Genomics of hybrid parallel origin in Aquilegia ecalcarata.

BACKGROUND: The parallel evolution of similar traits or species provides strong evidence for the role of natural selection in evolution. Traits or species that evolved repeatedly can be driven by separate de novo mutations or interspecific gene flow. Although parallel evolution has been reported in many studies, documented cases of parallel evolution caused by gene flow are scarce by comparison. Aquilegia ecalcarata and A. kansuensis belong to the genus of Aquilegia, and are the closest related sister species. Mutiple origins of A. ecalcarata have been reported in previous studies, but whether they have been driven by separate de novo mutations or gene flow remains unclear. RESULTS: In this study, We conducted genomic analysis from 158 individuals of two repeatedly evolving pairs of A. ecalcarata and A. kansuensis. All samples were divided into two distinct clades with obvious geographical distribution based on phylogeny and population structure. Demographic modeling revealed that the origin of the A. ecalcarata in the Eastern of China was caused by gene flow, and the Eastern A. ecalcarata occurred following introgression from Western A. ecalcarata population. Analysis of Treemix and D-statistic also revealed that a strong signal of gene flow was detected from Western A. ecalcarata to Eastern A. ecalcarata. Genetic divergence and selective sweep analyses inferred parallel regions of genomic divergence and identified many candidate genes associated with ecologically adaptive divergence between species pair. Comparative analysis of parallel diverged regions and gene introgression confirms that gene flow contributed to the parallel evolution of A. ecalcarata. CONCLUSIONS: Our results further confirmed the multiple origins of A. ecalcarata and highlighted the roles of gene flow. These findings provide new evidence for parallel origin after hybridization as well as insights into the ecological adaptation mechanisms underlying the parallel origins of species.

Characterizing complete mitochondrial genome of Aquilegia amurensis and its evolutionary implications.

BACKGROUND: Aquilegia is a model system for studying the evolution of adaptive radiation. However, very few studies have been conducted on the Aquilegia mitochondrial genome. Since mitochondria play a key role in plant adaptation to abiotic stress, analyzing the mitochondrial genome may provide a new perspective for understanding adaptive evolution. RESULTS: The Aquilegia amurensis mitochondrial genome was characterized by a circular chromosome and two linear chromosomes, with a total length of 538,736 bp; the genes included 33 protein-coding genes, 24 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. We subsequently conducted a phylogenetic analysis based on single nucleotide polymorphisms (SNPs) in the mitochondrial genomes of 18 Aquilegia species, which were roughly divided into two clades: the European-Asian clade and the North American clade. Moreover, the genes mttB and rpl5 were shown to be positively selected in European-Asian species, and they may help European and Asian species adapt to environmental changes. CONCLUSIONS: In this study, we assembled and annotated the first mitochondrial genome of the adaptive evolution model plant Aquilegia. The subsequent analysis provided us with a basis for further molecular studies on Aquilegia mitochondrial genomes and valuable information on adaptive evolution in Aquilegia.

Time-Course Transcriptome Analysis of Aquilegia vulgaris Root Reveals the Cell Wall's Roles in Salinity Tolerance.

Salt stress has a considerable impact on the development and growth of plants. The soil is currently affected by salinisation, a problem that is becoming worse every year. This means that a significant amount of salt-tolerant plant material needs to be added. Aquilegia vulgaris has aesthetically pleasing leaves, unique flowers, and a remarkable tolerance to salt. In this study, RNA-seq technology was used to sequence and analyse the transcriptome of the root of Aquilegia vulgaris seedlings subjected to 200 mM NaCl treatment for 12, 24, and 48 h. In total, 12 Aquilegia vulgaris seedling root transcriptome libraries were constructed. At the three time points of salt treatment compared with the control, 3888, 1907, and 1479 differentially expressed genes (DEGs) were identified, respectively. Various families of transcription factors (TFs), mainly AP2, MYB, and bHLH, were identified and might be linked to salt tolerance. Gene Ontology (GO) analysis of DEGs revealed that the structure and composition of the cell wall and cytoskeleton may be crucial in the response to salt stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs showed a significant enrichment of the pentose and glucuronate interconversion pathway, which is associated with cell wall metabolism after 24 and 48 h of salt treatment. Based on GO and KEGG analyses of DEGs, the pentose and glucuronate interconversion pathway was selected for further investigation. AP2, MYB, and bHLH were found to be correlated with the functional genes in this pathway based on a correlation network. This study provides the groundwork for understanding the key pathways and gene networks in response to salt stress, thereby providing a theoretical basis for improving salt tolerance in Aquilegia vulgaris.

