Evolutionary paths to new phenotypes.

Ecological model systems inform on innovative traits in plants and animals.

Convergence in carnivorous pitcher plants reveals a mechanism for composite trait evolution.

Composite traits involve multiple components that, only when combined, gain a new synergistic function. Thus, how they evolve remains a puzzle. We combined field experiments, microscopy, chemical analyses, and laser Doppler vibrometry with comparative phylogenetic analyses to show that two carnivorous Nepenthes pitcher plant species independently evolved similar adaptations in three distinct traits to acquire a new, composite trapping mechanism. Comparative analyses suggest that this new trait arose convergently through "spontaneous coincidence" of the required trait combination, rather than directional selection in the component traits. Our results indicate a plausible mechanism for composite trait evolution and highlight the importance of stochastic phenotypic variation as a facilitator of evolutionary novelty.

Draft genome and transcriptome of Nepenthes mirabilis, a carnivorous plant in China.

OBJECTIVES: Nepenthes belongs to the monotypic family Nepenthaceae, one of the largest carnivorous plant families. Nepenthes species show impressive adaptive radiation and suffer from being overexploited in nature. Nepenthes mirabilis is the most widely distributed species and the only Nepenthes species that is naturally distributed within China. Herein, we reported the genome and transcriptome assemblies of N. mirabilis. The assemblies will be useful resources for comparative genomics, to understand the adaptation and conservation of carnivorous species. DATA DESCRIPTION: This work produced ~ 139.5 Gb N. mirabilis whole genome sequencing reads using leaf tissues, and ~ 21.7 Gb and ~ 27.9 Gb of raw RNA-seq reads for its leaves and flowers, respectively. Transcriptome assembly obtained 339,802 transcripts, in which 79,758 open reading frames (ORFs) were identified. Function analysis indicated that these ORFs were mainly associated with proteolysis and DNA integration. The assembled genome was 691,409,685 bp with 159,555 contigs/scaffolds and an N50 of 10,307 bp. The BUSCO assessment of the assembled genome and transcriptome indicated 91.1% and 93.7% completeness, respectively. A total of 42,961 genes were predicted in the genome identified, coding for 45,461 proteins. The predicted genes were annotated using multiple databases, facilitating future functional analyses of them. This is the first genome report on the Nepenthaceae family.

Proof of anthocyanins in the carnivorous plant genus Nepenthes.

Yellow to red colored betalains are a chemotaxonomic feature of Caryophyllales, while in most other plant taxa, anthocyanins are responsible for these colors. The carnivorous plant family Nepenthaceae belongs to Caryophyllales; here, red-pigmented tissues seem to attract insect prey. Strikingly, the chemical nature of red color in Nepenthes has never been elucidated. Although belonging to Caryophyllales, in Nepenthes, some molecular evidence supports the presence of anthocyanins rather than betalains. However, there was previously no direct chemical proof of this. Using ultra-high-performance liquid chromatography-electrospray ionization-high-resolution mass spectrometry, we identified cyanidin glycosides in Nepenthes species and tissues. Further, we reveal the existence of a complete set of constitutively expressed anthocyanin biosynthetic genes in Nepenthes. Thus, here we finally conclude the long-term open question regarding red pigmentation in Nepenthaceae.

Stretch-activated ion channels identified in the touch-sensitive structures of carnivorous Droseraceae plants.

In response to touch, some carnivorous plants such as the Venus flytrap have evolved spectacular movements to capture animals for nutrient acquisition. However, the molecules that confer this sensitivity remain unknown. We used comparative transcriptomics to show that expression of three genes encoding homologs of the MscS-Like (MSL) and OSCA/TMEM63 family of mechanosensitive ion channels are localized to touch-sensitive trigger hairs of Venus flytrap. We focus here on the candidate with the most enriched expression in trigger hairs, the MSL homolog FLYCATCHER1 (FLYC1). We show that FLYC1 transcripts are localized to mechanosensory cells within the trigger hair, transfecting FLYC1 induces chloride-permeable stretch-activated currents in naïve cells, and transcripts coding for FLYC1 homologs are expressed in touch-sensing cells of Cape sundew, a related carnivorous plant of the Droseraceae family. Our data suggest that the mechanism of prey recognition in carnivorous Droseraceae evolved by co-opting ancestral mechanosensitive ion channels to sense touch.

In vitro plant regeneration and Agrobacterium-mediated genetic transformation of a carnivorous plant, Nepenthes mirabilis.

In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.

Structural Features of Carnivorous Plant (Genlisea, Utricularia) Tubers as Abiotic Stress Resistance Organs.

Carnivorous plants from the Lentibulariaceae form a variety of standard and novel vegetative organs and survive unfavorable environmental conditions. Within Genlisea, only G. tuberosa, from the Brazilian Cerrado, formed tubers, while Utricularia menziesii is the only member of the genus to form seasonally dormant tubers. We aimed to examine and compare the tuber structure of two taxonomically and phylogenetically divergent terrestrial carnivorous plants: Genlisea tuberosa and Utricularia menziesii. Additionally, we analyzed tubers of U. mannii. We constructed phylogenetic trees using chloroplast genes matK/trnK and rbcL and used studied characters for ancestral state reconstruction. All examined species contained mainly starch as histologically observable reserves. The ancestral state reconstruction showed that specialized organs such as turions evolved once and tubers at least 12 times from stolons in Lentibulariaceae. Different from other clades, tubers probably evolved from thick stolons for sect. Orchidioides and both structures are primarily water storage structures. In contrast to species from section Orchidioides, G. tuberosa, U. menziesii and U. mannii form starchy tubers. In G. tuberosa and U. menziesii, underground tubers provide a perennating bud bank that protects the species in their fire-prone and seasonally desiccating environments.

Genomes of the Venus Flytrap and Close Relatives Unveil the Roots of Plant Carnivory.

Most plants grow and develop by taking up nutrients from the soil while continuously under threat from foraging animals. Carnivorous plants have turned the tables by capturing and consuming nutrient-rich animal prey, enabling them to thrive in nutrient-poor soil. To better understand the evolution of botanical carnivory, we compared the draft genome of the Venus flytrap (Dionaea muscipula) with that of its aquatic sister, the waterwheel plant Aldrovanda vesiculosa, and the sundew Drosera spatulata. We identified an early whole-genome duplication in the family as source for carnivory-associated genes. Recruitment of genes to the trap from the root especially was a major mechanism in the evolution of carnivory, supported by family-specific duplications. Still, these genomes belong to the gene poorest land plants sequenced thus far, suggesting reduction of selective pressure on different processes, including non-carnivorous nutrient acquisition. Our results show how non-carnivorous plants evolved into the most skillful green hunters on the planet.