Co-evolution between Frankia populations and host plants in the family Casuarinaceae and consequent patterns of global dispersal

Symbioses between the root nodule-forming, nitrogen-fixing actinomycete Frankia and its angiospermous host plants are important in the nitrogen economies of numerous terrestrial ecosystems. Molecular characterization of Frankia strains using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses of the 16S rRNA-ITS gene and of the nifD-nifK spacer was conducted directly on root nodules collected worldwide from Casuarina and Allocasuarina trees. In their native habitats in Australia, host species contained seven distinctive sets of Frankia in seven different molecular phylogenetic groups. Where Casuarina and Allocasuarina trees are newly planted outside Australia, they do not normally nodulate unless Frankia is introduced with the host seedling. Nodules from Casuarina trees introduced outside Australia over the last two centuries were found to contain Frankia from only one of the seven phylogenetic groups associated with the host genus Casuarina in Australia. The phylogenetic group of Frankia found in Casuarina and Allocasuarina trees introduced outside Australia is the only group that has yielded isolates in pure culture, suggesting a greater ability to survive independently of a host. Furthermore, the Frankia species in this group are able to nodulate a wider range of host species than those in the other six groups. In baiting studies, Casuarina spp. are compatible with more Frankia microsymbiont groups than Allocasuarina host spp. adapted to drier soil conditions, and C. equisetifolia has broader microsymbiont compatibility than other Casuarina spp. Some Frankia associated with the nodular rhizosphere and rhizoplan, but not with the nodular tissue, of Australian hosts were able to nodulate cosmopolitan Myrica plants that have broad microsymbiont compatibility and, hence, are a potential host of Casuarinaceae-infective Frankia outside the hosts' native range. The results are consistent with the idea that Frankia symbiotic promiscuity and ease of isolation on organic substrates, suggesting saprophytic potential, are associated with increased microsymbiont ability to disperse and adapt to diverse new environments, and that both genetics and environment determine a host's nodular microsymbiont.     

Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.     

Life beneath the surface of the central Texan Balcones Escarpment: genus Anillinus Casey, 1918 (Coleoptera,Carabidae, Bembidiini): new species,a key to the Texas species,and notes about their way of life and evolution

The Texas fauna of the genus Anillinus Casey, 1918 includes three previously described species (A. affabilis (Brues), 1902, A. depressus (Jeannel), 1963 and A. sinuatus (Jeannel), 1963) and four new species here described: A. acutipennis Sokolov & Reddell, sp. n. (type locality: Fort Hood area, Bell County, Texas); A. comalensis Sokolov & Kavanaugh, sp. n. (type locality: 7 miles W of New Braunfels, Comal County, Texas); A. forthoodensis Sokolov & Reddell, sp. n. (type locality: Fort Hood area, Bell County, Texas); A. wisemanensis Sokolov & Kavanaugh, sp. n. (type locality: Wiseman Sink, Hays County, Texas). A key for identification of adults of these species is provided. The fauna includes both soil- and cave-inhabiting species restricted to the Balcones Fault Zone and Lampasas Cut Plain and adjacent areas underlain by the Edwards-Trinity Aquifer. Based on morphological and distributional data, we hypothesize that four lineages of endogean Anillinus species extended their geographical ranges from a source area in the Ouachita-Ozark Mountains to the Balconian region in central Texas. There the cavernous Edwards-Trinity aquifer system provided an excellent refugium as the regional climate in the late Tertiary and early Quaternary became increasingly drier, rendering life at the surface nearly impossible for small, litter-inhabiting arthropods. Isolated within the Edwards-Trinity aquifer system, these anilline lineages subsequently differentiated, accounting for the currently known diversity. The paucity of specimens and difficulty in collecting them suggest that additional undiscovered species remain to be found in the region.     

Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR and restriction fragment length polymorphism analysis with crushed root nodules.

