Does hybridization of endophytic symbionts in a native grass increase fitness in resource-limited environments?

Hybridization is common among plants, animals and microbes. However, the ecological consequences of hybridization for microbes are far less understood than for plants and animals. For symbiotic Epichloë fungi, hybridization is widespread and may augment the well-known benefits of the endophytes to their grass hosts, especially in stressful environments. We tested the hybrid fitness hypothesis (HFH) that hybrid endophytes enhance fitness in stressful environments relative to non-hybrid endophytes. In a long-term field experiment, we monitored growth and reproduction of hybrid-infected (H+), non-hybrid infected (NH+), naturally endophyte free (E-) plants and those plants from which the endophyte had been experimentally removed (H- and NH-) in resource-rich and resource-poor environments. Infection by both endophyte species enhanced growth and reproduction. H+ plants outperformed NH+ plants in terms of growth by the end of the experiment, supporting HFH. However, H+ plants only outperformed NH+ plants in the resource-rich treatment, contrary to HFH. Plant genotypes associated with each endophyte species had strong effects on growth and reproduction. Our results provide some support the HFH hypothesis but not based upon adaptation to stressful environments. Our results reinforce the notion of a complex interplay between endophyte and plant genotype and environmental factors that determine fitness of the symbiotum.

Alkaloid variation among epichloid endophytes of sleepygrass (Achnatherum robustum) and consequences for resistance to insect herbivores.

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloë funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

The effects of endophytes on seed production and seed predation of tall fescue and meadow fescue.

Fungal endophytes of grasses are often included in agricultural management and in ecological studies of natural grass populations. In European agriculture and ecological studies, however, grass endophytes are largely ignored. In this study, we determined endophyte infection frequencies of 13 European cultivars and 49 wild tall fescue (Schedonorus phoenix) populations in Northern Europe. We then examined seed production and seed predation of endophyte-infected (E+) and endophyte-free (E-) tall fescue (in wild grass populations and in a field experiment) and meadow fescue (Schedonorus pratensis; in a field experiment only). Endophytes were detected in only one of the 13 cultivars. In contrast, >90% of wild tall fescue plants harbored endophytes in 45 wild populations but were absent in three inland populations in Estonia. In three wild tall fescue study sites, 17%, 22%, and 56% of the seeds were preyed upon by the cocksfoot moth. Endophyte infection did not affect seed mass of tall fescue in the field experiment. However, seed predation was lower in E+ than E- grasses in the two tall fescue populations with higher predation rates. For meadow fescue, the mean number of seeds from E+ plants was higher than E- plants, but E- and E+ seeds had equal rates of predation by the moth. Our results suggest that the effects of grass endophytes on seed production and cocksfoot moth seed predation vary considerably among grass species, and the effects may depend on herbivore pressure and other environmental conditions.

Comparison of electrospray ionization and atmospheric pressure photoionization liquid chromatography mass spectrometry methods for analysis of ergot alkaloids from endophyte-infected sleepygrass (Achnatherum robustum).

Ergot alkaloids are mycotoxins with an array of biological effects. With this study, we investigated for the first time the application of atmospheric pressure photoionization (APPI) as an ionization method for LC-MS analysis of ergot alkaloids, and compared its performance to that of the more established technique of electrospray ionization (ESI). Samples of the grass Achnatherum robustum infected with the ergot producing Epichloë fungus were extracted using cold methanol and subjected to reserved-phase HPLC-ESI-MS and HPLC-APPI-MS analysis. The ergot alkaloids ergonovine and lysergic acid amide were detected in these samples, and quantified via external calibration. Validation parameters were recorded in accordance with ICH guidelines. A triple quadrupole MS operated in multiple reaction monitoring yielded the lowest detection limits. The performance of APPI and ESI methods was comparable. Both methods were subject to very little matrix interference, with percent recoveries ranging from 82% to 100%. As determined with HPLC-APPI-MS quantification, lysergic acid amide and ergonovine were extracted from an A. robustum sample infected with the Epichloë fungus at concentrations of 1.143±0.051 ppm and 0.2822±0.0071 ppm, respectively. There was no statistically significant difference between these concentrations and those determined using ESI for the same samples.

