Population studies of native grass-endophyte symbioses provide clues for the roles of host jumps and hybridization in driving their evolution.

Fungal endophytes in the genera Epichloë and Neotyphodium, collectively termed the epichloae, have fascinated biologists for decades. These intriguing fungi, also referred to as 'class 1 or clavicipitaceous endophytes', spend the large majority, or even their entire life cycle, within the tissues of their cool-season grass hosts without eliciting any symptoms of infection. While all epichloae reside within the intercellular spaces of aboveground vegetative grass tissues, the species at the symbiotic extreme are known as Neotyphodium, and the intimacy of their interaction extends to the reproductive (flowering) stage. At this point, fungal filaments (hyphae) nondestructively invade the developing ovaries of their host and are incorporated into perfectly viable, healthy seeds. Thus, these endophytes live solely within the tissues of their host plants and are transmitted maternally from generation to generation. A second life history characteristic of interest is that while all Epichloë and some Neotyphodium species are haploid, a great many of the strictly seed-transmitted Neotyphodium spp. are interspecific hybrids. This phenomenon may be critical for the success of these symbioses over longer spans of evolutionary time and will be discussed in greater detail below. A third characteristic, and one of the primary reasons these grass endophytes have received so much attention over the last three decades, is the strong mutualistic nature these relationships often exhibit. In exchange for photosynthetically derived carbon, the endophytes protect their cool-season grass hosts from grazing herbivores and a variety of abiotic stresses. It has been hypothesized that these three biological phenomena are related (Schardl & Craven 2003), perhaps with the former two driving the third, and it is here that the recent article in Molecular Ecology entitled 'Genetic diversity in epichloid endophytes of Hordelymus europaeus suggests repeated host jumps and interspecific hybridizations', by Oberhofer & Leuchtmann (2012), provides critical clues to linking these traits together. While the large majority of studies have focused on documenting the ever-increasing list of mutualistic qualities attributed to these fungi, very few have taken an exhaustive population-level approach to document plant and endophyte genotypes within a naturally occurring system (Faeth et al. 2010; Jani et al. 2010; Tintjer & Rudgers 2006). Such information is crucial to more fully elucidate the factors shaping grass-endophyte symbioses and those often driving these relationships to mutualistic extremes.

A novel test for host-symbiont codivergence indicates ancient origin of fungal endophytes in grasses.

Significant phylogenetic codivergence between plant or animal hosts (H) and their symbionts or parasites (P) indicates the importance of their interactions on evolutionary time scales. However, valid and realistic methods to test for codivergence are not fully developed. One of the systems where possible codivergence has been of interest involves the large subfamily of temperate grasses (Pooideae) and their endophytic fungi (epichloae). These widespread symbioses often help protect host plants from herbivory and stresses and affect species diversity and food web structures. Here we introduce the MRCALink (most-recent-common-ancestor link) method and use it to investigate the possibility of grass-epichloë codivergence. MRCALink applied to ultrametric H and P trees identifies all corresponding nodes for pairwise comparisons of MRCA ages. The result is compared to the space of random H and P tree pairs estimated by a Monte Carlo method. Compared to tree reconciliation, the method is less dependent on tree topologies (which often can be misleading), and it crucially improves on phylogeny-independent methods such as ParaFit or the Mantel test by eliminating an extreme (but previously unrecognized) distortion of node-pair sampling. Analysis of 26 grass species-epichloë species symbioses did not reject random association of H and P MRCA ages. However, when five obvious host jumps were removed, the analysis significantly rejected random association and supported grass-endophyte codivergence. Interestingly, early cladogenesis events in the Pooideae corresponded to early cladogenesis events in epichloae, suggesting concomitant origins of this grass subfamily and its remarkable group of symbionts. We also applied our method to the well-known gopher-louse data set.

Molecular identification of the turf grass rapid blight pathogen.

Rapid blight is a newly described disease on turf grasses, primarily found on golf courses using suboptimal water for irrigation purposes. On the basis of shared morphological characteristics, it has been proposed that the rapid blight pathogen belongs to a genus of stramenopiles, Labyrinthula, which had been known to cause disease of marine plants only. We have collected 10 isolates from four species of turf grass in five states and sequenced portions of the SSU (18S) rDNA gene from each to provide a definitive taxonomic placement for rapid blight pathogens. We also included sequences from Labyrinthuloides yorkensis, Schizochytrium aggregatum, Aplanochytrium sp., Thraustochytrium striatum, Achlya bisexualis and several nonturf-grass isolates of Labyrinthula. We found that rapid blight isolates indeed are placed firmly within the genus Labyrinthula and that they lack detectable genetic diversity in the 18S rDNA region. We propose that the rapid blight pathogens share a recent common ancestor and might have originated from a single, infected population.

Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses.