Starch and Sucrose Metabolism and Plant Hormone Signaling Pathways Play Crucial Roles in Aquilegia Salt Stress Adaption.

Salt stress is one of the main abiotic stresses that strongly affects plant growth. Clarifying the molecular regulatory mechanism in ornamental plants under salt stress is of great significance for the ecological development of saline soil areas. Aquilegia vulgaris is a perennial with a high ornamental and commercial value. To narrow down the key responsive pathways and regulatory genes, we analyzed the transcriptome of A. vulgaris under a 200 mM NaCl treatment. A total of 5600 differentially expressed genes were identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis pointed out that starch and sucrose metabolism and plant hormone signal transduction were significantly improved. The above pathways played crucial roles when A. vulgaris was coping with salt stress, and their protein-protein interactions (PPIs) were predicted. This research provides new insights into the molecular regulatory mechanism, which could be the theoretical basis for screening candidate genes in Aquilegia.

Characterization and expression analysis of bHLH transcription factors reveal their putative regulatory effects on nectar spur development in Aquilegia species.

Nectar spur is a hollow extension of certain flower parts and shows strikingly diverse size and shape in Aquilegia. Nectar spur development is involved in cell division and expansion processes. The basic helix-loop-helix (bHLH) transcription factors (TFs) control a diversity of organ morphogenesis, including cell division and cell expansion processes. However, the role of bHLH genes in nectar spur development in Aquilegia is mainly unknown. We conducted a genome-wide identification of the bHLH gene family in Aquilegia to determine structural characteristics and phylogenetic relationships, and to analyze expression profiles of these genes during the development of nectar spur in spurless and spurred species. A total of 120 AqbHLH genes were identified from the Aquilegia coerulea genome. The phylogenetic tree showed that AqbHLH proteins were divided into 15 subfamilies, among which S7 and S8 subfamilies occurred marked expansion. The AqbHLH genes in the same clade had similar motif composition and gene structure characteristics. Conserved residue analysis indicated nineteen residues with conservation of more than 50% were found in the four conserved regions. In the upstream sequence of AqbHLH genes, the light-responsive element was the most abundant cis-acting element. Eighteen AqbHLH genes showed syntenic relationships, and eight genes from four syntenic pairs underwent tandem duplications. According to the expression profiling analysis by public RNA-Seq data and qRT-PCR results, five AqbHLH genes, including AqbHLH027, AqbHLH046, AqbHLH082, AqbHLH083 and AqbHLH092, were differentially expressed between different tissues in A. coerulea at early developmental stages, as well as between spurless and spurred Aquilegia species. Of them, AqbHLH046 was not only highly expressed in spur compared with blade, but also showed higher expression levels in spurred species than spurless specie, suggesting it plays an essential role in the development of spur by regulating cell division. This study lays a foundation to investigate the function of AqbHLH genes family in nectar spur development, and has potential implications for speciation and genetic breeding in the genus Aquilegia.

Complex developmental and transcriptional dynamics underlie pollinator-driven evolutionary transitions in nectar spur morphology in Aquilegia (columbine).

PREMISE: Determining the developmental programs underlying morphological variation is key to elucidating the evolutionary processes that generated the stunning biodiversity of the angiosperms. Here, we characterized the developmental and transcriptional dynamics of the elaborate petal nectar spur of Aquilegia (columbine) in species with contrasting pollination syndromes and spur morphologies. METHODS: We collected petal epidermal cell number and length data across four Aquilegia species, two with short, curved nectar spurs of the bee-pollination syndrome and two with long, straight spurs of the hummingbird-pollination syndrome. We also performed RNA-seq on A. brevistyla (bee) and A. canadensis (hummingbird) distal and proximal spur compartments at multiple developmental stages. Finally, we intersected these data sets with a previous QTL mapping study on spur length and shape to identify new candidate loci. RESULTS: The differential growth between the proximal and distal surfaces of curved spurs is primarily driven by differential cell division. However, independent transitions to straight spurs in the hummingbird syndrome have evolved by increasing differential cell elongation between spur surfaces. The RNA-seq data reveal these tissues to be transcriptionally distinct and point to auxin signaling as being involved with the differential cell elongation responsible for the evolution of straight spurs. We identify several promising candidate genes for future study. CONCLUSIONS: Our study, taken together with previous work in Aquilegia, reveals the complexity of the developmental mechanisms underlying trait variation in this system. The framework we established here will lead to exciting future work examining candidate genes and processes involved in the rapid radiation of the genus.

Genetic architecture underlying variation in floral meristem termination in Aquilegia.

Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of an FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl number (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We explored potential candidate genes under each QTL and characterized novel expression patterns of select loci of interest using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated, and our results provide insight into how floral morphological diversity can be generated at the meristematic level.

Genetic and Epigenetic Signatures Associated with the Divergence of Aquilegia Species.

Widely grown in the Northern Hemisphere, the genus Aquilegia (columbine) is a model system in adaptive radiation research. While morphological variations between species have been associated with environmental factors, such as pollinators, how genetic and epigenetic factors are involved in the rapid divergence in this genus remains under investigated. In this study, we surveyed the genomes and DNA methylomes of ten Aquilegia species, representative of the Asian, European and North American lineages. Our analyses of the phylogeny and population structure revealed high genetic and DNA methylomic divergence across these three lineages. By multi-level genome-wide scanning, we identified candidate genes exhibiting lineage-specific genetic or epigenetic variation patterns that were signatures of inter-specific divergence. We demonstrated that these species-specific genetic variations and epigenetic variabilities are partially independent and are both functionally related to various biological processes vital to adaptation, including stress tolerance, cell reproduction and DNA repair. Our study provides an exploratory overview of how genetic and epigenetic signatures are associated with the diversification of the Aquilegia species.

Non-pollinator selection for a floral homeotic mutant conferring loss of nectar reward in Aquilegia coerulea.

Here, we describe a polymorphic population of Aquilegia coerulea with a naturally occurring floral homeotic mutant, A. coerulea var. daileyae, where the characteristic petals with nectar spurs are replaced with a second set of sepals. Although it would be expected that this loss of pollinator reward would be disadvantageous to the mutant, we find that it has reached relatively high frequency (∼25%) and is under strong, positive selection across multiple seasons (s = 0.17-0.3) primarily due to reduced floral herbivory. We identify the underlying locus (APETALA3-3) and multiple causal loss-of-function mutations indicating an ongoing soft sweep. Elevated linkage disequilibrium around the two most common causal alleles indicates that positive selection has been occurring for many generations. Lastly, genotypic frequencies at AqAP3-3 indicate a degree of positive assortative mating by morphology. Together, these data provide both a compelling example that large-scale discontinuous morphological changes differentiating taxa can occur due to single mutations and a particularly clear example of linking genotype, phenotype, and fitness.

Genetic architecture of floral traits in bee- and hummingbird-pollinated sister species of Aquilegia (columbine).

Interactions with animal pollinators have helped shape the stunning diversity of flower morphologies across the angiosperms. A common evolutionary consequence of these interactions is that some flowers have converged on suites of traits, or pollination syndromes, that attract and reward specific pollinator groups. Determining the genetic basis of these floral pollination syndromes can help us understand the processes that contributed to the diversification of the angiosperms. Here, we characterize the genetic architecture of a bee-to-hummingbird pollination shift in Aquilegia (columbine) using QTL mapping of 17 floral traits encompassing color, nectar composition, and organ morphology. In this system, we find that the genetic architectures underlying differences in floral color are quite complex, and we identify several likely candidate genes involved in anthocyanin and carotenoid floral pigmentation. Most morphological and nectar traits also have complex genetic underpinnings; however, one of the key floral morphological phenotypes, nectar spur curvature, is shaped by a single locus of large effect.

Conserved and non-conserved triggers of 24-nucleotide reproductive phasiRNAs in eudicots.

Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre-meiotic 21-nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non-canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a lineage-specific trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275-derived 24-nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24-nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24-nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs.

A reappraisal of the phylogenetic placement of the Aquilegia whole-genome duplication.

The accurate placement of an ancient whole-genome duplication (WGD) in relation to the lineage divergence is important. Here, we re-investigated the Aquilegia coerulea WGD and found it is more likely lineage-specific rather than shared by all eudicots.

Transcriptome profiling and weighted gene co-expression network analysis of early floral development in Aquilegia coerulea.

The earliest phases of floral development include a number of crucial processes that lay the foundation for the subsequent morphogenesis of floral organs and success in reproduction. Currently, key transcriptional changes during this developmental window have been characterized in the model species Arabidopsis thaliana, but little is known about how transcriptional dynamics change over the course of these developmental processes in other plant systems. Here, we have conducted the first in-depth transcriptome profiling of early floral development in Aquilegia at four finely dissected developmental stages, with eight biological replicates per stage. Using differential gene expression analysis and weighted gene co-expression network analysis, we identified both crucial genes whose expression changes mark the transitions between developmental stages and hub genes in co-expression modules. Our results support the potential functional conservation of key genes in early floral development that have been identified in other systems, but also reveal a number of previously unknown or overlooked loci that are worthy of further investigation. In addition, our results highlight not only the dynamics of transcriptional regulation during early floral development, but also the potential involvement of the complex, essential networks of small RNA and post-translational regulation to these developmental stages.