A new type F Wolbachia from Splendidofilariinae (Onchocercidae) supports the recent emergence of this supergroup.

Wolbachia are vertically transmitted endosymbiotic bacteria of arthropods and onchocercid nematodes. It is commonly accepted that they co-evolved with their filarial hosts, and have secondarily been lost in some species. However, most of the data on the Wolbachia/Onchocercidae relationship have been derived from studies on two subfamilies, the Dirofilariinae and the Onchocercinae, which harbour parasites of humans and domestic animals. Within the last few years, analyses of more diverse material have suggested that some groups of Onchocercidae do not have Wolbachia, such as recently studied Splendidofilariinae from birds. This study takes advantage of the analysis of additional Splendidofilariinae, Rumenfilaria andersoni from a Finnish reindeer and Madathamugadia hiepei from a South African gecko, using PCR, immunohistochemical staining and whole-mount fluorescent analysis to detect Wolbachia and describe its strains. A DNA barcoding approach and phylogenetic analyses were used to investigate the symbiosis between Wolbachia and the Onchocercidae. A new supergroup F Wolbachia was demonstrated in M. hiepei, representing the first filarial nematode harbouring Wolbachia described in a non-mammalian host. In the adult, Wolbachia infects the female germline but not the hypodermis, and intestinal cells are also infected. The phylogenetic analyses confirmed a recent emergence of supergroup F. They also suggested several events of horizontal transmission between nematodes and arthropods in this supergroup, and the existence of different metabolic interactions between the filarial nematodes and their symbionts.

Urban biodiversity: patterns and mechanisms.

The patterns of biodiversity changes in cities are now fairly well established, although diversity changes in temperate cities are much better studied than cities in other climate zones. Generally, plant species richness often increases in cities due to importation of exotic species, whereas animal species richness declines. Abundances of some groups, especially birds and arthropods, often increase in urban areas despite declines in species richness. Although several models have been proposed for biodiversity change, the processes underlying the patterns of biodiversity in cities are poorly understood. We argue that humans directly control plants but relatively few animals and microbes-the remaining biological community is determined by this plant "template" upon which natural ecological and evolutionary processes act. As a result, conserving or reconstructing natural habitats defined by vegetation within urban areas is no guarantee that other components of the biological community will follow suit. Understanding the human-controlled and natural processes that alter biodiversity is essential for conserving urban biodiversity. This urban biodiversity will comprise a growing fraction of the world's repository of biodiversity in the future.

Fungal-mediated multitrophic interactions--do grass endophytes in diet protect voles from predators?

Plant-associated micro-organisms such as mycotoxin-producing endophytes commonly have direct negative effects on herbivores. These effects may be carried over to natural enemies of the herbivores, but this has been rarely explored. We examined how feeding on Neotyphodium endophyte infected (E+) and endophyte free (E-) meadow ryegrass (Scherodonus pratensis) affects body mass, population size and mobility of sibling voles (Microtus levis), and whether the diet mediates the vulnerability of voles to least weasel (Mustela nivalis nivalis) predation. Because least weasels are known to be olfactory hunters, we also examined whether they are able to distinguish olfactory cues of voles fed on E+ and E- diets. Neither body mass of voles nor population size differed between diets. However, contrary to our prediction, least weasels preyed more often on voles fed with E- grass than on voles fed with E+ grass. The mobility of voles fed on E+ grass was reduced compared to voles fed on E- grass, but this effect was unrelated to risk of predation. Least weasels appeared unable to distinguish between excrement odours of voles between the two treatments. Our results suggest that consumption of endophytic grass is not directly deleterious to sibling voles. What's more, consumption of endophytes appears to be advantageous to voles by reducing risk of mammalian predation. Our study is thus the first to demonstrate an effect of plant-associated microbial symbionts on herbivore-predator interactions in vertebrate communities.