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.

The genome of jojoba (Simmondsia chinensis): A taxonomically isolated species that directs wax ester accumulation in its seeds.

Seeds of the desert shrub, jojoba (Simmondsia chinensis), are an abundant, renewable source of liquid wax esters, which are valued additives in cosmetic products and industrial lubricants. Jojoba is relegated to its own taxonomic family, and there is little genetic information available to elucidate its phylogeny. Here, we report the high-quality, 887-Mb genome of jojoba assembled into 26 chromosomes with 23,490 protein-coding genes. The jojoba genome has only the whole-genome triplication (γ) shared among eudicots and no recent duplications. These genomic resources coupled with extensive transcriptome, proteome, and lipidome data helped to define heterogeneous pathways and machinery for lipid synthesis and storage, provided missing evolutionary history information for this taxonomically segregated dioecious plant species, and will support efforts to improve the agronomic properties of jojoba.

A phylogenomic analysis of Nepenthes (Nepenthaceae).

Nepenthaceae is one of the largest carnivorous plant families and features ecological and morphological adaptations indicating an impressive adaptive radiation. However, investigation of evolutionary and taxonomic questions is hindered by poor phylogenetic understanding, with previous molecular studies based on limited loci and taxa. We use high-throughput sequencing with a target-capture methodology based on a 353-loci, probe set to recover sequences for 197 samples, representing 151 described or putative Nepenthes species. Phylogenetic analyses were performed using supermatrix and maximum quartet species tree approaches. Our analyses confirm five Western outlier taxa, followed by N. danseri, as successively sister to the remainder of the group. We also find mostly consistent recovery of two major Southeast Asian clades. The first contains common or widespread lowland species plus a Wallacean-New Guinean clade. Within the second clade, sects. Insignes and Tentaculatae are well supported, while geographically defined clades representing Sumatra, Indochina, Peninsular Malaysia, Palawan, Mindanao and Borneo are also consistently recovered. However, we find considerable conflicting signal at the site and locus level, and often unstable backbone relationships. A handful of Bornean taxa are inconsistently placed and require further investigation. We make further suggestions for a modified infra-generic classification of genus Nepenthes.

Comparative cytogenetics of the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae): a very diverse group of the suborder Cactineae, Caryophyllales.

The clade ACPT (Anacampserotaceae, Cactaceae, Portulacaceae, and Talinaceae) is the most diverse lineage of the subordem Cactineae. The relationships between these families are still uncertain, with different topologies suggested by phylogenetic analyses with several combinations of markers. Different basic numbers (x) have been suggested for each family and for the subord, often in a contestable way. Comparative cytogenetic has helped to understand the evolutionary relationships of phylogenetically poorly resolved groups, as well as their mechanisms of karyotype evolution. The karyotype evolution in representatives of Cactineae was analyzed, focusing on the ACPT clade, through the analysis of chromosome number in a phylogenetic bias. The phylogeny obtained showed a well-resolved topology with support for the monophyly of the five families. Although a chromosomal number is known for less than 30% of the Cactineae species, the analyses revealed a high karyotype variability, from 2n = 8 to 2n = 110. The analysis of character reconstruction of the ancestral haploid numbers (p) suggested p = 12 for Cactineae, with distinct basic numbers for the clade family ACPT: Cactaceae and Montiaceae (p = 11), Talinaceae (p = 12), and Anacampserotaceae and Portulacaceae (p = 9). Talinaceae, Anacampserotaceae, and Cactaceae were stable, while Portulaca and Montiaceae were karyotypically variable. The chromosome evolution of this group was mainly due to events of descending disploidy and poliploidy. Our data confirm that the low phylogenetic resolution among the families of the ACPT clade is due to a divergence of this clade in a short period of time. However, each of these families can be characterized by basic chromosome numbers and unique karyotype evolution events.

Digestive glands extraction and precise pigment analysis support the exclusion of the carnivorous plant Dionaea muscipula Ellis from the Caryophyllales order.

