Recent progress in understanding the evolution of carnivorous lentibulariaceae (lamiales)

Carnivorous plants have emerged as model systems for addressing many ecological and evolutionary questions, and since Lentibulariaceae comprise more than half of all known carnivorous species (325 spp.), they are of particular interest. Studies using various molecular markers have established that Lentibulariaceae and their three genera are monophyletic with Pinguicula being sister to a Genlisea-Utricularia-clade, while the closest relatives of the family remain uncertain. Character states of the carnivorous syndrome in related proto-carnivorous lamialean families apparently emerged independently. In Utricularia, the terrestrial habit has been reconstructed as plesiomorphic, and an extension of subgenus Polypompholyx is warranted. In the protozoan-attracting Genlisea, subgenus Tayloria is revealed as basal lineage. In Pinguicula, the six major lineages found reflect radiations in clearly defined geographic regions, whereas most previously recognized subgeneric taxa are non-monophyletic. Genlisea and Utricularia exhibit substitutional rates that rank among the highest in angiosperms for the molecular markers analyzed. One possible explanation for this lies in selective constraints on a wide range of genomic regions that may have been lowered due to the use of an alternative mode of acquiring nutrients.     

Smallest angiosperm genomes found in lentibulariaceae, with chromosomes of bacterial size

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.     

Functional utrastructure of Genlisea (Lentibulariaceae) digestive hairs

BACKGROUND AND AIMS: Digestive structures of carnivorous plants produce external digestive enzymes, and play the main role in absorption. In Lentibulariaceae, the ultrastructure of digestive hairs has been examined in some detail in Pinguicula and Utricularia, but the sessile digestive hairs of Genlisea have received very little attention so far. The aim of this study was to fill this gap by expanding their morphological, anatomical and histochemical characterization. METHODS: Several imaging techniques were used, including light, confocal and electron microscopy, to reveal the structure and function of the secretory hairs of Genlisea traps. This report demonstrates the application of cryo-SEM for fast imaging of whole, physically fixed plant secretory structures. KEY RESULTS AND CONCLUSION: The concentration of digestive hairs along vascular bundles in subgenus Genlisea is a primitive feature, indicating its basal position within the genus. Digestive hairs of Genlisea consist of three compartments with different ultrastructure and function. In subgenus Tayloria the terminal hair cells are transfer cells, but not in species of subgenus Genlisea. A digestive pool of viscous fluid occurs in Genlisea traps. In spite of their similar architecture, the digestive-absorptive hairs of Lentibulariaceae feature differences in morphology and ultrastructure.     

Female germ unit in Genlisea and Utricularia, with remarks about the evolution of the extra-ovular female gametophyte in members of Lentibulariaceae

Lentibulariaceae is the largest family among carnivorous plants which displays not only an unusual morphology and anatomy but also the special evolution of its embryological characteristics. It has previously been reported by authors that Utricularia species lack a filiform apparatus in the synergids. The main purposes of this study were to determine whether a filiform apparatus occurs in the synergids of Utricularia and its sister genus Genlisea, and to compare the female germ unit in these genera. The present studies clearly show that synergids in both genera possess a filiform apparatus; however, it seems that Utricularia quelchii synergids have a simpler structure compared to Genlisea aurea and other typical angiosperms. The synergids are located at the terminal position in the embryo sacs of Pinguicula, Genlisea and were probably also located in that position in common Utricularia ancestor. This ancestral characteristic still occurs in some species from the Bivalvaria subgenus. An embryo sac, which grows out beyond the limit of the integument and has contact with nutritive tissue, appeared independently in different Utricularia lineages and as a consequence of this, the egg apparatus changes position from apical to lateral.     

Oxygen concentrations inside the traps of the carnivorous plants Utricularia and Genlisea (Lentibulariaceae)

