Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

Many birds initiate incubation before clutch completion, which results in asynchronous hatching. The ensuing within-brood size disparity often places later-hatched nestlings at a developmental disadvantage, but the functional significance of the timing of the onset of incubation is poorly understood. Early incubation may serve to maintain the viability of early-laid eggs, which declines over time owing to the putative effects of ambient temperature. An unexplored risk to egg viability is trans-shell infection by micro-organisms. We experimentally investigated the rate and magnitude of microbial trans-shell infection of the egg, and the relative effects of ambient temperature and micro-organisms on hatching success. We show that infection of egg contents is prevalent and occurs within the time required to lay a clutch. The probability of infection depends on the climatic conditions, the exposure period and the phylogenetic composition of the eggshell microbiota. We also demonstrate that microbial infection and ambient temperature act independently to reduce egg viability considerably. Our results suggest that these two factors could affect the onset of avian incubation in a wide range of environments.

Many avian species initiate incubation before clutch completion, which causes eggs to hatch asynchronously. This influences brood competitive dynamics and often results in nestling mortality. The prevailing hypotheses contend that parents incubate early because asynchronous hatching provides fitness benefits to parents or surviving offspring. An alternative idea is that early incubation is the best of a bad job because of the costs of delaying incubation to the viability of first-laid eggs. To explore this, we examined the potential for microbial infection, and the relative effects of infection and suboptimal development temperatures on the viability of pearly-eyed thrasher (Margarops fuscatus) eggs. We exposed newly laid eggs for 5 days at either end of a tropical altitudinal gradient and cleaned shells of half the eggs to reduce microbial growth. Uncleaned eggs were infected more than were cleaned eggs, and infection was greater for eggs exposed at the cool, humid site than at the hot, less humid site. Parentally incubated eggs, however, were not infected, suggesting that incubation limits infection. The consequence of exposure to infection and high ambient temperatures was a dramatic reduction in viability; cleaned eggs held at the cool site had the highest hatching success, which was significantly greater than for uncleaned eggs at this site and for cleaned eggs held at the hot site. This provides the first evidence that microbes can infect unincubated eggs of a wild bird, and that infection and ambient temperature act independently to reduce hatching success. These factors could affect avian life-history strategies in diverse habitats.

Avian eggshells harbour microbes shortly after laying, and under appropriate ambient conditions they can multiply rapidly, penetrate through shell pores, infect egg contents and cause embryo mortality. We experimentally examined how incubation affects bacterial processes on the eggshells of pearl-eyed thrashers Margarops fuscatus nesting in tropical montane and lowland forests in Puerto Rico. Bacteria and fungi grew rapidly on shells of newly laid, unincubated eggs exposed to ambient conditions, but declined to low levels on shells of eggs incubated by thrashers. Divergence in bacterial growth between incubated and exposed eggs was more marked at the montane forest than at the lowland site. Pathogenic microorganisms became increasingly dominant on shells of exposed eggs, but these groups were relatively rare on incubated eggs, where more benign, less invasive groups prevailed. Some incubation during laying may be necessary to decrease the probability of trans-shell infection by reducing the growth of harmful bacteria and fungi on eggshells, although it may increase hatching asynchrony and the likelihood of brood reduction.

No abstract

6Lisbon Oceanarium, 1999-005 Lisbon, Portugal A global phylogeny for chelonid fibropapilloma-associated herpesvirus (CFPHV), the most likely aetiological agent of fibropapillomatosis (FP) in sea turtles, was inferred, using dated sequences, through Bayesian Markov chain Monte Carlo analysis and used to estimate the virus evolutionary rate independent of the evolution of the host, and to resolve the phylogenetic positions of new haplotypes from Puerto Rico and the Gulf of Guinea. Four phylogeographical groups were identified: eastern Pacific, western Atlantic/eastern Caribbean, mid-west Pacific and Atlantic. The latter comprises the Gulf of Guinea and Puerto Rico, suggesting recent virus gene flow between these two regions. One virus haplotype from Florida remained elusive, representing either an independent lineage sharing a common ancestor with all other identified virus variants or an Atlantic representative of the lineage giving rise to the eastern Pacific group. The virus evolutionary rate ranged from 1.62¾10 "4 to 2.22¾10 "4 substitutions per site per year, which is much faster than what is expected for a herpesvirus. The mean time for the most recent common ancestor of the modern virus variants was estimated at 192.90-429.71 years ago, which, although more recent than previous estimates, still supports an interpretation that the global FP pandemic is not the result of a recent acquisition of a virulence mutation(s). The phylogeographical pattern obtained seems partially to reflect sea turtle movements, whereas altered environments appear to be implicated in current FP outbreaks and in the modern evolutionary history of CFPHV. INTRODUCTIONChelonid fibropapilloma-associated herpesvirus (CFPHV) is the most likely aetiological agent of fibropapillomatosis (FP) (Arthur et al., 2008a;Greenblatt et al., 2005;Lackovich et al., 1999;Quackenbush et al., 1998), a neoplastic disease of sea turtles, characterized by recent outbreaks (Diez et al., 2010;Foley et al., 2005;Work & Balazs, 1999). The tumours can be both external and internal and, although benign, depending on location and size, they may obstruct crucial functions such as swimming, feeding and sight, or may impede organ function (Herbst, 1994;Herbst & Klein, 1995). Severe FP also leads to bacteraemia (Work et al., 2003). The most susceptible life stages appear to be neritic juveniles and subadults, whereas in adults the disease is rare (Ene et al., 2005;Herbst & Klein, 1995;Work et al., 2004). A high prevalence of FP is common in anthropogenically altered environments (Aguirre & Lutz, 2004;Herbst, 1994;Van Houtan et al., 2010), suggesting that factors in these environments promote disease outbreaks, for example, through the enhancement of virus transmissibility and/or the enhancement of disease expression via substances thatThe GenBank/EMBL/DDBJ accession numbers for the CFPHV sequences determined in this study are JN580279-JN580296 and JN625251-JN625262.Further details about samples are available with the online version of this paper. Phylogenies of herpesv...

The process of natural mummification is a rare and unique process from which little is known about the resulting microbial community structure. In the present study, we characterized the microbiome of paleofeces, and ascending, transverse and descending colon of an 11th century A.D. pre-Columbian Andean mummy by 16S rRNA gene high-throughput sequencing and metagenomics. Firmicutes were the most abundant bacterial group, with Clostridium spp. comprising up to 96.2% of the mummified gut, while Turicibacter spp. represented 89.2% of the bacteria identified in the paleofeces. Microbiome profile of the paleofeces was unique when compared to previously characterized coprolites that did not undergo natural mummification. We identified DNA sequences homologous to Clostridium botulinum, Trypanosoma cruzi and human papillomaviruses (HPVs). Unexpectedly, putative antibiotic-resistance genes including beta-lactamases, penicillin-binding proteins, resistance to fosfomycin, chloramphenicol, aminoglycosides, macrolides, sulfa, quinolones, tetracycline and vancomycin, and multi-drug transporters, were also identified. The presence of putative antibiotic-resistance genes suggests that resistance may not necessarily be associated with a selective pressure of antibiotics or contact with European cultures. Identification of pathogens and antibiotic-resistance genes in ancient human specimens will aid in the understanding of the evolution of pathogens as a way to treat and prevent diseases caused by bacteria, microbial eukaryotes and viruses.

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.