Identification and typification of Nepenthes blancoi,with N. abalata sp. nov. from the western Visayas,Philippines

Nepenthes blancoi Bl. is neotypified on material conspecific with N. alata Blanco. Material previously associated with N. blancoi is described as N. abalata Jebb & Cheek sp. nov. This is a grassland species seemingly related to N. philippinensis Macfarl. but with similarities to the grassland species of Indo‐China. Its conservation status is assessed as ‘Critically Endangered’ (CR).     

A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata

 Cytoplasmic somatic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Nicotiana alata were constructed. A total of 33 green regenerants were obtained after fusion of protoplasts from a tobacco cytoplasmic chlorophyll-deficient mutant and gamma irradiation-inactivated leaf protoplasts of N. alata. Twenty nine of them were male sterile and displayed an altered stamen morphology (formation of petaloid and stigmoid structures instead of stamens). Southern-blot analyses of eight CMS plants using N. alata-specific nuclear repetitive DNA and cpDNA probes revealed that they contained nuclear genetic material of N. tabacum and chloroplasts from N. alata. Restriction-enzyme analysis of mitochondrial DNAs of the cybrids in question showed different patterns consisting of an incomplete mix of mtDNA fragments from both parents, as well as new fragments. Southern-blot analysis of mtDNAs with a sunflower atpA probe gave the same recombinant hybridization pattern for all analyzed cybrids, indicating that high-frequency specific recombination occurs in the atpA region. Analysis of the progeny from three successive backcrosses of the studied cybrids with N. tabacum demonstrated a strict cytoplasmic inheritance of the male-sterile phenotype. Received: 10 May 1997 / Accepted: 31 March 1998     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Local and Regional-Scale Food Web Structure in Nepenthes alata Pitchers1

Tropical Nepenthes pitcher plants provide small, isolated aquatic habitats. We examined inter-pitcher variation in the community structure of the inhabitants of Nepenthes alata Blanco in West Sumatra, focusing on the conditions of the pitchers, bacterial density in the pitcher fluid, density and biomass of metazoan inhabitants, and the frequencies of interspecific encounters. Older pitchers contained more insect carcasses. The bacterial density increased with the age of the pitchers, but decreased in withered pitchers that contained finely decomposed detritus. In live pitchers, the bacterial density, the density, mass and species richness of metazoa, and the number of trophic levels per pitcher were positively correlated with detrital mass, which was correlated with volume of pitcher fluid. The metazoan fauna from N. alata consisted of 4 predators and 12 saprophages, among the richest known for Nepenthes species. However, each individual pitcher harbored a limited numbers of species, owing to (1) the low incidence of many species, and (2) the aggregated distribution and different temporal colonization pattern of major species. Six dipteran taxa (one predator and five saprophages) accounted for the bulk of metazoan inhabitant biomass. Of 48 combinations of predator-prey encountered, only four occurred frequently (in > 30% of pitchers), which included two predators and three saprophages. Thus, individual pitchers harbored relatively simple communities despite the regional species richness, and only limited kinds of predator-prey encounters seemed to occur frequently in the regional food web. The local-scale properties of the subdivided communities presented here provide the basic information for understanding the maintenance of regional species richness and food web complexity.     

The carnivorous plant described as Sarracenia alata contains two cryptic species

Modern methods for species delimitation provide biologists with the power to detect cryptic diversity in nearly any system. To illustrate the application of such methods, we collected data (21 sequence loci) from a carnivorous plant in southeastern North America and applied several recently developed methods (Gaussian clustering, Structurama, BPP, spedeSTEM). The pale pitcher plant Sarracenia alata inhabits the southeastern USA along the northern coast of the Gulf of Mexico. Sarracenia alata populations are separated by the Mississippi River and Atchafalaya Basin, a known biogeographical barrier in this region, but the cohesiveness of S. alata as currently classified has not been tested rigorously. Multiple analytical approaches (including allelic clustering and species trees methods) suggest that S. alata comprises two cryptic lineages that correspond to the eastern and western portions of the plant's distribution. That such clear genetic evidence for cryptic diversity exists within S. alata and is in conflict with other sources of data (e.g. morphology, environmental differentiation) illustrates a conundrum faced by those who investigate species boundaries: genetic data are often the first type of data to accumulate evidence of differentiation, but most existing taxonomic treatments are based on nongenetic data. Our results suggest that S. alata as currently described contains two cryptic species, and we recommend the elevation of the western populations to species status. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 737–746.     