In the order Caryophyllales, plants synthesize betalains instead of anthocyanins, with only two exceptions, the Caryophyllaceae and Molluginaceae. Dionaea muscipula Ellis was included in the Caryophyllales order but recent research based on genetic studies proposed the consideration of the Droseraceae family into the Nepenthales order. In this work we face the dilemma of the phylogenetic classification of Dionaea from a phytochemical point of view. Dionaea's pigments were analyzed by using techniques of structural analysis. Extracts from the leaves, mature stem and flowers of different specimens of Dionaea were analyzed, to find possible differences in the types of pigments or in their proportion in different parts of the plant. These extracts were analyzed by spectrophotometry, HPLC co-elution and ESI-MS/MS. In addition, digestive glands were extracted from the snap trap with minor sample manipulation and by reducing the non-pigmented plant tissue. Considering only the digestive glands instead of whole snap traps, the analyses allowed to quantitate and elucidate the structure of the compounds responsible for the red coloration: delphinidin-3-O-glucoside (myrtillin), cyanidin-3-O-glucoside (kuromanin) and a third compound, the aglycone cyanidin, detected in the species for the first time. The unambiguous results of the present work support the exclusion of Dionaea from the Caryophyllales.

Analyzing Contentious Relationships and Outlier Genes in Phylogenomics.

Recent studies have demonstrated that conflict is common among gene trees in phylogenomic studies, and that less than one percent of genes may ultimately drive species tree inference in supermatrix analyses. Herein, we examined two data sets where supermatrix and coalescent-based species trees conflict. We identified two highly influential "outlier" genes in each data set. When removed from each data set, the inferred supermatrix trees matched the topologies obtained from coalescent analyses. We also demonstrate that, while the outlier genes in the vertebrate data set have been shown in a previous study to be the result of errors in orthology detection, the outlier genes from a plant data set did not exhibit any obvious systematic error, and therefore, may be the result of some biological process yet to be determined. While topological comparisons among a small set of alternate topologies can be helpful in discovering outlier genes, they can be limited in several ways, such as assuming all genes share the same topology. Coalescent species tree methods relax this assumption but do not explicitly facilitate the examination of specific edges. Coalescent methods often also assume that conflict is the result of incomplete lineage sorting. Herein, we explored a framework that allows for quickly examining alternative edges and support for large phylogenomic data sets that does not assume a single topology for all genes. For both data sets, these analyses provided detailed results confirming the support for coalescent-based topologies. This framework suggests that we can improve our understanding of the underlying signal in phylogenomic data sets by asking more targeted edge-based questions.

Chloroplast Genome of the Folk Medicine and Vegetable Plant Talinum paniculatum (Jacq.) Gaertn.: Gene Organization, Comparative and Phylogenetic Analysis.

The complete chloroplast (cp) genome of Talinum paniculatum (Caryophyllale), a source of pharmaceutical efficacy similar to ginseng, and a widely distributed and planted edible vegetable, were sequenced and analyzed. The cp genome size of T. paniculatum is 156,929 bp, with a pair of inverted repeats (IRs) of 25,751 bp separated by a large single copy (LSC) region of 86,898 bp and a small single copy (SSC) region of 18,529 bp. The genome contains 83 protein-coding genes, 37 transfer RNA (tRNA) genes, eight ribosomal RNA (rRNA) genes and four pseudogenes. Fifty one (51) repeat units and ninety two (92) simple sequence repeats (SSRs) were found in the genome. The pseudogene rpl23 (Ribosomal protein L23) was insert AATT than other Caryophyllale species by sequence alignment, which located in IRs region. The gene of trnK-UUU (tRNA-Lys) and rpl16 (Ribosomal protein L16) have larger introns in T. paniculatum, and the existence of matK (maturase K) genes, which usually located in the introns of trnK-UUU, rich sequence divergence in Caryophyllale. Complete cp genome comparison with other eight Caryophyllales species indicated that the differences between T. paniculatum and P. oleracea were very slight, and the most highly divergent regions occurred in intergenic spacers. Comparisons of IR boundaries among nine Caryophyllales species showed that T. paniculatum have larger IRs region and the contraction is relatively slight. The phylogenetic analysis among 35 Caryophyllales species and two outgroup species revealed that T. paniculatum and P. oleracea do not belong to the same family. All these results give good opportunities for future identification, barcoding of Talinum species, understanding the evolutionary mode of Caryophyllale cp genome and molecular breeding of T. paniculatum with high pharmaceutical efficacy.

Disparity, diversity, and duplications in the Caryophyllales.

