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.     

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.     

Evolution of carnivory in Lentibulariaceae and the Lamiales

As a basis for analysing the evolution of the carnivorous syndrome in Lentibulariaceae (Lamiales), phylogenetic reconstructions were conducted based on coding and non-coding chloroplast DNA (matK gene and flanking trnK intron sequences, totalling about 2.4 kb). A dense taxon sampling including all other major lineages of Lamiales was needed since the closest relatives of Lentibulariaceae and the position of "proto-carnivores" were unknown. Tree inference using maximum parsimony, maximum likelihood, and Bayesian approaches resulted in fully congruent topologies within Lentibulariaceae, whereas relationships among the different lineages of Lamiales were only congruent between likelihood and Bayesian optimizations. Lentibulariaceae and their three genera (Pinguicula, Genlisea, and Utricularia) are monophyletic, with Pinguicula being sister to a Genlisea-Utricularia clade. Likelihood and Bayesian trees converge on Bignoniaceae as sister to Lentibulariaceae, albeit lacking good support. The "proto-carnivores" (Byblidaceae, Martyniaceae) are found in different positions among other Lamiales but not as sister to the carnivorous Lentibulariaceae, which is also supported by Khishino-Hasegawa tests. This implies that carnivory and its preliminary stages ("proto-carnivores") independently evolved more than once among Lamiales. Ancestral states of structural characters connected to the carnivorous syndrome are reconstructed using the molecular tree, and a hypothesis on the evolutionary pathway of the carnivorous syndrome in Lentibulariaceae is presented. Extreme DNA mutational rates found in Utricularia and Genlisea are shown to correspond to their unusual nutritional specialization, thereby hinting at a marked degree of carnivory in these two genera.     

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.     

Chromosome identification for the carnivorous plant <Emphasis Type="Italic">Genlisea margaretae</Emphasis>

Genlisea margaretae, subgenus Genlisea, section Recurvatae (184 Mbp/1C), belongs to a plant genus with a 25-fold genome size difference and an extreme genome plasticity. Its 19 chromosome pairs could be distinguished individually by an approach combining optimized probe pooling and consecutive rounds of multicolor fluorescence in situ hybridization (mcFISH) with bacterial artificial chromosomes (BACs) selected for repeat-free inserts. Fifty-one BACs were assigned to 18 chromosome pairs. They provide a tool for future assignment of genomic sequence contigs to distinct chromosomes as well as for identification of homeologous chromosome regions in other species of the carnivorous Lentibulariaceae family, and potentially of chromosome rearrangements, in cases where more than one BAC per chromosome pair was identified.     

Iridoid and caffeoyl phenylethanoid glycosides of the endangered carnivorous plant Pinguicula lusitanica L. (Lentibulariaceae)

This work reports for the first time the identification of the major compounds of Pinguicula lusitanica, an endangered carnivorous plant species, using minimal amounts of plant material. A methanol extract was prepared from in vitro cultured plantlets and analyzed by HPLC–SPE–NMR/HPLC–MS. Three iridoid and five caffeoyl phenylethanoid glycosides were identified. These groups of natural compounds were previously reported in the Lentibulariaceae family and have been used as chemotaxonomic markers in related families.     

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.     

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.     

Revision of the genus Genlisea (Lentibulariaceae) in Africa and Madagascar with notes on ecology and phytogeography

The African and Madagascan representatives of the carnivorous genus Genlisea are investigated. All African material belongs to subgenus Genlisea and can be assigned to 10 taxa, which mainly differ in indumentum of calyx, corolla and ovary. The former section Tayloria , restricted to South America, is raised to subgeneric rank. One species, Genlisea margaretae , occurs in South-East Africa and Madagascar as well. All species are restricted to specific oligotrophous habitats like granitic outcrops (inselbergs), ferricretes, white sands and swamps. Genlisea taylori from Angola is described as new. All species are described and illustrated and a key and distribution maps are presented.     

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.     

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.     

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.     

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.     

Reproductive biology and pollination of the carnivorous Genlisea violacea (Lentibulariaceae)

• Genlisea violacea is a Brazilian endemic carnivorous plant species distributed in the cerrado biome, mainly in humid environments, on sandy and oligotrophic soil or wet rocks. Studies on reproductive biology or pollination in the Lentibulariaceae are notably scarce; regarding the genus Genlisea, the current study is the first to show systematic and standardised research on reproductive biology from field studies to describe the foraging of visiting insects and determine the effective pollinators of Genlisea.
• We studied two populations of G. violacea through the observation of flower visitors for 4 months of the rainy and dry seasons. Stigmatic receptivity, pollen viability, and breeding system were evaluated together with histochemistry and morphological analyses of flowers.
• The flowers showed stigmatic receptivity of 100% in open buds and mature flowers, reducing to 80% for senescent flowers. Nearly 80% of pollen grains are viable, decreasing to 40–45% after 48 h. Nectar is produced by glandular trichomes inside the spur. Two bee species are effective pollinators: one of the genus Lasioglossum (subgenus Dialictus: Halictidae) and the other of the genus Ceratina (subgenus Ceratinula: family Apidae). Moreover, bee‐like flies of the Syrphidae family may also be additional pollinators.
• Genlisea violacea is an allogamous and self‐compatible species. The differences in flower‐visiting fauna for both populations can be attributed to factors such as climate, anthropogenic effect, seasonal factors related to insects and plants, as well as the morphological variation of flowers in both populations.