Effects of RNases on rejection of pollen from Nicotiana tabacum and N. plumbaginifolia

S-RNases are implicated in both inter- and intra-specific pollen rejection in Nicotiana. At least two mechanisms contribute to S-RNase dependent rejection of pollen from self compatilble species such as Nicotiana plumbaginifolia and N. tabacum. Cloned S-RNases from self incompatible N. alata expressed in styles of self compatible N. tabacum cause rejection of N. tabacum pollen through a factor-independent mechanism. However, rejection of N. plumbaginifolia pollen occurs only when S-RNases are expressed in conjunction with non-S-RNase factors from N. alata (factor-dependent pollen rejection). Here, we compared the pollen rejection activity of four RNases in these two systems. Recombinant RNase expression levels in the factor-dependent N. plumbaginifolia system were insufficient to cause pollen rejection. However, three S-RNases were active in the factor-independent N. tabacum pollen rejection system. This system shows the broadest specificity of any system so far examined. However, RNaseI from E. coli could not cause N. tabacum pollen rejection. Thus, RNase activity alone is not sufficient for pollen rejection. Our results suggest that S-RNases are specially adapted to function in pollen rejection. Received: 15 December 2000 / Accepted: 1 May 2001     

Isolation and characterization of microsatellite markers from the sweet passion fruit (Passiflora alata Curtis: Passifloraceae)

Passiflora alata is one of the commercialized species of the passion vines, grown for its edible fruits. It is found in South America, mainly Paraguay, Argentina, Peru and Brazil. The development of a set of microsatellite markers was proposed to provide tools for analysing both genetic structure of wild populations and the reproductive system of the species, which are poorly understood. Seven markers were developed from a P. alata‐genomic library enriched for simple sequence repeats (SSR). Twelve individuals were genotyped, and the levels of the expected heterozygosity (H_E) did show that those markers could be used to develop P. alata genetic studies.     

Revision of ploidy status of Dioscorea alata L. (Dioscoreaceae) by cytogenetic and microsatellite segregation analysis

Dioscorea alata is a polyploid species with several ploidy levels and its basic chromosome number has been considered by most authors to be x = 10. Standard chromosome counting and flow cytometry analysis were used to determine the chromosome number of 110 D. alata accessions of the CIRAD germplasm collection. The results revealed that 76% of accessions have 2n = 40 chromosomes, 7% have 2n = 60 chromosomes and 17% have 2n = 80 chromosomes. Progenies were produced from 2n = 40 types of D. alata and the segregation patterns of six microsatellite markers in four different progenies were analysed. The Bayesian method was used to test for diploid versus tetraploid (allo- and autotetraploid) modes of inheritance. The results provided the genetic evidence to establish the diploidy of plants with 2n = 40 chromosomes and to support the hypothesis that plants with 2n = 40, 60 and 80 chromosomes are diploids, triploids and tetraploids, respectively, and that the basic chromosome number of D. alata is x = 20. The findings obtained in the present study are significant for effective breeding programs, genetic diversity analysis and elucidation of the phylogeny and the species origin of D. alata.     

Regulation of LAT52 promoter activity during pollen tube growth through the pistil of Nicotiana alata.

