolution of reproductive isolation beyond the first barriers'.

Genomic footprints of repeated evolution of CAM photosynthesis in a Neotropical species radiation.

The adaptive radiation of Bromeliaceae (pineapple family) is one of the most diverse among Neotropical flowering plants. Diversification in this group was facilitated by shifts in several adaptive traits or "key innovations" including the transition from C3 to CAM photosynthesis associated with xeric (heat/drought) adaptation. We used phylogenomic approaches, complemented by differential gene expression (RNA-seq) and targeted metabolite profiling, to address the mechanisms of C3 /CAM evolution in the extremely species-rich bromeliad genus, Tillandsia, and related taxa. Evolutionary analyses of whole-genome sequencing and RNA-seq data suggest that evolution of CAM is associated with coincident changes to different pathways mediating xeric adaptation in this group. At the molecular level, C3 /CAM shifts were accompanied by gene expansion of XAP5 CIRCADIAN TIMEKEEPER homologs, a regulator involved in sugar- and light-dependent regulation of growth and development. Our analyses also support the re-programming of abscisic acid-related gene expression via differential expression of ABF2/ABF3 transcription factor homologs, and adaptive sequence evolution of an ENO2/LOS2 enolase homolog, effectively tying carbohydrate flux to abscisic acid-mediated abiotic stress response. By pinpointing different regulators of overlapping molecular responses, our results suggest plausible mechanistic explanations for the repeated evolution of correlated adaptive traits seen in a textbook example of an adaptive radiation.

Phenotypic expression of floral traits in hybrid zones provides insights into their genetic architecture.

Information on the genetic architecture of phenotypic traits is helpful for constructing and testing models of the ecoevolutionary dynamics of natural populations. For plant groups with long life cycles there is a lack of line cross experiments that can unravel the genetic architecture of loci underlying quantitative traits. To fill this gap, we propose the use of variation for phenotypic traits expressed in natural hybrid zones as an alternative approach. We used data from orchid hybrid zones and compared expected and observed patterns of phenotypic trait expression in different early-generation hybrid classes identified by molecular genetic markers. We found evidence of additivity, dominance, and epistatic interactions for different phenotypic traits. We discuss the potential of this approach along with its limitations and suggest that it may represent a realistic way to gain an initial insight into the heritability and genomic architecture of traits in organismal groups with complex life history, such as orchids and many others.

The low-copy nuclear gene Agt1 as a novel DNA barcoding marker for Bromeliaceae.

BACKGROUND: The angiosperm family Bromeliaceae comprises over 3.500 species characterized by exceptionally high morphological and ecological diversity, but a very low genetic variation. In many genera, plants are vegetatively very similar which makes determination of non flowering bromeliads difficult. This is particularly problematic with living collections where plants are often cultivated over decades without flowering. DNA barcoding is therefore a very promising approach to provide reliable and convenient assistance in species determination. However, the observed low genetic variation of canonical barcoding markers in bromeliads causes problems. RESULT: In this study the low-copy nuclear gene Agt1 is identified as a novel DNA barcoding marker suitable for molecular identification of closely related bromeliad species. Combining a comparatively slowly evolving exon sequence with an adjacent, genetically highly variable intron, correctly matching MegaBLAST based species identification rate was found to be approximately double the highest rate yet reported for bromeliads using other barcode markers. CONCLUSION: In the present work, we characterize Agt1 as a novel plant DNA barcoding marker to be used for barcoding of bromeliads, a plant group with low genetic variation. Moreover, we provide a comprehensive marker sequence dataset for further use in the bromeliad research community.

Estimating and accounting for genotyping errors in RAD-seq experiments.

In non-model organisms, evolutionary questions are frequently addressed using reduced representation sequencing techniques due to their low cost, ease of use, and because they do not require genomic resources such as a reference genome. However, evidence is accumulating that such techniques may be affected by specific biases, questioning the accuracy of obtained genotypes, and as a consequence, their usefulness in evolutionary studies. Here, we introduce three strategies to estimate genotyping error rates from such data: through the comparison to high quality genotypes obtained with a different technique, from individual replicates, or from a population sample when assuming Hardy-Weinberg equilibrium. Applying these strategies to data obtained with Restriction site Associated DNA sequencing (RAD-seq), arguably the most popular reduced representation sequencing technique, revealed per-allele genotyping error rates that were much higher than sequencing error rates, particularly at heterozygous sites that were wrongly inferred as homozygous. As we exemplify through the inference of genome-wide and local ancestry of well characterized hybrids of two Eurasian poplar (Populus) species, such high error rates may lead to wrong biological conclusions. By properly accounting for these error rates in downstream analyses, either by incorporating genotyping errors directly or by recalibrating genotype likelihoods, we were nevertheless able to use the RAD-seq data to support biologically meaningful and robust inferences of ancestry among Populus hybrids. Based on these findings, we strongly recommend carefully assessing genotyping error rates in reduced representation sequencing experiments, and to properly account for these in downstream analyses, for instance using the tools presented here.

Development of novel microsatellite markers for Alkanna tinctoria by comparative transcriptomics.

PREMISE: Alkanna tinctoria (Boraginaceae) is an important medicinal herb with its main distribution across the Mediterranean region. To reveal its genetic variation and population structure, microsatellite markers were developed and validated in four Greek populations. METHODS AND RESULTS: RNA-Seq data of the related species Arnebia euchroma and Echium plantagineum were assembled and mined to identify conserved ortholog sets containing simple sequence repeat motifs. Fifty potential loci were identified and then tested on A. tinctoria, of which 17 loci were polymorphic. The number of alleles ranged from one to nine, and the levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and 0.000 to 0.820, respectively. Most of these loci could be successfully amplified in eight other species of Boraginaceae (Alkanna graeca, A. hellenica, A. sfikasiana, Echium vulgare, E. plantagineum, Lithospermum officinale, Borago officinalis, and Anchusa officinalis). CONCLUSIONS: This study provides the first set of microsatellite loci for studying the genetic variation and population structure of A. tinctoria and shows their potential usefulness in other Boraginaceae species.