     

Reproductive ecology of the three Nordic Pinguicula species (Lentibulariaceae)

Reproductive ecology (pollination biology, breeding systems, and reproductive effort and success) of the three Nordic species of Pinguicula, P. alpina, P. villosa , and P. vulgaris (Lentibulariaceae), was investigated in a subarctic-subalpine area at Abisko, N Swedish Lapland. Additional studies were carried out at Latnjajaure Field Station, a subarctic-alpine tundra site in the Abisko mountains ( P. alpina, P. vulgaris ), and in W Greenland ( P. vulgaris ). At Abisko the species are sympatric and large populations of all three were found within a 50 × 50 m area. The three species are reproduc-tively isolated by internal barriers by occupying different ploidy levels. Pinguicula alpina and P. vulgaris thrive in base-rich habitats, whereas P. villosa is restricted to nutrient-poor Sphagnum bogs, but habitat separation alone is probably not sufficient to prevent illegitimate pollen flow among the species. However, results showed that there are large and consistent differences in pollination biology, flowering phenology, and breeding systems, and these factors interact to create a highly efficient reproductive isolation at all levels, pre-zygotic as well as post-zygotic. Pinguicula alpina is an early-flowering outbreeder, P. vulgaris is an opportunistic late-flowering inbreeder, and P. villosa is quite intermediate between the two extremes. The phenology-based life history strategies of the Pinguicula species were in accordance with a general model developed for arctic flowering plants, predicting maximized fitness through pollen or seed in early- and late-flowering species, respectively.     

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.     

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.     

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.     

Assessing genetic diversity for the USA endemic carnivorous plant <Emphasis Type="Italic">Pinguicula ionantha</Emphasis> R.K. Godfrey (Lentibulariaceae)

Understanding patterns of genetic diversity and population structure for rare, narrowly endemic plant species, such as Pinguicula ionantha (Godfrey’s butterwort; Lentibulariaceae), informs conservation goals and can directly affect management decisions. Pinguicula ionantha is a federally listed species endemic to the Florida Panhandle in the southeastern USA. The main goal of our study was to assess patterns of genetic diversity and structure in 17 P. ionantha populations, and to determine if diversity is associated with geographic location or population characteristics. We scored 240 individuals at a total of 899 AFLP markers (893 polymorphic markers). We found no relationship between the estimated population size with either of two measures of diversity (proportion of loci polymorphic, P = 0.37; Nei’s gene diversity, P = 0.50). We also found low levels of population genetic structure; there was no clear relationship of genetic isolation by distance (P = 0.23) and only a small (but significant) proportion of genetic variation was partitioned amongst regions (2.4 %, P = 0.02) or populations (20.8 %, P < 0.001). STRUCTURE analysis found that the model with two inferred clusters (K = 2) best described the AFLP data; the dominant cluster at each site corresponded to the results from PCoA and Nei’s genetic distance analyses. The observed patterns of genetic diversity suggest that although P. ionantha populations are isolated spatially by distance and both natural and anthropogenic barriers, some gene flow occurs among them or isolation has been too recent to leave a genetic signature. The relatively low level of genetic diversity associated with this species is a concern as it may impair fitness and evolutionary capability in a changing environment. The results of this study provide the foundation for the development of management practices that will assist in the protection of this rare carnivorous plant.     

Evidence for the Polyphyly of Haworthia (Asphodelaceae Subfamily Alooideae; Asparagales) Inferred from Nucleotide Sequences of rbcL, matK, ITS1 and Genomic Fingerprinting with ISSR-PCR

Abstract: Four molecular markers have been studied to examine the phylogenetic position of the South African plant genus Haworthia Duval within the succulent Asphodelaceae. Sequence data of the chloroplast genes mat K and rbc L were compared to the nuclear markers ITS1 and ISSR (Inter Simple Sequence Repeat) analysis. Both lines of molecular data, chloroplast and nuclear DNA, indicate that Haworthia is polyphyletic, forming two distinct clades. Most taxa previously combined as Haworthia subgenus Haworthia branch off early in the alooid chloroplast trees forming a strongly monophyletic group, whereas subgenus Hexangulares forms a polyphyletic assemblage comprising other alooid genera. The nuclear markers ITS1 and ISSR fingerprinting support the two groups as distinctly different, therefore confirming the division seen in chloroplast DNA. The practical implication is that the generic concept of Haworthia may have to be restricted to H. subgenus Haworthia or alternatively, that the groups of Haworthia be treated as infrageneric taxa within a broadened (Linnaean) concept of Aloe.