The pollen-specific promoter of the LAT52 gene is known to direct expression of marker proteins during the last stages of pollen maturation and in very early pollen tube growth.We have examined the expression of LAT52-GUS during later stages of pollen tube growth in style and ovary of the relatively long-styled species Nicotiana alata. GUS activity was detected histochemically and found to be present in germinating pollen grains of N. alata and in tubes growing through the upper part of the style. No GUS activity was detected in 99% of the pollen tubes growing through the lower part of the style, but activity was present in tubes within the ovary. This finding indicates that the LAT52 promoter is regulated in growing pollen tubes, and is most active during the earliest and latest stages of pollen tube growth. GUS activity was also detected in some ovules, where it presumably marked the release of pollen tube cytoplasm into the ovule. The distribution of ovules with GUS activity within the ovary is not consistent with high-precision pollen tube guidance to the ovule. Received: 16 August 1999 / Revision accepted: 20 December 1999     

Molecular and genetic characterization of novel S-RNases from a natural population of Nicotiana alata

Self-incompatibility in the Solanaceae is mediated by S-RNase alleles expressed in the style, which confer specificity for pollen recognition. Nicotiana alata has been successfully used as an experimental model to elucidate cellular and molecular aspects of S-RNase-based self-incompatibility in Solanaceae. However, S-RNase alleles of this species have not been surveyed from natural populations and consequently the S-haplotype diversity is poorly known. Here the molecular and functional characterization of seven S-RNase candidate sequences, identified from a natural population of N. alata, are reported. Six of these candidates, S ₅ , S ₂₇ , S ₇₀ , S ₇₅ , S ₁₀₇ , and S ₂₁₀ , showed plant-specific amplification in the natural population and style-specific expression, which increased gradually during bud maturation, consistent with the reported S-RNase expression. In contrast, the S ₆₃ ribonuclease was present in all plants examined and was ubiquitously expressed in different organs and bud developmental stages. Genetic segregation analysis demonstrated that S ₂₇ , S ₇₀ , S ₇₅ , S ₁₀₇ , and S ₂₁₀ alleles were fully functional novel S-RNases, while S ₅ and S ₆₃ resulted to be non-S-RNases, although with a clearly distinct pattern of expression. These results reveal the importance of performing functional analysis in studies of S-RNase allelic diversity. Comparative phylogenetic analysis of six species of Solanaceae showed that N. alata S-RNases were included in eight transgeneric S-lineages. Phylogenetic pattern obtained from the inclusion of the novel S-RNase alleles confirms that N. alata represents a broad sample of the allelic variation at the S-locus of the Solanaceae.     

Plastid phylogenetics of Oceania yams (Dioscorea spp., Dioscoreaceae) reveals natural interspecific hybridization of the greater yam (D. alata)

Phylogenetic relationships of Oceanian staple yams (species of Dioscorea section Enantiophyllum) were investigated using plastid trnL‐F and rpl32‐trnL^(UAG) sequences and nine nuclear co‐dominant microsatellites. Analysis of herbarium specimens, used as taxonomic references, allowed the comparison with samples collected in the field. It appears that D. alata, D. transversa and D. hastifolia are closely related species. This study does not support a direct ancestry from D. nummularia to D. alata as previously hypothesized. The dichotomy in D. nummularia previously described by farmers in semi‐perennial and annual types was reflected by molecular markers, but the genetic structure of D. nummularia appears more complex. Dioscorea nummularia displayed two haplotypes, each corresponding to a different genetic group. One, including a D. nummularia voucher from New Guinea, is closer to D. tranversa, D. alata and D. hastifolia and encompasses only semi‐perennial types. The second group is composed of semi‐perennial and annual yams. However, some of these annual yams also displayed D. alata haplotypes. Nuclear markers revealed that some annual yams shared alleles with D. alata and semi‐perennial D. nummularia, suggesting a hybrid origin, which may explain their intermediate morphotypes and the difficulty met in classifying them.     

Structure and development of the pitchers from the carnivorous plantNepenthes alata (Nepenthaceae)

The pitchers of the tropical carnivorous plant Nepenthes alata are highly specialized organs for the attraction and capture of insects and absorption of nutrients from them. This study examined the structure and development of these pitchers, with particular focus on the nectaries and digestive glands. Immature pitchers developed at the tips of tendrils and were tightly sealed by a lid structure that opened during the end of pitcher elongation. Opened pitchers exposed a ridged peristome containing large nectaries. Like other members of the genus, a thick coating of epicuticular waxy scales covered the upper one-third of the pitcher. Scattered within this zone were cells resembling a stomatal complex with a protruding ridge. Cross sections showed that this ridge was formed by asymmetric divisions of the epidermal cells and lacked an underlying pore. The basal region of the trap had large multicellular glands that developed from single epidermal cells. These glands were closely associated with underlying vascular traces and provided a mechanism for supplying fluid to closed immature pitchers.     