DNA extracted directly from nodules was used to assess the genetic diversity of Frankia strains symbiotically associated with two species of the genus Casuarina and two of the genus Allocasuarina naturally occurring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of Casuarina-infective Frankia strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the nif operon. PCR products were then analyzed by using a set of restriction endonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of the eight unisolated Frankia strains from Casuarina equisetifolia in Australia. Group 2 consisted of two unisolated Frankia strains from C. equisetifolia, whereas groups 3 to 5 comprised all unisolated strains from Casuarina cunninghamiana, Allocasuarina torulosa, and Allocasuarina littoralis, respectively. These results demonstrate that, contrary to the results of previous molecular studies of isolated strains, there is genetic diversity among Frankia strains that infect members of the family Casuarinacaeae. The apparent high homogeneity of Frankia strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia.     

The occurrence of haemoglobin and hydrogenase in nodules of twelve Casuarina-Frankia symbiotic associations

Seedlings of Casuarina cunninghamiana Miq., C. equisetifolia L. and C. glauca Sieber growing in N-deficient potting mix were inoculated with Frankia sp. from each of 4 different sources. After ca 4 months, plants were harvested and nodules from the 12 Casuarina-Frankia combinations evaluated for (1) concentrations of haemoglobin (measured as CO-reactive haem) and (2) occurrence of hydrogenase. The aim was to determine if these factors were related to nitrogen accumulation and biomass production. There were marked host-Frankia source interactions with up to 10-fold differences in plant dry weight and 50-fold differences in the efficiency of nitrogen fixation (as estimated by N₂ accumulated mg^?1 nodule dry weight). Differences in plant growth and nitrogen accumulation were apparently related to nodule specific activity, because the 12 associations had similar nodulation characteristics, e.g. time for nodulation to occur. The concentration of haemoglobin in Casuarina nodules ranged from 0 to 27 nmol haem (g FW)^?1. There was a strong linear correlation between concentrations of haemoglobin and dry weights of the whole plants (r=0.77, 0.92 and 0.97, P≤0.05) for C. cunninghamiana, C. equisetifolia and C. glauca symbiotic associations, respectively. However, the linear correlation between concentration of haemoglobin and nitrogen content of whole plant was lower (r=0.60, 0.64 and 0.71, P≤0.05) for the three Casuarina symbioses, respectively, and there was only a poor correlation between haemoglobin concentration in nodules and the rate of nitrogen accumulation on nodule weight basis. This indicates that haemoglobin concentration is not the sole physiological determinant of nitrogen fixation in Casuarina. All the Casuarina-Frankia symbiotic associations studied also showed the presence of a hydrogen uptake enzyme. The activity of the enzyme ranged from 5.1 to 34.1 μmol H₂ (g FW)^?1 h^?1, and hydrogen uptake was not correlated with plant dry weight, nitrogen content or the rate of nitrogen fixation. Hydrogen evolution could not be detected in any of the associations.     

Do single nodules of Casuarinaceae contain more than oneFrankia strain?

Summary Studies using inoculum suspensions prepared from single nodules of Casuarinaceae suggest that more than oneFrankia strain may be found in some nodules. Evidence is also presented to support the concept of intergeneric specificity in host-Frankia relations in the family Casuarinaceae.     

Floral attraction and flower visitors of a subcanopy,tropical rainforest tree,Fontainea picrosperma

1. Flowering plants in tropical rainforests rely heavily on pollen vectors for successful reproduction. Research into pollination systems in tropical rainforests is dominated by canopy species, while subcanopy plant–pollinator interactions remain under‐represented. The microclimate beneath the rainforest canopy is characterized by low light levels and is markedly different from the canopy environment that receives more light energy.
2. We studied the floral attractants and floral visitors of a dioecious, subcanopy tree, Fontainea picrosperma (Euphorbiaceae), in the Wet Tropics bioregion of northern Queensland, Australia.
3. We found that wind pollination is rare and male and female flowers do not produce nectar. Female flowers are likely pollinated due to their perceptual similarity to pollen‐offering male flowers. Female flowers had the same scent profile as male flowers, and floral scent was an important floral attractant that acted to regulate pollinator behavior. The two most abundant scent compounds present in the floral bouquet were benzyl alcohol and 4‐oxoisophorone. These compounds are ubiquitous in nature and are known to attract a wide variety of insects. Both day‐time and night‐time pollinators contributed to successful pollen deposition on the stigma, and diurnal flower visitors were identified from several orders of insects including beetles, flies, predatory wasps, and thrips. Fontainea picrosperma is therefore likely to be pollinated by a diverse array of small insects.
4. Synthesis. Our data indicate that F. picrosperma has a generalist, entomophilous pollination syndrome. The rainforest subcanopy is a distinctive environment characterized by low light levels, low or turbulent wind speeds, and relatively high humidity. Female flowers of F. picrosperma exhibit cost‐saving strategies by not producing nectar and mimicking the smell of reward‐offering male flowers. Insects opportunistically forage on or inhabit flowers, and pollination occurs from a pool of small insects with low energy requirements that are found beneath the rainforest canopy.