BACKGROUND AND AIMS: Species of Utricularia and Genlisea (Lentibulariaceae) are carnivorous, capturing small prey in traps which are physiologically very active, with abundant quadrifid and bifid glands. Traps of Utricularia have walls composed of two cell layers, and are filled with water. Diverse communities of commensal microorganisms often live inside the traps. Genlisea forms long, hollow subterranean traps of foliar origin, growing in anoxic wet substrate. Knowledge of the O(2) concentrations inside Utricularia and Genlisea traps is vital for understanding their physiological functioning and conditions for the life of commensals. To test the hypothesis that prey are killed by anoxia inside the traps, and to measure respiration of traps, [O(2)] was measured in the fluid in mature traps of these species. METHODS: Oxygen concentration and electrical redox potential were measured using a small Clark-type oxygen sensor and a miniature platinum electrode, respectively, in the fluid of excised and intact traps of six aquatic Utricularia species and in Genlisea hispidula traps. KEY RESULTS: Steady-state [O(2)] in the traps of both genera always approached zero (median 0.0-4.7 microm). The [O(2)] decreased after electrodes were inserted into Utricularia traps at a rate which ranged from 0.09 to 1.23 mm h(-1) and was lower in traps of irradiated and intact shoots with higher [O(2)] in shoot tissues. Redox potential ranged from -24 to -105 mV in the traps, confirming the very small or zero [O(2)]. CONCLUSIONS: Very small or zero [O(2)], effectively anoxia, is demonstrated in Utricularia and Genlisea traps. This is probably below the critical [O(2)] for prey survival, and causes captured prey to die of suffocation. Internal trap glands and trap commensals are considered to be adapted to facultative anoxia interrupted by limited periods of higher [O(2)] after firings.     

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants

Abstract: A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as “proto‐carnivores”, lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional “carnivorous organ”, which can trap a prey, digest it, and finally absorb available nutrients.     

Morphology and nrITS phylogeny of the genus Pinguicula L. (Lentibulariaceae), with special attention to embryo evolution

The genus Pinguicula (Lentibulariaceae) is unusual within the dicot order Lamiales because of the occurrence of both embryos with two cotyledons and those with just one cotyledon. In order to elucidate the infrageneric relationships and the evolutionary history of the embryo, we analysed (1) the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA (nrITS) of 29 Old and New World taxa of Pinguicula, and (2) the morphological and anatomical characters of the seeds. We suggest that the cotyledon number and spermoderm structure were quite unstable in the evolution of Pinguicula. Although basal nodes of the nrITS tree are sensitive to taxon sampling, all tree topologies found in this study imply homoplasy in the cotyledon number.     

Floral micromorphology and nectar composition of the early evolutionary lineage Utricularia (subgenus Polypompholyx,Lentibulariaceae)

Protoplasma - Utricularia (Lentibulariaceae) is a genus comprising around 240 species of herbaceous, carnivorous plants. Utricularia is usually viewed as an insect-pollinated genus, with the...     

Microsatellite markers developed for Utricularia reniformis (Lentibulariaceae)

? Premise of the study: Microsatellite markers were developed to study the genetic diversity and population structure of the carnivorous bladderwort Utricularia reniformis, which is endemic to the Atlantic Forest of southern and southeastern Brazil. Cross-species amplification was tested in U. gibba, U. neottioides, U. subulata, and Pinguicula benedicta. ? Methods and Results: The U. reniformis genome was sequenced in a 454 GS FLX sequencer, and eight primer sets were developed based on the microsatellites identified from the reads. All loci are polymorphic, showing 1.6 to 4.8 alleles per population. Preliminary results show that primer sets are suitable for population-level studies. Cross-species amplification was successful in three other Utricularia species and one Pinguicula species. ? Conclusions: Markers developed in this study provide tools for analyses of intra- and interpopulation genetic diversity in Utricularia and Pinguicula.     

Les flavonoïdes de deux Lentibulariacées: Pinguicula vulgaris et Utricularia vulgaris

Apigenin, isoscutellarein, scutellarein, luteolin, 6-hydroxyluteolin and hypolaetin have been identified in fresh leaves of Pinguicula vulgaris; apigenin, luteolin, 6-hydroxyluteolin and diosmetin were found in Utricularia vulgaris. These identifications, based on spectral characteristics, support a relationship between Lentibulariaceae and Tubiflorae.     

Iridoid glucosides from Utricularia australis and Pinguicula vulgaris (lentibulariaceae)

Utricularia australis contained 6-deoxycatalpol, a new iridoid glucoside, besides aucubin, gardoside and mussaenosidic acid. From Pinguicula vulgaris was isolated catalpol, globularin and 10-(Z)-cinnamoyl-catalpol, the latter being a new compound. Thus, the iridoids found in Lentibulariaceae belong to structural types which are common in Scrophulariaceae and related families.     