High bioassay values in Terminalia alata leaves: indication of Cu mineralisation in Malanjkhand Granitoid,Central India

Abstract

Eighteen plant species belonging to 11 families commonly growing in the Pathratola, Dhorli and Pipardhar areas of the Malanjkhand Copper Province were studied. High Cu values were observed in the leaves of the abundant species of Terminalia alata in Pipardhar area. The leaves of T. alata also show wide pre- and post-monsoon seasonal variations in their Cu contents (average: 593-1,713 μg g^-1). Amongst several samples from various species, most of the leaf samples of T. alata, collected from the Pipardhar area showed high Cu values that lie within and occupy the high ends of cumulative probability curves. Anomalous Cu values of > 200 and > 497 μg g^-1were obtained for most of the T. alata leaves of the Pipradhar area for pre- and post-monsoon periods, respectively. The correlation coefficient values between Cu contents of T. alata leaves and substrate soil do not show a significant relationship for any area in either season. However, t- test results between Cu contents of T. alata leaves and substrate soil samples indicated a significant relationship for the samples collected from the Pipardhar and Pathratola areas during the pre-monsoon period. For post-monsoon period, the t-test results show non-significant relationship in the Pipardhar area. Another sampling area selected adjacent to the Pipardhar area had significance in the t-test results between the two sample periods. Polynomial curves revealed that 465 and 480 μg g^-1 of Cu in the soil are the “toxicity threshold limits” for the pre- and post-monsoon periods, respectively. Possible reasons attributed to the high bioassay values found in the T. alata leaves have been discussed in the paper.     

Phylogeographic concordance factors quantify phylogeographic congruence among co‐distributed species in the Sarracenia alata pitcher plant system

Comparative phylogeographic investigations have identified congruent phylogeographic breaks in co‐distributed species in nearly every region of the world. The qualitative assessments of phylogeographic patterns traditionally used to identify such breaks, however, are limited because they rely on identifying monophyletic groups across species and do not account for coalescent stochasticity. Only long‐standing phylogeographic breaks are likely to be obvious; many species could have had a concerted response to more recent landscape events, yet possess subtle signs of phylogeographic congruence because ancestral polymorphism has not completely sorted. Here, we introduce Phylogeographic Concordance Factors (PCFs), a novel method for quantifying phylogeographic congruence across species. We apply this method to the Sarracenia alata pitcher plant system, a carnivorous plant with a diverse array of commensal organisms. We explore whether a group of ecologically associated arthropods have co‐diversified with the host pitcher plant, and identify if there is a positive correlation between ecological interaction and PCFs. Results demonstrate that multiple arthropods share congruent phylogeographic breaks with S. alata, and provide evidence that the level of ecological association can be used to predict the degree of similarity in the phylogeographic pattern. This study outlines an approach for quantifying phylogeographic congruence, a central concept in biogeographic research.     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.     

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata

Defensins are a large family of small, cysteine‐rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C‐terminal pro‐peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt‐thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC₅₀) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5–10 times less effective at killing necrotrophs, but only two‐fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad‐spectrum resistance to pathogens.     

Polyploidy and sterility in relation to sex in Dioscorea alata L. (Dioscoreaceae)

Chromosome numbers and fertility studies of 73 male and 30 female flowering germplasm accessions of Dioscorea alata L. were carried out. All males were tetraploids showing the same chromosome number (n=20 or 2n=40) and were pollen fertile (10.9–96.2%), most of them being highly fertile. Among the female the majority were higher ploids (hexa-and octoploids; 2n=60 and 80) and sexually completely sterile. There were only two tetraploid female accessions which were sexually fertile. Pollination studies revealed that seed sterility in D. alata was due to female sterility associated with the occurrence of higher levels of ploidy. The female sex-limited occurrence of higher polyploidy and sterility observed in D. alata is a curious situation among dioecious higher plants.