POPOVICH, encoding a C2H2 zinc-finger transcription factor, plays a central role in the development of a key innovation, floral nectar spurs, in Aquilegia.

The evolution of novel features, such as eyes or wings, that allow organisms to exploit their environment in new ways can lead to increased diversification rates. Therefore, understanding the genetic and developmental mechanisms involved in the origin of these key innovations has long been of interest to evolutionary biologists. In flowering plants, floral nectar spurs are a prime example of a key innovation, with the independent evolution of spurs associated with increased diversification rates in multiple angiosperm lineages due to their ability to promote reproductive isolation via pollinator specialization. As none of the traditional plant model taxa have nectar spurs, little is known about the genetic and developmental basis of this trait. Nectar spurs are a defining feature of the columbine genus Aquilegia (Ranunculaceae), a lineage that has experienced a relatively recent and rapid radiation. We use a combination of genetic mapping, gene expression analyses, and functional assays to identify a gene crucial for nectar spur development, POPOVICH (POP), which encodes a C2H2 zinc-finger transcription factor. POP plays a central role in regulating cell proliferation in the Aquilegia petal during the early phase (phase I) of spur development and also appears to be necessary for the subsequent development of nectaries. The identification of POP opens up numerous avenues for continued scientific exploration, including further elucidating of the genetic pathway of which it is a part, determining its role in the initial evolution of the Aquilegia nectar spur, and examining its potential role in the subsequent evolution of diverse spur morphologies across the genus.

Identification of the target genes of AqAPETALA3-3 (AqAP3-3) in Aquilegia coerulea (Ranunculaceae) helps understand the molecular bases of the conserved and nonconserved features of petals.

Identification and comparison of the conserved and variable downstream genes of floral organ identity regulators are critical to understanding the mechanisms underlying the commonalities and peculiarities of floral organs. Yet, because of the lack of studies in nonmodel species, a general picture of the regulatory evolution between floral organ identity genes and their targets is still lacking. Here, by conducting extensive chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), electrophoretic mobility shift assay and bioinformatic analyses, we identify and predict the target genes of a petal identity gene, AqAPETALA3-3 (AqAP3-3), in Aquilegia coerulea (Ranunculaceae) and compare them with those of its counterpart in Arabidopsis thaliana, AP3. In total, 7049 direct target genes are identified for AqAP3-3, of which 2394 are highly confident and 1085 are shared with AP3. Gene Ontology enrichment analyses further indicate that conserved targets are largely involved in the formation of identity-related features, whereas nonconserved targets are mostly required for the formation of species-specific features. These results not only help understand the molecular bases of the conserved and nonconserved features of petals, but also pave the way to studying the regulatory evolution between floral organ identity genes and their targets.

Developmental and molecular characterization of novel staminodes in Aquilegia.

BACKGROUND AND AIMS: The ranunculid model system Aquilegia is notable for the presence of a fifth type of floral organ, the staminode, which appears to be the result of sterilization and modification of the two innermost whorls of stamens. Previous studies have found that the genetic basis for the identity of this new organ is the result of sub- and neofunctionalization of floral organ identity gene paralogues; however, we do not know the extent of developmental and molecular divergence between stamens and staminodes. METHODS: We used histological techniques to describe the development of the Aquilegia coerulea 'Origami' staminode relative to the stamen filament. These results have been compared with four other Aquilegia species and the closely related genera Urophysa and Semiaquilegia. As a complement, RNA sequencing has been conducted at two developmental stages to investigate the molecular divergence of the stamen filaments and staminodes in A. coerulea 'Origami'. KEY RESULTS: Our developmental study has revealed novel features of staminode development, most notably a physical interaction along the lateral margin of adjacent organs that appears to mediate their adhesion. In addition, patterns of abaxial/adaxial differentiation are observed in staminodes but not stamen filaments, including asymmetric lignification of the adaxial epidermis in the staminodes. The comparative transcriptomics are consistent with the observed lignification of staminodes and indicate that stamen filaments are radialized due to overexpression of adaxial identity, while the staminodes are expanded due to the balanced presence of abaxial identity. CONCLUSIONS: These findings suggest a model in which the novel staminode identity programme interacts with the abaxial/adaxial identity pathways to produce two whorls of laterally expanded organs that are highly differentiated along their abaxial/adaxial axis. While the ecological function of Aquilegia staminodes remains to be determined, these data are consistent with a role in protecting the early carpels from herbivory and/or pathogens.