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a 'one-step operation'. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513 Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars 'Smooth Cayenne' and 'Queen' exhibited ancient and recent admixture, while 'Singapore Spanish' supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.

Mating system variation in hybrid zones: facilitation, barriers and asymmetries to gene flow.

Plant mating systems play a key role in structuring genetic variation both within and between species. In hybrid zones, the outcomes and dynamics of hybridization are usually interpreted as the balance between gene flow and selection against hybrids. Yet, mating systems can introduce selective forces that alter these expectations; with diverse outcomes for the level and direction of gene flow depending on variation in outcrossing and whether the mating systems of the species pair are the same or divergent. We present a survey of hybridization in 133 species pairs from 41 plant families and examine how patterns of hybridization vary with mating system. We examine if hybrid zone mode, level of gene flow, asymmetries in gene flow and the frequency of reproductive isolating barriers vary in relation to mating system/s of the species pair. We combine these results with a simulation model and examples from the literature to address two general themes: (1) the two-way interaction between introgression and the evolution of reproductive systems, and (2) how mating system can facilitate or restrict interspecific gene flow. We conclude that examining mating system with hybridization provides unique opportunities to understand divergence and the processes underlying reproductive isolation.

Targeted Capture of Hundreds of Nuclear Genes Unravels Phylogenetic Relationships of the Diverse Neotropical Palm Tribe Geonomateae.

The tribe Geonomateae is a widely distributed group of 103 species of Neotropical palms which contains six ecologically important understory or subcanopy genera. Although it has been the focus of many studies, our understanding of the evolutionary history of this group, and in particular of the taxonomically complex genus Geonoma, is far from complete due to a lack of molecular data. Specifically, the previous Sanger sequencing-based studies used a few informative characters and partial sampling. To overcome these limitations, we used a recently developed Arecaceae-specific target capture bait set to undertake a phylogenomic analysis of the tribe Geonomateae. We sequenced 3,988 genomic regions for 85% of the species of the tribe, including 84% of the species of the largest genus, Geonoma. Phylogenetic relationships were inferred using both concatenation and coalescent methods. Overall, our phylogenetic tree is highly supported and congruent with taxonomic delimitations although several morphological taxa were revealed to be non-monophyletic. It is the first time that such a large genomic dataset is provided for an entire tribe within the Arecaceae. Our study lays the groundwork not only for detailed macro- and micro-evolutionary studies within the group, but also sets a workflow for understanding other species complexes across the tree of life.

Admixture mapping in interspecific Populus hybrids identifies classes of genomic architectures for phytochemical, morphological and growth traits.

The genomic architecture of functionally important traits is key to understanding the maintenance of reproductive barriers and trait differences when divergent populations or species hybridize. We conducted a genome-wide association study (GWAS) to study trait architecture in natural hybrids of two ecologically divergent Populus species. We genotyped 472 seedlings from a natural hybrid zone of Populus alba and Populus tremula for genome-wide markers from reduced representation sequencing, phenotyped the plants in common gardens for 46 phytochemical (phenylpropanoid), morphological and growth traits, and used a Bayesian polygenic model for mapping. We detected three classes of genomic architectures: traits with finite, detectable associations of genetic loci with phenotypic variation in addition to highly polygenic heritability; traits with indications for polygenic heritability only; and traits with no detectable heritability. For the first class, we identified genome regions with plausible candidate genes for phenylpropanoid biosynthesis or its regulation, including MYB transcription factors and glycosyl transferases. GWAS in natural, recombinant hybrids represent a promising step towards resolving the genomic architecture of phenotypic traits in long-lived species. This facilitates the fine-mapping and subsequent functional characterization of genes and networks causing differences in hybrid performance and fitness.

A dedicated target capture approach reveals variable genetic markers across micro- and macro-evolutionary time scales in palms.

Understanding the genetics of biological diversification across micro- and macro-evolutionary time scales is a vibrant field of research for molecular ecologists as rapid advances in sequencing technologies promise to overcome former limitations. In palms, an emblematic, economically and ecologically important plant family with high diversity in the tropics, studies of diversification at the population and species levels are still hampered by a lack of genomic markers suitable for the genotyping of large numbers of recently diverged taxa. To fill this gap, we used a whole genome sequencing approach to develop target sequencing for molecular markers in 4,184 genome regions, including 4,051 genes and 133 non-genic putatively neutral regions. These markers were chosen to cover a wide range of evolutionary rates allowing future studies at the family, genus, species and population levels. Special emphasis was given to the avoidance of copy number variation during marker selection. In addition, a set of 149 well-known sequence regions previously used as phylogenetic markers by the palm biological research community were included in the target regions, to open the possibility to combine and jointly analyse already available data sets with genomic data to be produced with this new toolkit. The bait set was effective for species belonging to all three palm sub-families tested (Arecoideae, Ceroxyloideae and Coryphoideae), with high mapping rates, specificity and efficiency. The number of high-quality single nucleotide polymorphisms (SNPs) detected at both the sub-family and population levels facilitates efficient analyses of genomic diversity across micro- and macro-evolutionary time scales.

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.