     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Dispersal of Spores of Mycorrhizal Fungi in Scats of Native Mammals in Tropical Forests of Northeastern Australia

One hundred and thirty-eight scat (faecal) samples from 17 mammal species native to forests of northeastern Queensland were examined for the presence of spores of both ectomycorrhizal and arbuscular mycorrhizal fungi. Spores of mycorrhizal fungi were found in 57 percent of scat samples representing 12 animal species (Aepyprymnus rufescens, Antechinus godmani, Bettongia tropica, Hypsiprymnodon moschatus, Isoodon macrourus, Melomys ceruinipes, Perameles nasuta, Rattus fuscipes, R. tunneyi, Thylogale stigmatica, Trichourur uulperula, Uromys caudimaculatus). Spores were absent in scats of Antechinus stuartii, Dasyurus hallucatus, Dendrolagus lumholtzi, Petaurus australis and Mesembriomys gouldii. Spores of ectomycorrhizal fungi occurred in 38 percent of scats, and all but one of these samples were from Eucalyptus-dominated sclerophyll forests. Based on the frequency and abundance of spores in scats, five mammals were considered active consumers of hypogeous mycorrhizal sporocarps in sclerophyll forests (A. rufescens, B. tropica, I. macrourus, P. nasuta, and U. caudimaculatus). Individual scats of these animals generally contained a range of distinctive spore types. Spores of arbuscular mycorrhizal fungi were found in low abundance in almost 40 percent of scat samples collected, from both sclerophyll forest and rainforest habitats. We suggest that the majoriry of these spores were acquired incidentally through ingestion of soil during foraging activities on the forest floor. Glasshouse inoculation experiments in which seedlings of Eucalyptus grandis and Sorghum bicolor were inoculated with scat material from several species of mammal demonstrated that the spores of ectomycorrhizal and arbuscular mycorrhizal fungi retained some viability and colonized the roots of host-plant seedlings. Insufficient information is known of the ecology of mycorrhizal fungi in Australia's tropical forests to speculate as to the implications of these findings for forest conservation and rehabilitation.     

The North American obligate cave fauna: regional patterns

The obligate cave faunas of nine regions of the United States –Florida Lime Sinks, Appalachians, Interior Low Plateaus, Ozarks, Driftless Area,Edwards Aquifer/Balcones Escarpment, Guadalupe Mountains, Black Hills, andMother Lode – are described and compared. The number of aquatic(stygobitic) species ranged from zero (Black Hills) to 82 (Appalachians), andthe number of terrestrial (troglobitic) species ranged from zero (Florida LimeSinks) to 256 (Interior Low Plateau). Even at the level of genus, overlapbetween regions is low. Several predictor variables (karst area, number ofcaves, number of long caves, number of deep caves, distance from the Pleistoceneice margin, distance from the late Cretaceous Sea, and vegetation type – asurrogate for productivity) were assessed using rank order statistics,especially rank order multiple regression with a backward elimination procedure.For both stygobites and troglobites, only number of caves was a significantpredictor. The absence of a karst area effect suggested that the degree of karstdevelopment is better described by the number of caves rather than area ofkarst. There was no evidence that distance to Pleistocene glacial boundaries wasimportant, but there was some support for the importance of distance from late Cretaceous sea margins, a potential source of aquatic subterranean colonists. Finally,there was no indication that surface productivity had an effect on speciesrichness. Analysis was complicated by correlations among predictor variables.