The Aquilegia genome reveals a hybrid origin of core eudicots.

BACKGROUND: Whole-genome duplications (WGDs) have dominated the evolutionary history of plants. One consequence of WGD is a dramatic restructuring of the genome as it undergoes diploidization, a process under which deletions and rearrangements of various sizes scramble the genetic material, leading to a repacking of the genome and eventual return to diploidy. Here, we investigate the history of WGD in the columbine genus Aquilegia, a basal eudicot, and use it to illuminate the origins of the core eudicots. RESULTS: Within-genome synteny confirms that columbines are ancient tetraploids, and comparison with the grape genome reveals that this tetraploidy appears to be shared with the core eudicots. Thus, the ancient gamma hexaploidy found in all core eudicots must have involved a two-step process: first, tetraploidy in the ancestry of all eudicots, then hexaploidy in the ancestry of core eudicots. Furthermore, the precise pattern of synteny sharing suggests that the latter involved allopolyploidization and that core eudicots thus have a hybrid origin. CONCLUSIONS: Novel analyses of synteny sharing together with the well-preserved structure of the columbine genome reveal that the gamma hexaploidy at the root of core eudicots is likely a result of hybridization between a tetraploid and a diploid species.

Developmental and Molecular Changes Underlying the Vernalization-Induced Transition to Flowering in Aquilegia coerulea (James).

Reproductive success in plants is dependent on many factors but the precise timing of flowering is certainly among the most crucial. Perennial plants often have a vernalization or over-wintering requirement in order to successfully flower in the spring. The shoot apical meristem undergoes drastic developmental and molecular changes as it transitions into inflorescence meristem (IM) identity, which then gives rise to floral meristems (FMs). In this study, we have examined the developmental and gene expression changes underlying the transition from the vegetative to reproductive phases in the basal eudicot Aquilegia coerulea, which has evolved a vernalization response independently relative to other established model systems. Results from both our histology and scanning electron studies demonstrate that developmental changes in the meristem occur gradually during the third and fourth weeks of vernalization. Based on RNAseq data and cluster analysis, several known flowering time loci, including AqFT and AqFL1, exhibit dramatic changes in expression during the fourth week. Further consideration of candidate gene homologs as well as unexpected loci of interest creates a framework in which we can begin to explore the genetic basis of the flowering time transition in Aquilegia.

Comparative transcriptomics of early petal development across four diverse species of Aquilegia reveal few genes consistently associated with nectar spur development.

BACKGROUND: Petal nectar spurs, which facilitate pollination through animal attraction and pollen placement, represent a key innovation promoting diversification in the genus Aquilegia (Ranunculaceae). Identifying the genetic components that contribute to the development of these three-dimensional structures will inform our understanding of the number and types of genetic changes that are involved in the evolution of novel traits. In a prior study, gene expression between two regions of developing petals, the laminar blade and the spur cup, was compared at two developmental stages in the horticultural variety A. coerulea 'Origami'. Several hundred genes were differentially expressed (DE) between the blade and spur at both developmental stages. In order to narrow in on a set of genes crucial to early spur formation, the current study uses RNA sequencing (RNAseq) to conduct comparative expression analyses of petals from five developmental stages between four Aquilegia species, three with morphologically variable nectar spurs, A. sibirica, A. formosa, and A. chrysantha, and one that lacks nectar spurs, A. ecalcarata. RESULTS: Petal morphology differed increasingly between taxa across the developmental stages assessed, with petals from all four taxa being indistinguishable pre-spur formation at developmental stage 1 (DS1) and highly differentiated by developmental stage 5 (DS5). In all four taxa, genes involved in mitosis were down-regulated over the course of the assessed developmental stages, however, many genes involved in mitotic processes remained expressed at higher levels later in development in the spurred taxa. A total of 690 genes were identified that were consistently DE between the spurred taxa and A. ecalcarata at all five developmental stages. By comparing these genes with those identified as DE between spur and blade tissue in A. coerulea 'Origami', a set of only 35 genes was identified that shows consistent DE between petal samples containing spur tissue versus those without spur tissue. CONCLUSIONS: The results of this study suggest that expression differences in very few loci are associated with the presence and absence of spurs. In general, it appears that the spurless petals of A. ecalcarata cease cell divisions and enter the cell differentiation phase at an earlier developmental time point than those that produce spurs. This much more tractable list of 35 candidates genes will greatly facilitate targeted functional studies to assess the genetic control and evolution of petal spurs in Aquilegia.