Epichloë endophytes are fungal symbionts of grasses that span a continuum including asexual mutualists that are vertically transmitted, obligately sexual pathogens that are horizontally transmitted, and mixed-strategy symbionts with both mutualistic and pathogenic capabilities. Here we show that processes of genome evolution differ markedly for the different symbiont types. Genetic and phylogenetic analysis was conducted of a broad taxonomic, ecological and geographical sample of sexual and asexual isolates, in which were identified and sequenced alleles of genes for beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1), and microsatellite alleles were identified by length polymorphisms. The majority of asexual isolates had two or three alleles of most loci, but every sexual isolate had only single alleles for each locus. Phylogenetic analysis of tub2 and tef1 indicated that in all instances of multiple alleles in an isolate, the alleles were derived from different sexual species. It is concluded that, whereas horizontally transmissible species had haploid genomes and speciation occurred cladistically, most of the strictly seedborne mutualists were interspecific hybrids with heteroploid (aneuploid or polyploid) genomes. Furthermore, the phylogenetic evidence indicated that, in at least some instances, hybridization followed rather than caused evolution of the strictly seedborne habit. Therefore, the abundance of hybrid species among grass endophytes, and their prevalence in many host populations suggests a selective advantage of hybridization for the mutualistic endophytes.

Interspecific hybridization in plant-associated fungi and oomycetes: a review.

Fungi (kingdom Mycota) and oomycetes (kingdom Stramenopila, phylum Oomycota) are crucially important in the nutrient cycles of the world. Their interactions with plants sometimes benefit and sometimes act to the detriment of humans. Many fungi establish ecologically vital mutualisms, such as in mycorrhizal fungi that enhance nutrient acquisition, and endophytes that combat insects and other herbivores. Other fungi and many oomycetes are plant pathogens that devastate natural and agricultural populations of plant species. Studies of fungal and oomycete evolution were extraordinarily difficult until the advent of molecular phylogenetics. Over the past decade, researchers applying these new tools to fungi and oomycetes have made astounding new discoveries, among which is the potential for interspecific hybridization. Consequences of hybridization among pathogens include adaptation to new niches such as new host species, and increased or decreased virulence. Hybrid mutualists may also be better adapted to new hosts and can provide greater or more diverse benefits to host plants.

Extrapericardial and esophageal pressures with positive end-expiratory pressure in dogs.

Using flat balloon techniques to minimize distortion and artifacts, we studied the effect of positive end-expiratory pressure (PEEP) on local surface pressures between the lung and pericardium overlying the right (R) and left (L) ventricles of ventilated closed-chest anesthetized dogs in right lateral decubitus position. To test the hypothesis that local extrapericardial [Pep(L) and Pep(R)] and average pleural pressures change equally with PEEP, we also measured esophageal pressure (Pes). When 10-cmH2O PEEP was applied, mean increases in Pes, Pep(L), and Pep(R) were 6.2, 5.6, and 5.3 cmH2O, respectively. When PEEP was raised to 20 cmH2O, further average increases in Pes, Pep(L), and Pep(R) were 5.8, 5.0, and 5.4 cmH2O. At each level of PEEP, volume infusion was used to increase stroke volume. Volume infusion at 20-cmH2O PEEP was associated with small 1.0- and 1.5-cmH2O increases in Pep but no change in Pes. Analysis of confidence limits showed that application of up to 20-cmH2O PEEP, with or without volume infusion to restore stroke volume, is associated with nearly equal changes in esophageal and local extrapericardial pressures.

Effects of contusion and flail chest on pulmonary perfusion and oxygen exchange.

Localized pulmonary contusions were produced in the right lower lobes (RLL) of 12 anesthetized ventilated dogs, 6 of which had a flail segment in the chest wall over the RLL. Pulmonary oxygen exchange during ventilation with air and oxygen, and the lobar distribution of pulmonary perfusion by radioactive microsphere techniques were measured before and 3 h after contusion, and again after thoracotomy. These were compared to 12 noncontused dogs, 6 of which had a flail segment. Contusion produced an average decrease of 20 Torr in Pao2 during air breathing and an average increase in Qs/Qt of less than 5%, surprisingly small given the doubled weight and average 44% shunt calculated in the contused lobe after thoracotomy. No significant effect of flail or thoracotomy was found, indicating that the presence of an intact chest wall and lung-chest wall interdependence was not a major factor preventing a larger increase in intact whole-animal shunt of contused dogs. Rather, the small effect of this severe lobar injury on whole-animal shunt was due to a 30% decrease in RLL relative perfusion. This reduction was demonstrated to be localized to a smaller hemorrhagic subsection of the contused lobe.

Pathophysiology of pulmonary contusion in dogs.

We produced a localized right lower lobe (RLL) contusion in 14 anesthetized ventilated dogs, 7 of which were treated with positive end-expiratory pressure (PEEP group). We measured gas exchange, pulmonary mechanics, and regional function before and 5 h after the contusion. Arterial PO2 decreased by 20 Torr and venous admixture doubled in both groups during air breathing. The shunt fraction (Qs/Qt) was minimally increased, despite a large lobar Qs/Qt (0.43) in the contused RLL. These results were explained by reduced ventilation per unit volume (VA/V), and ventilation-to-perfusion ratios of the contused RLL measured with 133Xe technique. We conclude that pulmonary contusion causes a leak of blood and plasma, flooding 25% of the air spaces of the RLL at FRC, reducing the compliance of adjacent air spaces, and resulting in a reduced VA/V and a large RLL Qs/Qt. These results are consistent with the observed reduction in regional volume and perfusion in the contused RLL, and suggest that Qs/Qt was not increased because blood flow was markedly reduced to flooded air spaces. PEEP reduced the hypoxemia, but increased the contusion.