Developmental origins of precocial forelimbs in marsupial neonates.

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.

From offshore to onshore: multiple origins of shallow-water corals from deep-sea ancestors.

Shallow-water tropical reefs and the deep sea represent the two most diverse marine environments. Understanding the origin and diversification of this biodiversity is a major quest in ecology and evolution. The most prominent and well-supported explanation, articulated since the first explorations of the deep sea, holds that benthic marine fauna originated in shallow, onshore environments, and diversified into deeper waters. In contrast, evidence that groups of marine organisms originated in the deep sea is limited, and the possibility that deep-water taxa have contributed to the formation of shallow-water communities remains untested with phylogenetic methods. Here we show that stylasterid corals (Cnidaria: Hydrozoa: Stylasteridae)--the second most diverse group of hard corals--originated and diversified extensively in the deep sea, and subsequently invaded shallow waters. Our phylogenetic results show that deep-water stylasterid corals have invaded the shallow-water tropics three times, with one additional invasion of the shallow-water temperate zone. Our results also show that anti-predatory innovations arose in the deep sea, but were not involved in the shallow-water invasions. These findings are the first robust evidence that an important group of tropical shallow-water marine animals evolved from deep-water ancestors.

High-throughput isolation and mapping of C. elegans mutants susceptible to pathogen infection.

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5-8 megabase sub-chromosomal region.

C. elegans germline-deficient mutants respond to pathogen infection using shared and distinct mechanisms.

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including gram positive and gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Independent evolutionary origins of landlocked alewife populations and rapid parallel evolution of phenotypic traits.

Alewife, Alosa pseudoharengus, populations occur in two discrete life-history variants, an anadromous form and a landlocked (freshwater resident) form. Landlocked populations display a consistent pattern of life-history divergence from anadromous populations, including earlier age at maturity, smaller adult body size, and reduced fecundity. In Connecticut (USA), dams constructed on coastal streams separate anadromous spawning runs from lake-resident landlocked populations. Here, we used sequence data from the mtDNA control region and allele frequency data from five microsatellite loci to ask whether coastal Connecticut landlocked alewife populations are independently evolved from anadromous populations or whether they share a common freshwater ancestor. We then used microsatellite data to estimate the timing of the divergence between anadromous and landlocked populations. Finally, we examined anadromous and landlocked populations for divergence in foraging morphology and used divergence time estimates to calculate the rate of evolution for foraging traits. Our results indicate that landlocked populations have evolved multiple times independently. Tests of population divergence and estimates of gene flow show that landlocked populations are genetically isolated, whereas anadromous populations exchange genes. These results support a 'phylogenetic raceme' model of landlocked alewife divergence, with anadromous populations forming an ancestral core from which landlocked populations independently diverged. Divergence time estimates suggest that landlocked populations diverged from a common anadromous ancestor no longer than 5000 years ago and perhaps as recently as 300 years ago, depending on the microsatellite mutation rate assumed. Examination of foraging traits reveals landlocked populations to have significantly narrower gapes and smaller gill raker spacings than anadromous populations, suggesting that they are adapted to foraging on smaller prey items. Estimates of evolutionary rates (in haldanes) indicate rapid evolution of foraging traits, possibly in response to changes in available resources.

The evolutionary origins of human patience: temporal preferences in chimpanzees, bonobos, and human adults.

To make adaptive choices, individuals must sometimes exhibit patience, forgoing immediate benefits to acquire more valuable future rewards [1-3]. Although humans account for future consequences when making temporal decisions [4], many animal species wait only a few seconds for delayed benefits [5-10]. Current research thus suggests a phylogenetic gap between patient humans and impulsive, present-oriented animals [9, 11], a distinction with implications for our understanding of economic decision making [12] and the origins of human cooperation [13]. On the basis of a series of experimental results, we reject this conclusion. First, bonobos (Pan paniscus) and chimpanzees (Pan troglodytes) exhibit a degree of patience not seen in other animals tested thus far. Second, humans are less willing to wait for food rewards than are chimpanzees. Third, humans are more willing to wait for monetary rewards than for food, and show the highest degree of patience only in response to decisions about money involving low opportunity costs. These findings suggest that core components of the capacity for future-oriented decisions evolved before the human lineage diverged from apes. Moreover, the different levels of patience that humans exhibit might be driven by fundamental differences in the mechanisms representing biological versus abstract rewards.

Biogeographic analysis of the woody plants of the Southern Appalachians: Implications for the origins of a regional flora.

PREMISE OF THE STUDY: We investigated the origins of 252 Southern Appalachian woody species representing 158 clades to analyze larger patterns of biogeographic connectivity around the northern hemisphere. We tested biogeographic hypotheses regarding the timing of species disjunctions to eastern Asia and among areas of North America. METHODS: We delimited species into biogeographically informative clades, compiled sister-area data, and generated graphic representations of area connections across clades. We calculated taxon diversity within clades and plotted divergence times. KEY RESULTS: Of the total taxon diversity, 45% were distributed among 25 North American endemic clades. Sister taxa within eastern North America and eastern Asia were proportionally equal in frequency, accounting for over 50% of the sister-area connections. At increasing phylogenetic depth, connections to the Old World dominated. Divergence times for 65 clades with intercontinental disjunctions were continuous, whereas 11 intracontinental disjunctions to western North America and nine to eastern Mexico were temporally congruent. CONCLUSIONS: Over one third of the clades have likely undergone speciation within the region of eastern North America. The biogeographic pattern for the region is asymmetric, consisting of mostly mixed-aged, low-diversity clades connecting to the Old World, and a minority of New World clades. Divergence time data suggest that climate change in the Late Miocene to Early Pliocene generated disjunct patterns within North America. Continuous splitting times during the last 45 million years support the hypothesis that widespread distributions formed repeatedly during favorable periods, with serial cooling trends producing pseudocongruent area disjunctions between eastern North America and eastern Asia.

Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.

Pathogen-Specific Adaptations to Conserved Signaling Pathways in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.

Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.

The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.

To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.