Phylogenetic analysis of Pinguicula (Lentibulariaceae): chloroplast DNA sequences and morphology support several geographically distinct radiations

The genus Pinguicula is one of the three genera of the carnivorous Lentibulariaceae, comprising approximately 80 species. Phylogeny inference using nucleotide sequences of the chloroplast gene matK and the trnK group II intron, as well as a set of 32 morphological characters revealed five well-supported, major lineages within the genus. These lineages largely reflect radiations in clearly defined geographic regions, whereas most previously recognized sections of the genus are shown to be para- or polyphyletic. A species-rich Mexican-Central American-Caribbean clade has the Eurasian P. alpina and an East Asian clade as successive sisters. All three are characterized by a production of flower buds on winter-resting plants, a specific corolla hair structure and a very large corolla lower central lobe. Another diverse clade is composed of species with primarily European distribution including the widespread type species P. vulgaris. For this clade, vegetative reproduction during dormancy is synapomorphic. Species native to SE North America and the South American Andes and a group of Mediterranean and NE Atlantic coast species together appear in a fifth well-supported clade, that is characterized by a tropical-type growth habit. It is the only clade that has reached temperate zones of the southern hemisphere.     

Sequences of 72 plastid genes support the early divergence of Cornales in the asterids

Recent phylogenetic analyses of molecular data have supported different hypotheses of relationships among Cornales,Ericales,and core asterids.Such hypotheses have implications for the evolution of important morphological and embryological features of asterids.In this study we generated plastid genome-scale data of Davidia (Cornales) and Franklinia (Ericales) and combined them with published sequence data of eudicots.Our maximum parsimony,maximum likelihood,and Bayesian analyses generated strongly supported and congruent phylogenetic relationships among the three major lineages of the asterids.Cornales diverges first in asterids; Ericales is sister to the core asterids.Adding two more taxa helps mitigate long branch attraction in parsimony analyses.Sampling 26-28 plastid protein-coding genes may provide satisfactory resolution and support for relationships of eudicots including basal lineages of asterids.     

Development of microsatellite markers for the carnivorous plant <Emphasis Type="Italic">Genlisea aurea</Emphasis> (Lentibulariaceae) using genomics data of NGS

Genlisea aurea A.St.-Hil. is a carnivorous plant endemic species to Brazil in the Lentibulariaceae family. Very few studies have addressed the genetic structure and conservation status of G. aurea and the Lentibulariaceae. Microsatellites markers are advantageous tools that can be employed to predict the vulnerability of Lentibulariaceae species. Therefore, the development of molecular markers focusing the population analyses of Genlisea for future genetic studies and conservation actions are essential. Thus, we developed simple sequence repeats (SSRs) based on in silico analyses of G. aurea draft genome assembly. We characterized 40 individuals from several populations and identified 12 loci that were polymorphic, with heterozygosity between 0.123 and 0.650. We demonstrated that the G. aurea SSR markers work cross-species in Genlisea filiformis, G. repens, G. tuberosa and G. violacea. These markers will be important for future population, phylogeographic and conservation studies in G. aurea and other Genlisea species.     

Soil fertilization enhances growth of the carnivorous plant Genlisea violacea

In a 116-d greenhouse growth experiment on a terrestrial carnivorous plant Genlisea violacea (Lentibulariaceae), mild fertilization of a peaty soil led to a 2.4 fold increase in total plant biomass as compared to the controls. Tissue P and K content in fertilized plants was significantly higher than that in the controls.     

Patterns of prey capture and prey availability among populations of the carnivorous plant Pinguicula moranensis (Lentibulariaceae) along an environmental gradient

In this study we explored the effect of the physical environment and the availability of prey (biomass and taxonomic composition) on the patterns of prey capture and reproduction on five populations of Pinguicula moranensis (Lentibulariaceae) in areas ranging from pine-oak forests to desert scrublands. Environmental variation was summarized using principal factor analysis. Prey availability and prey capture increased toward the shadiest, most humid, and fertile population. The probability of reproduction and average bud production per population did not follow the same tendency because both fitness components peaked at the middle of the environmental gradient. These results suggest that the benefits derived from carnivory are maximized at sites fulfilling a trade-off between light, moisture, and prey availability. We also found that the taxonomic composition of both the available prey and that of the prey captured by plants varied among populations. The results also indicated that the prey captured by plants are not a random sample of prey available within populations. Overall, the results from this study revealed a marked amount of heterogeneity in the physical and biotic environment among the populations of P. moranensis, which has the potential to affect the outcome of the interaction between this carnivorous species and its prey.     

Towards resolving Lamiales relationships: insights from rapidly evolving chloroplast sequences

Background  

In the large angiosperm order Lamiales, a diverse array of highly specialized life strategies such as carnivory, parasitism, epiphytism, and desiccation tolerance occur, and some lineages possess drastically accelerated DNA substitutional rates or miniaturized genomes. However, understanding the evolution of these phenomena in the order, and clarifying borders of and relationships among lamialean families, has been hindered by largely unresolved trees in the past.     