The role played by whole genome duplication (WGD) in plant evolution is actively debated. WGDs have been associated with advantages such as superior colonization, various adaptations, and increased effective population size. However, the lack of a comprehensive mapping of WGDs within a major plant clade has led to uncertainty regarding the potential association of WGDs and higher diversification rates. Using seven chloroplast and nuclear ribosomal genes, we constructed a phylogeny of 5036 species of Caryophyllales, representing nearly half of the extant species. We phylogenetically mapped putative WGDs as identified from analyses on transcriptomic and genomic data and analyzed these in conjunction with shifts in climatic occupancy and lineage diversification rate. Thirteen putative WGDs and 27 diversification shifts could be mapped onto the phylogeny. Of these, four WGDs were concurrent with diversification shifts, with other diversification shifts occurring at more recent nodes than WGDs. Five WGDs were associated with shifts to colder climatic occupancy. While we find that many diversification shifts occur after WGDs, it is difficult to consider diversification and duplication to be tightly correlated. Our findings suggest that duplications may often occur along with shifts in either diversification rate, climatic occupancy, or rate of evolution.

Caryophyllales are the main hosts of a unique set of ectomycorrhizal fungi in a Neotropical dry forest.

The ectomycorrhizal symbiosis was long thought to be restricted to temperate forests. However, as tropical forests have been explored, it has become clear that these habitats host unique ectomycorrhizal (ECM) fungi. We have been exploring tropical dry forests (TDF), which are endangered terrestrial ecosystems and hotspots of endemism. Since Fabaceae is the main plant family in this environment, we hypothesized that trees in this lineage would be the main ECM hosts. We sequenced the ITS rDNA region from fungi and both rbcL and trnL cpDNA from plants to identify both symbiotic partners from root tips. The systematic position of each symbiont was confirmed by Bayesian phylogenetic inference. We identified 20 plant species belonging to 10 families that hosted 19 unique ECM fungal species from 5 lineages. Most ECM fungi were associated with Caryophyllales, not with Fabaceae. Achatocarpus and Guapira, the main hosts, are scattered throughout the forest and are not in monodominant patches. The low ECM fungal diversity can be explained by the low density of host plants and their high specificity. Our results indicate that Caryophyllales is an important order of tropical ECM hosts with at least four independent evolutionary lineages that have evolved the ability to form ectomycorrhizae.

Targeted Enrichment of Large Gene Families for Phylogenetic Inference: Phylogeny and Molecular Evolution of Photosynthesis Genes in the Portullugo Clade (Caryophyllales).

Hybrid enrichment is an increasingly popular approach for obtaining hundreds of loci for phylogenetic analysis across many taxa quickly and cheaply. The genes targeted for sequencing are typically single-copy loci, which facilitate a more straightforward sequence assembly and homology assignment process. However, this approach limits the inclusion of most genes of functional interest, which often belong to multi-gene families. Here, we demonstrate the feasibility of including large gene families in hybrid enrichment protocols for phylogeny reconstruction and subsequent analyses of molecular evolution, using a new set of bait sequences designed for the "portullugo" (Caryophyllales), a moderately sized lineage of flowering plants (~ 2200 species) that includes the cacti and harbors many evolutionary transitions to C$_{\mathrm{4}}$ and CAM photosynthesis. Including multi-gene families allowed us to simultaneously infer a robust phylogeny and construct a dense sampling of sequences for a major enzyme of C$_{\mathrm{4}}$ and CAM photosynthesis, which revealed the accumulation of adaptive amino acid substitutions associated with C$_{\mathrm{4}}$ and CAM origins in particular paralogs. Our final set of matrices for phylogenetic analyses included 75-218 loci across 74 taxa, with ~ 50% matrix completeness across data sets. Phylogenetic resolution was greatly improved across the tree, at both shallow and deep levels. Concatenation and coalescent-based approaches both resolve the sister lineage of the cacti with strong support: Anacampserotaceae $+$ Portulacaceae, two lineages of mostly diminutive succulent herbs of warm, arid regions. In spite of this congruence, BUCKy concordance analyses demonstrated strong and conflicting signals across gene trees. Our results add to the growing number of examples illustrating the complexity of phylogenetic signals in genomic-scale data.