Phylogeny and classification of the leafhopper subfamily Eurymelinae (Hemiptera: Cicadellidae) inferred from molecules and morphology

Phylogenetic analysis of the globally distributed arboreal leafhopper subfamily Eurymelinae was conducted based on DNA sequence data from three nuclear and two mitochondrial genes in addition to 86 discrete morphological characters. The analysis included 89 species representing 61 genera from all major biogeographic regions including six species from outgroups, Megophthalminae and Ulopinae. Trees resulting from partitioned Bayesian and maximum likelihood analyses of the combined data were well resolved and largely congruent, differing mainly in the relationships among the earliest diverging lineages. The results are consistent with an expanded concept of Eurymelinae, including tribes Austroagalloidini and Macropsini. Six monophyletic groups are recognized as new tribes, Balocerini, Chiasmodolini, Chileanoscopini, Idioceroidini, Kopamerrini and Nesocerini, tribe n. , and the previously recognized tribes Eurymelini, Idiocerini and Megipocerini are redefined. A new synonym, Busonini Zhang & Li, 2015 syn.n. is proposed here for Megipocerini Isaev, 1988. Molecular divergence time estimates were calibrated using two fossil taxa and suggested that the earliest divergences occurred in the Lower Cretaceous and that most major lineages of this group arose during the Cretaceous. Reconstruction of ancestral areas revealed considerable continental-scale biogeographical structure. The place of origin of Eurymelinae is equivocal but major lineages arose in the Neotropical, Australian and Afrotropical regions. A key to tribes and a checklist of genera showing current tribal placements are provided.     

Relationships of the temperate Australasian labrid fish tribe Odacini (Perciformes; Teleostei)

The labrid tribe Odacini comprises four genera and 12 species of fishes that inhabit shallow kelp forest and seagrass areas in temperate waters of Australia and New Zealand. Odacines are morphologically disparate, but share synapomorphies in fin structure and fusion of teeth into a beak-like oral jaw. A phylogenetic analysis of odacines was conducted to investigate their relationships to other labrid fishes, the relationships of species within the tribe, and the evolution of herbivory within the group. Fragments from two mitochondrial genes, 12S rDNA and 16S rDNA, and two nuclear genes, Tmo4C4 and RAG2, were sequenced for seven odacine species (representing all four genera), eight species representing the other major labrid lineages, and three outgroup species. Maximum likelihood and maximum parsimony analyses on the resulting 2338 bp of DNA sequence produced nearly identical topologies differing only in the placement of a clade containing the cheiline Cheilinus fasciatus and the scarine Cryptotomus roseus. The remaining clades received strong bootstrap support under maximum parsimony, and all clades in the maximum likelihood analysis received high bootstrap proportions and high posterior probabilities. The hypsigenyine labrid Choerodon anchorago formed the sister group to the odacines. Within the odacines, Odax cyanoallix+Odax pullus formed the sister to the remaining odacines, with Odax acroptilus, Odax cyanomelas, and Siphonognathus argyrophanes forming successively closer sister groups to the clade Haletta semifasciatus+Neoodax balteatus. Either herbivory evolved twice in the odacines, or herbivory evolved once with two reversions to carnivory. The latter hypothesis appears more likely in the light of odacine feeding biology.     

Functional anatomy of the ovule in Genlisea with remarks on ovule evolution in Lentibulariaceae

The Lentibulariaceae are highly evolved and specialized carnivorous angiosperms displaying not only unusual morphology and embryology but also specific changes in the genome and chromosomes as large as bacterial chromosomes. Comparative study of the morphology and detailed anatomy of the ovule in the genera Genlisea, Utricularia, and Pinguicula should shed new light on the phylogeny of this family. The clade Genlisea + Utricularia is sister to the genus Pinguicula, which is considered the most primitive taxon within Lentibulariaceae. Thus we should expect the ovules of Genlisea to be more similar to those of the more closely related genus Utricularia than to Pinguicula. Surprisingly, the ovules of Genlisea retain characters (free funiculus, ES remaining in the ovule) in common with Pinguicula, presumably inherited from a common ancestor. Genlisea ovules have only one main character in common with subgenus Polypompholyx (Utricularia): a well-developed funiculus. There are differences between the ovules of the subgenera Genlisea and Tayloria. In subgenus Genlisea the micropyle tends to be closer to the funiculus and the ovule forms an unusual jacket-like nutritive tissue of integumental origin. The most specialized ovules in Lentibulariaceae evolved in the genus Utricularia. The special chalazal nutritive tissue in Genlisea and Utricularia is simply a hypostase.